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1
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Duggirala S, Balasubramanian V, Seetharaman A, Murugan S, Roy J, Hassan S, V DR, Venkatraman G, Rayala SK. Transcriptome Analysis of Human Pancreatic Stellate Cells Co-cultured With PAK1-Modulated Cells Revealed the Role of Cytokine Pathway in Tumor Microenvironment. Pancreas 2025; 54:e414-e422. [PMID: 40314739 DOI: 10.1097/mpa.0000000000002450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2024] [Accepted: 11/26/2024] [Indexed: 05/03/2025]
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high metastatic ability, poor prognosis, and resistant to treatment. Tumor microenvironment plays a major role in the complexity of PDAC. OBJECTIVE OF THE STUDY The aim of the study was to examine the role of P21 activated kinase-1 (PAK1) in the sustenance of tumor microenvironment to enable tumor growth and progression. METHODOLOGY The effect of PAK1 in the tumor microenvironment was analyzed using a novel co-culture method involving pancreatic cancer cells and pancreatic stellate cells. The 2 cell types were grown in both direct and indirect cell culture models to facilitate the juxtracrine signaling and establish a secretome network. The established network was studied using the transcriptome sequencing of PAK1-modulated MIA PaCa-2 cells co-cultured with stellate cells. RESULTS The results showed that PAK1 influenced cells have increased interferon pathway when compared to PAK1 depleted cells. The levels of chemokine CCL3 was altered in PAK1-modulated cells as evidenced by the bioinformatic, QPCR, and ELISA analysis. The pathway and interactome analysis showed that CCL3 promotes interferon activation and myofibroblast differentiation in pancreatic cancer microenvironment. These results might help in identifying the PAK1 induced metastatic network in pancreatic cancer. Further investigation will provide adequate evidence of CCL3 and PAK1 in pancreatic carcinogenesis and metastasis. CONCLUSIONS The present study provides an understanding of tumor microenvironment and involvement of inflammatory cytokines in a juxtacrine mechanism to aggravate and accelerate pancreatic adenocarcinoma.
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Affiliation(s)
- Sridevi Duggirala
- Department of Biotechnology, Indian Institute of Technology Madras (IIT Madras), Chennai, Tamil Nadu, India
- Department of Cancer Biology & Molecular Diagnostics, Cancer Institute (W.I.A), Chennai, India
| | - Vaishnavi Balasubramanian
- Department of Human Genetics, Sri Ramachandra Institute of Higher education and Research, Porur, Chennai, India
| | - Abirami Seetharaman
- Department of Biotechnology, Indian Institute of Technology Madras (IIT Madras), Chennai, Tamil Nadu, India
| | - Sowmiya Murugan
- Department of Biotechnology, Indian Institute of Technology Madras (IIT Madras), Chennai, Tamil Nadu, India
| | - Joydeep Roy
- Department of Biotechnology, Indian Institute of Technology Madras (IIT Madras), Chennai, Tamil Nadu, India
| | | | - Devi Rajeswari V
- Department of Bio-Medical Sciences, School of Bio Sciences & Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India
| | - Ganesh Venkatraman
- Department of Bio-Medical Sciences, School of Bio Sciences & Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India
| | - Suresh Kumar Rayala
- Department of Biotechnology, Indian Institute of Technology Madras (IIT Madras), Chennai, Tamil Nadu, India
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2
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Verloy R, Privat-Maldonado A, Van Audenaerde J, Rovers S, Zaryouh H, De Waele J, Quatannens D, Peeters D, Roeyen G, Deben C, Smits E, Bogaerts A. Capturing the Heterogeneity of the PDAC Tumor Microenvironment: Novel Triple Co-Culture Spheroids for Drug Screening and Angiogenic Evaluation. Cells 2025; 14:450. [PMID: 40136699 PMCID: PMC11940881 DOI: 10.3390/cells14060450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2025] [Revised: 03/07/2025] [Accepted: 03/12/2025] [Indexed: 03/27/2025] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) presents significant treatment challenges due to its desmoplastic reaction, which impedes therapeutic effectiveness, highlighting the need for advanced vitro models to better mimic the complex tumor environment. The current three-dimensional co-culture models of fibroblasts and endothelial cells are lacking, which presents a challenge for performing more comprehensive in vitro research. Our study developed triple co-culture spheroid models using MiaPaCa-2 and BxPC-3 cancer cell lines, with RLT-PSC and hPSC21 pancreatic stellate cell lines and the endothelial cell line HMEC-1. These models were assessed through growth assays, multicolor flow cytometry to optimize cell ratios, cell viability assays to evaluate drug responses, and a tube formation assay with a spheroid-conditioned medium to examine angiogenesis. Our triple co-culture spheroids effectively replicate the PDAC microenvironment, showing significant variations in drug responses influenced by cellular composition, density, and spatial arrangement. The tube formation assay showcased the potential of our models to quantitatively assess a treatment-induced angiogenic response. These cost-effective triple-co-culture in vitro spheroid models provide vital insights into the PDAC microenvironment, significantly improving the quality of the in vitro evaluation of treatment responses.
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MESH Headings
- Humans
- Tumor Microenvironment/drug effects
- Coculture Techniques/methods
- Spheroids, Cellular/pathology
- Spheroids, Cellular/drug effects
- Cell Line, Tumor
- Pancreatic Neoplasms/pathology
- Pancreatic Neoplasms/drug therapy
- Pancreatic Neoplasms/blood supply
- Carcinoma, Pancreatic Ductal/pathology
- Carcinoma, Pancreatic Ductal/drug therapy
- Carcinoma, Pancreatic Ductal/blood supply
- Neovascularization, Pathologic/pathology
- Neovascularization, Pathologic/drug therapy
- Drug Screening Assays, Antitumor/methods
- Cell Survival/drug effects
- Drug Evaluation, Preclinical/methods
- Cell Proliferation/drug effects
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Affiliation(s)
- Ruben Verloy
- Research Group PLASMANT, Department of Chemistry, University of Antwerp, 2610 Antwerp, Belgium; (A.P.-M.)
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Angela Privat-Maldonado
- Research Group PLASMANT, Department of Chemistry, University of Antwerp, 2610 Antwerp, Belgium; (A.P.-M.)
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Jonas Van Audenaerde
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Sophie Rovers
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Hannah Zaryouh
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Jorrit De Waele
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Delphine Quatannens
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Dieter Peeters
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
- Department of Pathology, University Hospital Antwerp (UZA), 2650 Antwerp, Belgium
| | - Geert Roeyen
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
- Department of Hepatobiliary Transplantation and Endocrine Surgery, University Hospital Antwerp (UZA), 2650 Antwerp, Belgium
| | - Christophe Deben
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Evelien Smits
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), University of Antwerp, 2610 Antwerp, Belgium
| | - Annemie Bogaerts
- Research Group PLASMANT, Department of Chemistry, University of Antwerp, 2610 Antwerp, Belgium; (A.P.-M.)
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3
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Brichkina A, Ems M, Suezov R, Singh R, Lutz V, Picard FSR, Nist A, Stiewe T, Graumann J, Daude M, Diederich WE, Finkernagel F, Chung HR, Bartsch DK, Roth K, Keber C, Denkert C, Huber M, Gress TM, Lauth M. DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer. Gut 2024; 73:1684-1701. [PMID: 38834297 PMCID: PMC11420735 DOI: 10.1136/gutjnl-2023-331854] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Accepted: 05/15/2024] [Indexed: 06/06/2024]
Abstract
OBJECTIVE Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.
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Affiliation(s)
- Anna Brichkina
- Department of Gastroenterology Endocrinology and Metabolism, Center for Tumor and Immune Biology, Marburg, Germany
- Present address: Institute of Systems Immunology, Center for Tumor and Immune Biology, Marburg, Germany
| | - Miriam Ems
- Department of Gastroenterology Endocrinology and Metabolism, Center for Tumor and Immune Biology, Marburg, Germany
| | - Roman Suezov
- Department of Gastroenterology Endocrinology and Metabolism, Center for Tumor and Immune Biology, Marburg, Germany
| | - Rajeev Singh
- Department of Gastroenterology Endocrinology and Metabolism, Center for Tumor and Immune Biology, Marburg, Germany
| | - Veronika Lutz
- Institute of Systems Immunology, Philipps-Universitat Marburg, Marburg, Hessen, Germany
| | - Felix S R Picard
- Institute of Systems Immunology, Philipps-Universitat Marburg, Marburg, Hessen, Germany
| | - Andrea Nist
- Genomics Core Facility, Philipps University Marburg, Marburg, Germany
| | - Thorsten Stiewe
- Genomics Core Facility, Philipps University Marburg, Marburg, Germany
- Institute for Molecular Oncology, German Center for Lung Research (DZL), Marburg, Germany
| | - Johannes Graumann
- Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
- Institute of Translational Proteomics, Philipps University, Marburg, Germany
| | - Michael Daude
- Medicinal Chemistry Core Facility, Philipps University Marburg, Marburg, Germany
| | - Wibke E Diederich
- Medicinal Chemistry Core Facility, Philipps University Marburg, Marburg, Germany
- Department of Medicinal chemistry, Center for Tumor and Immune Biology, Marburg, Germany
| | - Florian Finkernagel
- Bioinformatics Core Facility, Center for Tumor and Immune Biology, Marburg, Germany
| | - Ho-Ryun Chung
- Institute for Medical Bioinformatics and Biostatistics, Institute for Molecular Biology and Tumor Research, Marburg, Germany
| | - Detlef K Bartsch
- Department of Visceral, Thoracic and Vascular Surgery, Philipps-University Marburg, Marburg, Germany
| | - Katrin Roth
- Cell Imaging Core Facility, Center for Tumor Biology and Immunology, Philipps-University Marburg, Marburg, Hessen, Germany
| | - Corinna Keber
- Institute of Pathology, University Hospital of Giessen-Marburg, Marburg, Germany
| | - Carsten Denkert
- Institute of Pathology, University Hospital of Giessen-Marburg, Marburg, Germany
| | - Magdalena Huber
- Institute of Systems Immunology, Philipps-Universitat Marburg, Marburg, Hessen, Germany
| | - Thomas M Gress
- Department of Gastroenterology, Endocrinology and Metabolism, Center for Tumor and Immune Biology, Marburg, Germany
| | - Matthias Lauth
- Department of Gastroenterology Endocrinology and Metabolism, Center for Tumor and Immune Biology, Marburg, Germany
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4
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Löhr JM. Pancreas 2000. My journey with the central organ. Pancreatology 2024; 24:671-676. [PMID: 38641487 DOI: 10.1016/j.pan.2024.03.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2024] [Accepted: 03/27/2024] [Indexed: 04/21/2024]
Abstract
The European Pancreatic Club Lifetime Achievement Award is a distinction awarded for research on the pancreas and service to European Pancreatology. It comes with the obligation to submit a review article to our society's journal, Pancreatology. It was awarded to me 2023 and I take this opportunity to highlight my journey with the central organ AKA the pancreas, that is inseparatable from "Pancreas 2000" - an educational program for future pancreatologists, inaugurated by Karolinska Institutet.
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Affiliation(s)
- J-Matthias Löhr
- Karolinska Institutet, Alfred Nobels Allé 8, S-141 86, Stockholm, Sweden.
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5
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Magni L, Yu H, Christensen NM, Poulsen MH, Frueh A, Deshar G, Johansen AZ, Johansen JS, Pless SA, Jørgensen NR, Novak I. Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer. Cancer Cell Int 2024; 24:148. [PMID: 38664691 PMCID: PMC11044319 DOI: 10.1186/s12935-024-03339-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2024] [Accepted: 04/18/2024] [Indexed: 04/29/2024] Open
Abstract
BACKGROUND The purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC. MATERIALS AND METHODS Blood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay. RESULTS Risk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1β, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects. CONCLUSION In conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.
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Affiliation(s)
- Lara Magni
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen Ø, Denmark
| | - Haoran Yu
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen Ø, Denmark
| | - Nynne M Christensen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen Ø, Denmark
| | - Mette H Poulsen
- Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
| | - Alexander Frueh
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen Ø, Denmark
| | - Ganga Deshar
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen Ø, Denmark
| | - Astrid Z Johansen
- Department of Oncology, Copenhagen University Hospital - Herlev and Gentofte Hospital, Herlev, Denmark
| | - Julia S Johansen
- Department of Oncology, Copenhagen University Hospital - Herlev and Gentofte Hospital, Herlev, Denmark
- Department of Medicine, Copenhagen University Hospital - Herlev and Gentofte, Herlev, Denmark
| | - Stephan A Pless
- Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
| | - Niklas R Jørgensen
- Department of Clinical Biochemistry, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark
- Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Ivana Novak
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen Ø, Denmark.
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Van den Eynde A, Gehrcken L, Verhezen T, Lau HW, Hermans C, Lambrechts H, Flieswasser T, Quatannens D, Roex G, Zwaenepoel K, Marcq E, Joye P, Cardenas De La Hoz E, Deben C, Gasparini A, Montay-Gruel P, Le Compte M, Lion E, Lardon F, Van Laere S, Siozopoulou V, Campillo-Davo D, De Waele J, Pauwels P, Jacobs J, Smits E, Van Audenaerde JRM. IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts. J Hematol Oncol 2024; 17:8. [PMID: 38331849 PMCID: PMC10854128 DOI: 10.1186/s13045-024-01525-w] [Citation(s) in RCA: 31] [Impact Index Per Article: 31.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2023] [Accepted: 01/11/2024] [Indexed: 02/10/2024] Open
Abstract
BACKGROUND It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma. METHODS RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs. RESULTS In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner. CONCLUSIONS We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.
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Affiliation(s)
- Astrid Van den Eynde
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium.
| | - Laura Gehrcken
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Tias Verhezen
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Ho Wa Lau
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Christophe Hermans
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Hilde Lambrechts
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Tal Flieswasser
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Delphine Quatannens
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Gils Roex
- Laboratory of Experimental Hematology (LEH), Vaccine and Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium
| | - Karen Zwaenepoel
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- Department of Pathology, Antwerp University Hospital, Edegem, Belgium
| | - Elly Marcq
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- Lab of Dendritic Cell Biology and Cancer Immunotherapy, VIB Center for Inflammation Research, Brussels, Belgium
- Brussels Center for Immunology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Philippe Joye
- Molecular Imaging Center Antwerp (MICA), University of Antwerp, Wilrijk, Belgium
| | | | - Christophe Deben
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Alessia Gasparini
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- Iridium Netwerk, Radiation Oncology, Antwerp, Belgium
| | - Pierre Montay-Gruel
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- Iridium Netwerk, Radiation Oncology, Antwerp, Belgium
| | - Maxim Le Compte
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Eva Lion
- Laboratory of Experimental Hematology (LEH), Vaccine and Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium
- Center for Cell Therapy and Regenerative Medicine (CCRG), Antwerp University Hospital, Edegem, Belgium
| | - Filip Lardon
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Steven Van Laere
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- Department of Pathology, Antwerp University Hospital, Edegem, Belgium
| | - Vasiliki Siozopoulou
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- University Hospital Saint-Luc, University of Louvain, Brussels, Belgium
| | - Diana Campillo-Davo
- Laboratory of Experimental Hematology (LEH), Vaccine and Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, Edegem, Belgium
| | - Jorrit De Waele
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Patrick Pauwels
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
- Department of Pathology, Antwerp University Hospital, Edegem, Belgium
| | - Julie Jacobs
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Evelien Smits
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Jonas R M Van Audenaerde
- Center for Oncological Research (CORE), Integrated Personalized and Precision Oncology Network (IPPON), Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
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7
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Schwörer S, Cimino FV, Ros M, Tsanov KM, Ng C, Lowe SW, Carmona-Fontaine C, Thompson CB. Hypoxia Potentiates the Inflammatory Fibroblast Phenotype Promoted by Pancreatic Cancer Cell-Derived Cytokines. Cancer Res 2023; 83:1596-1610. [PMID: 36912618 PMCID: PMC10658995 DOI: 10.1158/0008-5472.can-22-2316] [Citation(s) in RCA: 61] [Impact Index Per Article: 30.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2022] [Revised: 01/19/2023] [Accepted: 03/09/2023] [Indexed: 03/14/2023]
Abstract
Cancer-associated fibroblasts (CAF) are a major cell type in the stroma of solid tumors and can exert both tumor-promoting and tumor-restraining functions. CAF heterogeneity is frequently observed in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by a dense and hypoxic stroma that features myofibroblastic CAFs (myCAF) and inflammatory CAFs (iCAF) that are thought to have opposing roles in tumor progression. While CAF heterogeneity can be driven in part by tumor cell-produced cytokines, other determinants shaping CAF identity and function are largely unknown. In vivo, we found that iCAFs displayed a hypoxic gene expression and biochemical profile and were enriched in hypoxic regions of PDAC tumors, while myCAFs were excluded from these regions. Hypoxia led fibroblasts to acquire an inflammatory gene expression signature and synergized with cancer cell-derived cytokines to promote an iCAF phenotype in a HIF1α-dependent fashion. Furthermore, HIF1α stabilization was sufficient to induce an iCAF phenotype in stromal cells introduced into PDAC organoid cocultures and to promote PDAC tumor growth. These findings indicate hypoxia-induced HIF1α as a regulator of CAF heterogeneity and promoter of tumor progression in PDAC. SIGNIFICANCE Hypoxia in the tumor microenvironment of pancreatic cancer potentiates the cytokine-induced inflammatory CAF phenotype and promotes tumor growth. See related commentary by Fuentes and Taniguchi, p. 1560.
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Affiliation(s)
- Simon Schwörer
- Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, Illinois
| | - Francesco V Cimino
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York
| | - Manon Ros
- Center for Genomics and Systems Biology, New York University, New York, New York
| | - Kaloyan M Tsanov
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York
| | - Charles Ng
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York
| | - Scott W Lowe
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York
- Howard Hughes Medical Institute, Chevy Chase, Maryland
| | | | - Craig B Thompson
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York
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8
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Lottini T, Duranti C, Iorio J, Martinelli M, Colasurdo R, D’Alessandro FN, Buonamici M, Coppola S, Devescovi V, La Vaccara V, Coppola A, Coppola R, Lastraioli E, Arcangeli A. Combination Therapy with a Bispecific Antibody Targeting the hERG1/β1 Integrin Complex and Gemcitabine in Pancreatic Ductal Adenocarcinoma. Cancers (Basel) 2023; 15:2013. [PMID: 37046674 PMCID: PMC10093586 DOI: 10.3390/cancers15072013] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2023] [Revised: 03/10/2023] [Accepted: 03/26/2023] [Indexed: 03/30/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) represents an unmet medical need. Difficult/late diagnosis as well as the poor efficacy and high toxicity of chemotherapeutic drugs result in dismal prognosis. With the aim of improving the treatment outcome of PDAC, we tested the effect of combining Gemcitabine with a novel single chain bispecific antibody (scDb) targeting the cancer-specific hERG1/β1 integrin complex. First, using the scDb (scDb-hERG1-β1) in immunohistochemistry (IHC), Western blot (WB) analysis and immunofluorescence (IF), we confirmed the presence of the hERG1/β1 integrin complex in primary PDAC samples and PDAC cell lines. Combining Gemcitabine with scDb-hERG1-β1 improved its cytotoxicity on all PDAC cells tested in vitro. We also tested the combination treatment in vivo, using an orthotopic xenograft mouse model involving ultrasound-guided injection of PDAC cells. We first demonstrated good penetration of the scDb-hERG1-β1 conjugated with indocyanine green (ICG) into tumour masses by photoacoustic (PA) imaging. Next, we tested the effects of the combination at either therapeutic or sub-optimal doses of Gemcitabine (25 or 5 mg/kg, respectively). The combination of scDb-hERG1-β1 and sub-optimal doses of Gemcitabine reduced the tumour masses to the same extent as the therapeutic doses of Gemcitabine administrated alone; yielded increased survival; and was accompanied by minimised side effects (toxicity). These data pave the way for a novel therapeutic approach to PDAC, based on the combination of low doses of a chemotherapeutic drug (to minimize adverse side effects and the onset of resistance) and the novel scDb-hERG1-β1 targeting the hERG1/β1 integrin complex as neoantigen.
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Affiliation(s)
- Tiziano Lottini
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
| | - Claudia Duranti
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
| | - Jessica Iorio
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
| | - Michele Martinelli
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
- Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy
| | - Rossella Colasurdo
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
- Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy
| | - Franco Nicolás D’Alessandro
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
- Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy
| | - Matteo Buonamici
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
| | - Stefano Coppola
- Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands
| | - Valentina Devescovi
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
| | - Vincenzo La Vaccara
- General Surgery Unit, Department of Medicine, Fondazione Policlinico Universitario Campus Bio-Medico, Via Alvaro del Portillo, 00128 Rome, Italy
| | | | - Roberto Coppola
- General Surgery Unit, Department of Medicine, Fondazione Policlinico Universitario Campus Bio-Medico, Via Alvaro del Portillo, 00128 Rome, Italy
| | - Elena Lastraioli
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
| | - Annarosa Arcangeli
- Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, 50134 Firenze, Italy
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9
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Vom Stein AF, Rebollido-Rios R, Lukas A, Koch M, von Lom A, Reinartz S, Bachurski D, Rose F, Bozek K, Abdallah AT, Kohlhas V, Saggau J, Zölzer R, Zhao Y, Bruns C, Bröckelmann PJ, Lohneis P, Büttner R, Häupl B, Oellerich T, Nguyen PH, Hallek M. LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1. Nat Commun 2023; 14:1330. [PMID: 36899005 PMCID: PMC10006233 DOI: 10.1038/s41467-023-36824-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Accepted: 02/14/2023] [Indexed: 03/12/2023] Open
Abstract
Microenvironmental bystander cells are essential for the progression of chronic lymphocytic leukemia (CLL). We have discovered previously that LYN kinase promotes the formation of a microenvironmental niche for CLL. Here we provide mechanistic evidence that LYN regulates the polarization of stromal fibroblasts to support leukemic progression. LYN is overexpressed in fibroblasts of lymph nodes of CLL patients. LYN-deficient stromal cells reduce CLL growth in vivo. LYN-deficient fibroblasts show markedly reduced leukemia feeding capacity in vitro. Multi-omics profiling reveals that LYN regulates the polarization of fibroblasts towards an inflammatory cancer-associated phenotype through modulation of cytokine secretion and extracellular matrix composition. Mechanistically, LYN deletion reduces inflammatory signaling including reduction of c-JUN expression, which in turn augments the expression of Thrombospondin-1, which binds to CD47 thereby impairing CLL viability. Together, our findings suggest that LYN is essential for rewiring fibroblasts towards a leukemia-supportive phenotype.
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Affiliation(s)
- Alexander F Vom Stein
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Rocio Rebollido-Rios
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Anna Lukas
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Maximilian Koch
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Anton von Lom
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
- Mildred Scheel School of Oncology Aachen Bonn Cologne Düsseldorf, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
| | - Sebastian Reinartz
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Daniel Bachurski
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
- Mildred Scheel School of Oncology Aachen Bonn Cologne Düsseldorf, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
| | - France Rose
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- University of Cologne, Institute for Biomedical Informatics, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
| | - Katarzyna Bozek
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
- University of Cologne, Institute for Biomedical Informatics, Faculty of Medicine and University Hospital Cologne, Cologne, Germany
| | - Ali T Abdallah
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Viktoria Kohlhas
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Julia Saggau
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Rebekka Zölzer
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
| | - Yue Zhao
- Faculty of Medicine and University Hospital Cologne, Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany
| | - Christiane Bruns
- Faculty of Medicine and University Hospital Cologne, Department of General, Visceral and Cancer Surgery, University of Cologne, Cologne, Germany
| | - Paul J Bröckelmann
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany
- Mildred Scheel School of Oncology Aachen Bonn Cologne Düsseldorf, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany
- Max-Planck Institute for the Biology of Ageing, Cologne, Germany
| | - Philipp Lohneis
- Reference Centre for Lymph Node Pathology and Hematopathology, Hämatopathologie Lübeck, Lübeck, Germany
- Faculty of Medicine and University Hospital Cologne, Department of Pathology, University of Cologne, Cologne, Germany
| | - Reinhard Büttner
- Faculty of Medicine and University Hospital Cologne, Department of Pathology, University of Cologne, Cologne, Germany
| | - Björn Häupl
- Department of Hematology/Oncology, Johann Wolfgang Goethe University, Frankfurt, Germany
- German Cancer Consortium (DKTK), Heidelberg, Germany
- German Cancer Research Center (DKFZ), Heidelberg, Germany
- Frankfurt Cancer Institute, Goethe University Frankfurt, Frankfurt, Germany
| | - Thomas Oellerich
- Department of Hematology/Oncology, Johann Wolfgang Goethe University, Frankfurt, Germany
- German Cancer Consortium (DKTK), Heidelberg, Germany
- German Cancer Research Center (DKFZ), Heidelberg, Germany
- Frankfurt Cancer Institute, Goethe University Frankfurt, Frankfurt, Germany
| | - Phuong-Hien Nguyen
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany.
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.
| | - Michael Hallek
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, Cologne, Germany.
- Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
- CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.
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10
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Geyer M, Schreyer D, Gaul LM, Pfeffer S, Pilarsky C, Queiroz K. A microfluidic-based PDAC organoid system reveals the impact of hypoxia in response to treatment. Cell Death Dis 2023; 9:20. [PMID: 36681673 PMCID: PMC9867742 DOI: 10.1038/s41420-023-01334-z] [Citation(s) in RCA: 30] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 01/11/2023] [Accepted: 01/12/2023] [Indexed: 01/22/2023]
Abstract
Pancreatic Ductal Adenocarcinoma (PDAC) is estimated to become the second leading cause of cancer-related deaths by 2030 with mortality rates of up to 93%. Standard of care chemotherapeutic treatment only prolongs the survival of patients for a short timeframe. Therefore, it is important to understand events driving treatment failure in PDAC as well as identify potential more effective treatment opportunities. PDAC is characterized by a high-density stroma, high interstitial pressure and very low oxygen tension. The aim of this study was to establish a PDAC platform that supported the understanding of treatment response of PDAC organoids in mono-, and co-culture with pancreatic stellate cells (PSCs) under hypoxic and normoxic conditions. Cultures were exposed to Gemcitabine in combination with molecules targeting relevant molecular programs that could explain treatment specific responses under different oxygen pressure conditions. Two groups of treatment responses were identified, showing either a better effect in monoculture or co-culture. Moreover, treatment response also differed between normoxia and hypoxia. Modulation of response to Gemcitabine was also observed in presence of a Hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) inhibitor and HIF inhibitors. Altogether this highlights the importance of adjusting experimental conditions to include relevant oxygen levels in drug response studies in PDAC.
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Affiliation(s)
- Marlene Geyer
- grid.474144.60000 0004 9414 4776MIMETAS BV, De Limes 7, 2342DH Oegstgeest, The Netherlands
| | - Daniel Schreyer
- grid.8756.c0000 0001 2193 314XSchool of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Bearsden, G61 1BD Glasgow, United Kingdom
| | - Lisa-Marie Gaul
- grid.474144.60000 0004 9414 4776MIMETAS BV, De Limes 7, 2342DH Oegstgeest, The Netherlands
| | - Susanne Pfeffer
- grid.411668.c0000 0000 9935 6525Universitätsklinikum Erlangen, Schwabachanlage 12, 91054 Erlangen, Germany
| | - Christian Pilarsky
- grid.411668.c0000 0000 9935 6525Universitätsklinikum Erlangen, Schwabachanlage 12, 91054 Erlangen, Germany
| | - Karla Queiroz
- grid.474144.60000 0004 9414 4776MIMETAS BV, De Limes 7, 2342DH Oegstgeest, The Netherlands
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11
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Geyer M, Gaul LM, D`Agosto SL, Corbo V, Queiroz K. The tumor stroma influences immune cell distribution and recruitment in a PDAC-on-a-chip model. Front Immunol 2023; 14:1155085. [PMID: 37205118 PMCID: PMC10185841 DOI: 10.3389/fimmu.2023.1155085] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Accepted: 03/30/2023] [Indexed: 05/21/2023] Open
Abstract
The dense tumor stroma of pancreatic ductal adenocarcinoma (PDAC) and its secreted immune active molecules provide a barrier for chemotherapy treatment as well as for immune cell infiltration to the tumor core, providing a challenge for immunotherapeutic strategies. Consequently, the investigation of processes underlying the interaction between the tumor stroma, particularly activated pancreatic stellate cells (PSCs), and immune cells may offer new therapeutic approaches for PDAC treatment. In this study, we established a 3D PDAC model cultured under flow, consisting of an endothelial tube, PSCs and PDAC organoids. This was applied to study the role of the tumor microenvironment (TME) on immune cell recruitment and its effect on partly preventing their interaction with pancreatic cancer cells. We observed that stromal cells form a physical barrier, partly shielding the cancer cells from migrating immune cells, as well as a biochemical microenvironment, that seems to attract and influence immune cell distribution. In addition, stromal targeting by Halofuginone led to an increase in immune cell infiltration. We propose that the here developed model setups will support the understanding of the cellular interplay influencing the recruitment and distribution of immune cells, and contribute to the identification of key players in the PDAC immunosuppressive TME as well as support the discovery of new strategies to treat this immune unresponsive tumor.
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Affiliation(s)
| | | | | | - Vincenzo Corbo
- Department of Diagnostic and Public Health, University of Verona, Verona, Italy
| | - Karla Queiroz
- Mimetas B.V., Oegstgeest, Netherlands
- *Correspondence: Karla Queiroz,
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12
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Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells. STAR Protoc 2022; 3:101869. [PMID: 36595896 PMCID: PMC9692066 DOI: 10.1016/j.xpro.2022.101869] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Revised: 10/03/2022] [Accepted: 10/31/2022] [Indexed: 11/23/2022] Open
Abstract
Ex vivo organ culture can be a useful alternative to in vivo models, which can be time-, labor-, and cost-intensive. Here we describe a step-by-step protocol to use de-epithelialized porcine urinary bladders as scaffolds in air-liquid interface in vitro culture systems for a variety of pluripotent stem-cell-derived and patient-derived pancreatic cells and organoids. The scaffold can trigger cell maturation and enable cell-cell interaction and invasion capacity studies. However, this model is limited by the lack of functional vasculature. For complete details on the use and execution of this protocol, please refer to Melzer et al. (2022),1 Breunig et al. (2021),2 and Breunig et al. (2021).3.
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13
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Zhao T, Xiao D, Jin F, Sun X, Yu J, Wang H, Liu J, Cai W, Huang C, Wang X, Gao S, Liu Z, Yang S, Gao C, Hao J. ESE3-positive PSCs drive pancreatic cancer fibrosis, chemoresistance and poor prognosis via tumour-stromal IL-1β/NF-κB/ESE3 signalling axis. Br J Cancer 2022; 127:1461-1472. [PMID: 35986089 PMCID: PMC9553871 DOI: 10.1038/s41416-022-01927-y] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2021] [Revised: 07/08/2022] [Accepted: 07/15/2022] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Desmoplastic stroma, a feature of pancreatic ductal adenocarcinoma (PDAC), contains abundant activated pancreatic stellate cells (PSCs). How PSCs promote PDAC progression remains incompletely understood. METHODS Effect of epithelium-specific E-twenty six factor 3 (ESE3)-positive PSCs on PDAC fibrosis and chemoresistance was examined by western blot, RT-PCR, immunofluorescence, flow cytometry assay, chromatin immunoprecipitation, luciferase assay, immunohistochemistry and subcutaneous pancreatic cancer mouse model. RESULTS ESE3 expression increased in PSCs in PDAC tissues compared with those in normal PSCs. Clinical data showed that ESE3 upregulation in PSCs was positively correlated with tumour size, pTNM stage, CA19-9, carcinoembryonic antigen and serum CA242 level. ESE3 overexpression in PSCs was an independent negative prognostic factor for disease-free survival and overall survival amongst patients with PDAC. Mechanistically, the conditional medium from the loss and gain of ESE3-expressing PSCs influenced PDAC chemoresistance and tumour growth. ESE3 directly induced the transcription of α-SMA, collagen-I and IL-1β by binding to ESE3-binding sites on their promoters to activate PSCs. IL-1β upregulated ESE3 in PSCs through NF-κB activation, and ESE3 was required for PSC activation by tumour cell-derived IL-1β. CONCLUSION Inhibiting the IL-1β/ESE3 (PSCs)/IL-1β-positive feedback loop is a promising therapeutic strategy to reduce tumour fibrosis and increase chemotherapeutic efficacy in PDAC.
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Affiliation(s)
- Tiansuo Zhao
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Di Xiao
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Fanjie Jin
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Xugang Sun
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Jie Yu
- grid.452461.00000 0004 1762 8478Hepatopancreatobiliary Surgery Department, First Hospital of Shanxi Medical University, Taiyuan, PR China
| | - Hongwei Wang
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Jing Liu
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Wenrun Cai
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Chongbiao Huang
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Xiuchao Wang
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Song Gao
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Zhe Liu
- grid.265021.20000 0000 9792 1228Department of Immunology, Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, PR China
| | - Shengyu Yang
- grid.240473.60000 0004 0543 9901Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA USA
| | - Chuntao Gao
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Jihui Hao
- grid.411918.40000 0004 1798 6427Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
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14
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TRPM7 Modulates Human Pancreatic Stellate Cell Activation. Cells 2022; 11:cells11142255. [PMID: 35883700 PMCID: PMC9316618 DOI: 10.3390/cells11142255] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 07/10/2022] [Accepted: 07/19/2022] [Indexed: 01/27/2023] Open
Abstract
Pancreatic diseases, such as pancreatitis or pancreatic ductal adenocarcinoma, are characterized by the presence of activated pancreatic stellate cells (PSCs). These cells represent key actors in the tumor stroma, as they actively participate in disease development and progression: reprograming these PSCs into a quiescent phenotype has even been proposed as a promising strategy for restoring the hallmarks of a healthy pancreas. Since TRPM7 channels have been shown to regulate hepatic stellate cells proliferation and survival, we aimed to study the role of these magnesium channels in PSC activation and proliferation. PS-1 cells (isolated from a healthy pancreas) were used as a model of healthy PSCs: quiescence or activation were induced using all-trans retinoic acid or conditioned media of pancreatic cancer cells, respectively. The role of TRPM7 was studied by RNA silencing or by pharmacological inhibition. TRPM7 expression was found to be correlated with the activation status of PS-1 cells. TRPM7 expression was able to regulate proliferation through modulation of cell cycle regulators and most importantly p53, via the PI3K/Akt pathway, in a magnesium-dependent manner. Finally, the analysis of TCGA database showed the overexpression of TRPM7 in cancer-associated fibroblasts. Taken together, we provide strong evidences that TRPM7 can be considered as a marker of activated PSCs.
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15
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Melzer MK, Breunig M, Arnold F, Wezel F, Azoitei A, Roger E, Krüger J, Merkle J, Schütte L, Resheq Y, Hänle M, Zehe V, Zengerling F, Azoitei N, Klein L, Penz F, Singh SK, Seufferlein T, Hohwieler M, Bolenz C, Günes C, Gout J, Kleger A. Organoids at the PUB: The Porcine Urinary Bladder Serves as a Pancreatic Niche for Advanced Cancer Modeling. Adv Healthc Mater 2022; 11:e2102345. [PMID: 35114730 PMCID: PMC11468201 DOI: 10.1002/adhm.202102345] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Revised: 12/17/2021] [Indexed: 12/17/2022]
Abstract
Despite intensive research and progress in personalized medicine, pancreatic ductal adenocarcinoma remains one of the deadliest cancer entities. Pancreatic duct-like organoids (PDLOs) derived from human pluripotent stem cells (PSCs) or pancreatic cancer patient-derived organoids (PDOs) provide unique tools to study early and late stage dysplasia and to foster personalized medicine. However, such advanced systems are neither rapidly nor easily accessible and require an in vivo niche to study tumor formation and interaction with the stroma. Here, the establishment of the porcine urinary bladder (PUB) is revealed as an advanced organ culture model for shaping an ex vivo pancreatic niche. This model allows pancreatic progenitor cells to enter the ductal and endocrine lineages, while PDLOs further mature into duct-like tissue. Accordingly, the PUB offers an ex vivo platform for earliest pancreatic dysplasia and cancer if PDLOs feature KRASG12D mutations. Finally, it is demonstrated that PDOs-on-PUB i) resemble primary pancreatic cancer, ii) preserve cancer subtypes, iii) enable the study of niche epithelial crosstalk by spiking in pancreatic stellate and immune cells into the grafts, and finally iv) allow drug testing. In summary, the PUB advances the existing pancreatic cancer models by adding feasibility, complexity, and customization at low cost and high flexibility.
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Affiliation(s)
- Michael Karl Melzer
- Department of UrologyUlm UniversityUlm89081Germany
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Markus Breunig
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Frank Arnold
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Felix Wezel
- Department of UrologyUlm UniversityUlm89081Germany
| | - Anca Azoitei
- Department of UrologyUlm UniversityUlm89081Germany
| | - Elodie Roger
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Jana Krüger
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Jessica Merkle
- Department of Internal Medicine IUlm UniversityUlm89081Germany
- Core Facility OrganoidsUlm UniversityUlm89081Germany
| | - Lena Schütte
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Yazid Resheq
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Mark Hänle
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Viktor Zehe
- Department of UrologyUlm UniversityUlm89081Germany
| | | | - Ninel Azoitei
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Lukas Klein
- Department of GastroenterologyGastrointestinal Oncology and EndocrinologyUniversity Medicine GoettingenGoettingen37075Germany
| | - Frederike Penz
- Department of GastroenterologyGastrointestinal Oncology and EndocrinologyUniversity Medicine GoettingenGoettingen37075Germany
| | - Shiv K. Singh
- Department of GastroenterologyGastrointestinal Oncology and EndocrinologyUniversity Medicine GoettingenGoettingen37075Germany
| | | | - Meike Hohwieler
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | | | | | - Johann Gout
- Department of Internal Medicine IUlm UniversityUlm89081Germany
| | - Alexander Kleger
- Department of Internal Medicine IUlm UniversityUlm89081Germany
- Core Facility OrganoidsUlm UniversityUlm89081Germany
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16
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Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells. Cancers (Basel) 2022; 14:cancers14102508. [PMID: 35626109 PMCID: PMC9139966 DOI: 10.3390/cancers14102508] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Revised: 05/11/2022] [Accepted: 05/13/2022] [Indexed: 11/17/2022] Open
Abstract
Simple Summary How a single cytokine can induce a variety of cellular responses in the same cell or in different cells is a longstanding question. Transforming growth factor β (TGF-β) is a prototypical multifunctional cytokine of which biological responses are highly dependent on in a cellular context. TGF-β signals via intracellular SMAD transcription factors, and the duration and intensity of SMAD activation are key determinants for the responses that are elicited by TGF-β. To visualize the TGF-β signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional reporter using a quickly folded and highly unstable green florescent protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β-induced responses in cells cultured on plastic dishes, and in living animals. This tool allows for the analysis of TGF-β signaling at a single living cell level, and allows for the discovery of dynamic TGF-β SMAD- induced transcriptional responses in multi-step biological processes. Abstract Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.
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17
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Bots ST, Kemp V, Cramer SJ, van den Wollenberg DJ, Hornsveld M, Lamfers ML, van der Pluijm G, Hoeben RC. Nonhuman Primate Adenoviruses of the Human Adenovirus B Species Are Potent and Broadly Acting Oncolytic Vector Candidates. Hum Gene Ther 2022; 33:275-289. [PMID: 34861769 PMCID: PMC8972008 DOI: 10.1089/hum.2021.216] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 11/24/2021] [Indexed: 12/11/2022] Open
Abstract
The use of human adenoviruses (hAds) as oncolytic agents has demonstrated considerable potential. However, their efficacy in clinical studies is generally moderate and often varies between patients. This may, in part, be attributable to variable pre-existing neutralizing immunity in patients, which can impact the antitumor efficacy and lead to response heterogeneity. Our aim was to isolate new Ads for the development of oncolytic vectors with low prevalence of neutralizing immunity in the human population. To this end, we isolated a collection of new nonhuman primate (nhp) Ads from stool samples of four great ape species held captive. We elected 12 isolates comprising the broadest genetic variability for further characterization. For three new nhpAds, all classified as the human adenovirus B (HAdV-B) species, no neutralizing activity could be detected when exposed to a preparation of immunoglobulins isolated from a pool of >1,000 donors as a surrogate of population immunity. In addition, the nhpAds of the HAdV-B species showed enhanced oncolytic potency compared to nhpAds of the HAdV-C species as well as to human adenovirus type 5 (HAdV-C5) in vitro when tested in a panel of 29 human cancer cell lines. Next-generation sequencing of the viral genomes revealed higher sequence similarity between hAds and nhpAds of HAdV-B compared to HAdV-C, which might underlie the differences in oncolytic ability. As a proof-of-concept, the Rb-binding domain of the E1A protein of the gorilla-derived HAdV-B nhpAd-lumc007 was deleted, thereby creating a new oncolytic derivative, which demonstrated increased oncolytic potential compared to HAdV-C5. Collectively, our data demonstrate that nhpAds of the HAdV-B species can serve as an alternative for the development of potent oncolytic Ad vectors with limited pre-existing neutralizing immunity in humans.
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Affiliation(s)
- Selas T.F. Bots
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
| | - Vera Kemp
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
| | - Steve J. Cramer
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
| | | | - Marten Hornsveld
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
| | - Martine L.M. Lamfers
- Department of Neurosurgery, Brain Tumor Center, Erasmus Medical Center, Rotterdam, the Netherlands
| | - Gabri van der Pluijm
- Department of Urology, Leiden University Medical Center, Leiden, the Netherlands
| | - Rob C. Hoeben
- Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
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18
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Privat-Maldonado A, Verloy R, Cardenas Delahoz E, Lin A, Vanlanduit S, Smits E, Bogaerts A. Cold Atmospheric Plasma Does Not Affect Stellate Cells Phenotype in Pancreatic Cancer Tissue in Ovo. Int J Mol Sci 2022; 23:ijms23041954. [PMID: 35216069 PMCID: PMC8878510 DOI: 10.3390/ijms23041954] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2022] [Revised: 02/04/2022] [Accepted: 02/08/2022] [Indexed: 02/06/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a challenging neoplastic disease, mainly due to the development of resistance to radio- and chemotherapy. Cold atmospheric plasma (CAP) is an alternative technology that can eliminate cancer cells through oxidative damage, as shown in vitro, in ovo, and in vivo. However, how CAP affects the pancreatic stellate cells (PSCs), key players in the invasion and metastasis of PDAC, is poorly understood. This study aims to determine the effect of an anti-PDAC CAP treatment on PSCs tissue developed in ovo using mono- and co-cultures of RLT-PSC (PSCs) and Mia PaCa-2 cells (PDAC). We measured tissue reduction upon CAP treatment and mRNA expression of PSC activation markers and extracellular matrix (ECM) remodelling factors via qRT-PCR. Protein expression of selected markers was confirmed via immunohistochemistry. CAP inhibited growth in Mia PaCa-2 and co-cultured tissue, but its effectiveness was reduced in the latter, which correlates with reduced ki67 levels. CAP did not alter the mRNA expression of PSC activation and ECM remodelling markers. No changes in MMP2 and MMP9 expression were observed in RLT-PSCs, but small changes were observed in Mia PaCa-2 cells. Our findings support the ability of CAP to eliminate PDAC cells, without altering the PSCs.
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Affiliation(s)
- Angela Privat-Maldonado
- PLASMANT, Chemistry Department, Faculty of Sciences, University of Antwerp, 2610 Antwerp, Belgium; (R.V.); (A.L.); (A.B.)
- Solid Tumor Immunology Group, Center for Oncological Research, Integrated Personalized and Precision Oncology Network, Department of Molecular Imaging, Pathology, Radiotherapy and Oncology, University of Antwerp, 2610 Antwerp, Belgium;
- Correspondence: ; Tel.: +32-3265-25-76
| | - Ruben Verloy
- PLASMANT, Chemistry Department, Faculty of Sciences, University of Antwerp, 2610 Antwerp, Belgium; (R.V.); (A.L.); (A.B.)
- Solid Tumor Immunology Group, Center for Oncological Research, Integrated Personalized and Precision Oncology Network, Department of Molecular Imaging, Pathology, Radiotherapy and Oncology, University of Antwerp, 2610 Antwerp, Belgium;
| | - Edgar Cardenas Delahoz
- Industrial Vision Lab InViLab, Faculty of Applied Engineering, University of Antwerp, 2610 Antwerp, Belgium; (E.C.D.); (S.V.)
| | - Abraham Lin
- PLASMANT, Chemistry Department, Faculty of Sciences, University of Antwerp, 2610 Antwerp, Belgium; (R.V.); (A.L.); (A.B.)
- Solid Tumor Immunology Group, Center for Oncological Research, Integrated Personalized and Precision Oncology Network, Department of Molecular Imaging, Pathology, Radiotherapy and Oncology, University of Antwerp, 2610 Antwerp, Belgium;
| | - Steve Vanlanduit
- Industrial Vision Lab InViLab, Faculty of Applied Engineering, University of Antwerp, 2610 Antwerp, Belgium; (E.C.D.); (S.V.)
| | - Evelien Smits
- Solid Tumor Immunology Group, Center for Oncological Research, Integrated Personalized and Precision Oncology Network, Department of Molecular Imaging, Pathology, Radiotherapy and Oncology, University of Antwerp, 2610 Antwerp, Belgium;
| | - Annemie Bogaerts
- PLASMANT, Chemistry Department, Faculty of Sciences, University of Antwerp, 2610 Antwerp, Belgium; (R.V.); (A.L.); (A.B.)
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19
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Zhou T, Liu J, Xie Y, Yuan S, Guo Y, Bai W, Zhao K, Jiang W, Wang H, Wang H, Zhao T, Huang C, Gao S, Wang X, Yang S, Hao J. ESE3/EHF, a promising target of rosiglitazone, suppresses pancreatic cancer stemness by downregulating CXCR4. Gut 2022; 71:357-371. [PMID: 33674341 PMCID: PMC9422994 DOI: 10.1136/gutjnl-2020-321952] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/23/2020] [Revised: 02/02/2021] [Accepted: 02/20/2021] [Indexed: 02/03/2023]
Abstract
BACKGROUND AND AIMS The crosstalk between cancer stem cells (CSCs) and their niche is required for the maintenance of stem cell-like phenotypes of CSCs. Here, we identified E26 transformation-specific homologous factor (EHF) as a key molecule in decreasing the sensitivity of pancreatic cancer (PC) cells to CSCs' niche stimulus. We also explored a therapeutic strategy to restore the expression of EHF. DESIGN We used a LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse model and samples from patients with PC. Immunostaining, flow cytometry, sphere formation assays, anchorage-independent growth assay, in vivo tumourigenicity, reverse transcription PCR, chromatin immunoprecipitation (ChIP) and luciferase analyses were conducted in this study. RESULTS CXCL12 derived from pancreatic stellate cells (PSCs) mediates the crosstalk between PC cells and PSCs to promote PC stemness. Tumorous EHF suppressed CSC stemness by decreasing the sensitivity of PC to CXCL12 stimulus and inhibiting the crosstalk between PC and CSC-supportive niches. Mechanically, EHF suppressed the transcription of the CXCL12 receptor CXCR4. EHF had a cell autonomous role in suppressing cancer stemness by inhibiting the transcription of Sox9, Sox2, Oct4 and Nanog. Rosiglitazone suppressed PC stemness and inhibited the crosstalk between PC and PSCs by upregulating EHF. Preclinical KPC mouse cohorts demonstrated that rosiglitazone sensitised PDAC to gemcitabine therapy. CONCLUSIONS EHF decreased the sensitivity of PC to the stimulus from PSC-derived CSC-supportive niche by negatively regulating tumorous CXCR4. Rosiglitazone could be used to target PC stem cells and the crosstalk between CSCs and their niche by upregulating EHF.
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Affiliation(s)
- Tianxing Zhou
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Jing Liu
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China.,Department of Breast Oncoplastic Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, China
| | - Yongjie Xie
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Shuai Yuan
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Yu Guo
- State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, 38 Tongyan Road, Tianjin, 300071, China
| | - Weiwei Bai
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Kaili Zhao
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Wenna Jiang
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Hongwei Wang
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Haotian Wang
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Tiansuo Zhao
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Chongbiao Huang
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Song Gao
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Xiuchao Wang
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, PR China
| | - Shengyu Yang
- Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA
| | - Jihui Hao
- Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin, PR China
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20
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Schuurman M, Wallace M, Sahi G, Barillaro M, Zhang S, Rahman M, Sawyez C, Borradaile N, Wang R. N-acetyl-L-cysteine treatment reduces beta-cell oxidative stress and pancreatic stellate cell activity in a high fat diet-induced diabetic mouse model. Front Endocrinol (Lausanne) 2022; 13:938680. [PMID: 36093092 PMCID: PMC9452715 DOI: 10.3389/fendo.2022.938680] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Accepted: 08/08/2022] [Indexed: 11/13/2022] Open
Abstract
Obesity plays a major role in type II diabetes (T2DM) progression because it applies metabolic and oxidative stress resulting in dysfunctional beta-cells and activation of intra-islet pancreatic stellate cells (PaSCs) which cause islet fibrosis. Administration of antioxidant N-acetyl-L-cysteine (NAC) in vivo improves metabolic outcomes in diet-induced obese diabetic mice, and in vitro inhibits PaSCs activation. However, the effects of NAC on diabetic islets in vivo are unknown. This study examined if dosage and length of NAC treatment in HFD-induced diabetic mice effect metabolic outcomes associated with maintaining healthy beta-cells and quiescent PaSCs, in vivo. Male C57BL/6N mice were fed normal chow (ND) or high-fat (HFD) diet up to 30 weeks. NAC was administered in drinking water to HFD mice in preventative treatment (HFDpNAC) for 23 weeks or intervention treatment for 10 (HFDiNAC) or 18 (HFDiNAC+) weeks, respectively. HFDpNAC and HFDiNAC+, but not HFDiNAC, mice showed significantly improved glucose tolerance and insulin sensitivity. Hyperinsulinemia led by beta-cell overcompensation in HFD mice was significantly rescued in NAC treated mice. A reduction of beta-cell nuclear Pdx-1 localization in HFD mice was significantly improved in NAC treated islets along with significantly reduced beta-cell oxidative stress. HFD-induced intra-islet PaSCs activation, labeled by αSMA, was significantly diminished in NAC treated mice along with lesser intra-islet collagen deposition. This study determined that efficiency of NAC treatment is beneficial at maintaining healthy beta-cells and quiescent intra-islet PaSCs in HFD-induced obese T2DM mouse model. These findings highlight an adjuvant therapeutic potential in NAC for controlling T2DM progression in humans.
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Affiliation(s)
- Meg Schuurman
- Children’s Health Research Institute, London, ON, Canada
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
| | - Madison Wallace
- Children’s Health Research Institute, London, ON, Canada
- Department of Pathology and Laboratory Medicine, University of Western Ontario, London, ON, Canada
| | - Gurleen Sahi
- Children’s Health Research Institute, London, ON, Canada
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
| | - Malina Barillaro
- Children’s Health Research Institute, London, ON, Canada
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
| | - Siyi Zhang
- Children’s Health Research Institute, London, ON, Canada
| | - Mushfiqur Rahman
- Children’s Health Research Institute, London, ON, Canada
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
| | - Cynthia Sawyez
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
| | - Nica Borradaile
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
| | - Rennian Wang
- Children’s Health Research Institute, London, ON, Canada
- Department of Physiology & Pharmacology, University of Western Ontario, London, ON, Canada
- *Correspondence: Rennian Wang,
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21
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Schwörer S, Pavlova NN, Cimino FV, King B, Cai X, Sizemore GM, Thompson CB. Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment. Nat Metab 2021; 3:1484-1499. [PMID: 34764457 PMCID: PMC8606002 DOI: 10.1038/s42255-021-00480-x] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 09/14/2021] [Indexed: 12/27/2022]
Abstract
The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.
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Affiliation(s)
- Simon Schwörer
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Natalya N Pavlova
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Francesco V Cimino
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Bryan King
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Xin Cai
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Gina M Sizemore
- The Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA
- Department of Radiation Oncology, The Ohio State University, Columbus, OH, USA
| | - Craig B Thompson
- Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
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22
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Li X, Nania S, Kleiter I, Löhr JM, Heuchel RL. Targeting of Smad7 in Mesenchymal Cells Does Not Exacerbate Fibrosis During Experimental Chronic Pancreatitis. Pancreas 2021; 50:1427-1434. [PMID: 35041343 DOI: 10.1097/mpa.0000000000001951] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
OBJECTIVES Transforming growth factor-β (TGF-β)-mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of pancreatitis, and their profibrotic ability is mainly mediated by TGF-β signaling. To specifically address the role of fibrogenic cells in experimental pancreatic fibrosis, we deleted Smad7, the main feedback inhibitor of TGF-β signaling in this cell type in mice. METHODS A mouse strain harboring a conditional knockout allele of Smad7 (Smad7fl/fl) with the tamoxifen-inducible inducible Col1a2-CreERT allele was generated and compared with wild-type mice challenged with the cerulein-based model of chronic pancreatitis. RESULTS Pancreatic stellate cells lacking Smad7 had significantly increased collagen I and fibronectin production and showed a higher activation level in vitro. Surprisingly, the fibrotic index in the pancreata of treated conditional knockout mice was only slightly increased, without statistical significance. Except for fibronectin, the expression of different extracellular matrix proteins and the numbers of fibroblasts and inflammatory cells were similar between Smad7-mutant and control mice. CONCLUSIONS There was no clear evidence that the lack of Smad7 in pancreatic stellate cells plays a major role in experimental pancreatitis, at least in the mouse model investigated here.
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Affiliation(s)
- Xuan Li
- From the Pancreas Cancer Research (PaCaRes) Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden
| | - Salvatore Nania
- From the Pancreas Cancer Research (PaCaRes) Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden
| | - Ingo Kleiter
- Department of Neurology, Ruhr-Universität Bochum, Bochum, Germany
| | - J-Matthias Löhr
- From the Pancreas Cancer Research (PaCaRes) Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden
| | - Rainer L Heuchel
- From the Pancreas Cancer Research (PaCaRes) Lab, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden
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23
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IL-17B/RB Activation in Pancreatic Stellate Cells Promotes Pancreatic Cancer Metabolism and Growth. Cancers (Basel) 2021; 13:cancers13215338. [PMID: 34771503 PMCID: PMC8611647 DOI: 10.3390/cancers13215338] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2021] [Revised: 09/25/2021] [Accepted: 10/08/2021] [Indexed: 12/13/2022] Open
Abstract
Simple Summary Pancreatic cancer has the lowest survival rate of all malignancies. Understanding the interplay between tumor and stroma could lead to the development of new therapies. The metabolic role of interleukin 17B/interleukin 17B receptor (IL-17B/RB) has not been adequately studied in pancreatic cancer and is poorly understood. Here, we investigate the IL-17B/RB-mediated interactions between the tumor and the stroma. We analyze murine as well as human stromal and tumor cells, animal experiments with immunocompromised mice, and human cell lines with overexpression and knockdown of IL-17RB. We report that aberrant expression of IL-17B/RB in stromal pancreatic stellate cells (PSCs) accelerates tumor cell growth. IL-17B/RB-signaling supplies energy by increased oxidative phosphorylation (OXPHOS). Blocking IL-17B/RB to inhibit the tumor to stroma crosstalk could be a potential targeted therapy for pancreatic cancer. Abstract In pancreatic ductal adenocarcinoma (PDAC), the tumor stroma constitutes most of the cell mass and contributes to therapy resistance and progression. Here we show a hitherto unknown metabolic cooperation between pancreatic stellate cells (PSCs) and tumor cells through Interleukin 17B/Interleukin 17B receptor (IL-17B/IL-17RB) signaling. Tumor-derived IL-17B carrying extracellular vesicles (EVs) activated stromal PSCs and induced the expression of IL-17RB. PSCs increased oxidative phosphorylation while reducing mitochondrial turnover. PSCs activated tumor cells in a feedback loop. Tumor cells subsequently increased oxidative phosphorylation and decreased glycolysis partially via IL-6. In vivo, IL-17RB overexpression in PSCs accelerated tumor growth in a co-injection xenograft mouse model. Our results demonstrate a tumor-to-stroma feedback loop increasing tumor metabolism to accelerate tumor growth under optimal nutritional conditions.
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Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche. Proc Natl Acad Sci U S A 2021; 118:2105367118. [PMID: 34588305 DOI: 10.1073/pnas.2105367118] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/01/2021] [Indexed: 01/04/2023] Open
Abstract
Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV-focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.
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Sharma S, Pande G, Rai MK, Agarwal V. Indigenous Primary Culture Protocols for Human Adult Skin Fibroblast, Pancreatic Stellate Cells, and Peritoneal Fibroblasts. INDIAN JOURNAL OF RHEUMATOLOGY 2021; 16:298-303. [DOI: 10.4103/injr.injr_160_20] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Affiliation(s)
| | - Gaurav Pande
- Departments of Medical Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
| | - Mohit Kumar Rai
- Departments of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
- Both authors contributed equally to this work
| | - Vikas Agarwal
- Departments of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
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Wiedenmann S, Breunig M, Merkle J, von Toerne C, Georgiev T, Moussus M, Schulte L, Seufferlein T, Sterr M, Lickert H, Weissinger SE, Möller P, Hauck SM, Hohwieler M, Kleger A, Meier M. Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip. Nat Biomed Eng 2021; 5:897-913. [PMID: 34239116 PMCID: PMC7611572 DOI: 10.1038/s41551-021-00757-2] [Citation(s) in RCA: 60] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Accepted: 06/01/2021] [Indexed: 02/06/2023]
Abstract
Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.
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Affiliation(s)
- Sandra Wiedenmann
- Helmholtz Pioneer Campus, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany
| | - Markus Breunig
- Department of Internal Medicine I, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Jessica Merkle
- Department of Internal Medicine I, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Christine von Toerne
- Research Unit Protein Science, Helmholtz Zentrum München, Heidemannstraße 1, 80939 Müunich, Germany
| | - Tihomir Georgiev
- Helmholtz Pioneer Campus, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany
| | - Michel Moussus
- Helmholtz Pioneer Campus, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany
| | - Lucas Schulte
- Department of Internal Medicine I, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Thomas Seufferlein
- Department of Internal Medicine I, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Michael Sterr
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany,German Center for Diabetes Research (DZD), Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany,German Center for Diabetes Research (DZD), Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany,Institute of Stem Cell Research, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany,Technical University of Munich, School of Medicine, Ismaninger Straße 22, 81675 Munich, Germany
| | | | - Peter Möller
- Institute for Pathology, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Stefanie M. Hauck
- Research Unit Protein Science, Helmholtz Zentrum München, Heidemannstraße 1, 80939 Müunich, Germany
| | - Meike Hohwieler
- Department of Internal Medicine I, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany,Corresponding authors: ; ;
| | - Alexander Kleger
- Department of Internal Medicine I, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany,Corresponding authors: ; ;
| | - Matthias Meier
- Helmholtz Pioneer Campus, Helmholtz Zentrum München, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany,Technical University of Munich, School of Medicine, Ismaninger Straße 22, 81675 Munich, Germany,Corresponding authors: ; ;
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Cannon A, Thompson CM, Bhatia R, Armstrong KA, Solheim JC, Kumar S, Batra SK. Molecular mechanisms of pancreatic myofibroblast activation in chronic pancreatitis and pancreatic ductal adenocarcinoma. J Gastroenterol 2021; 56:689-703. [PMID: 34279724 PMCID: PMC9052363 DOI: 10.1007/s00535-021-01800-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Accepted: 06/15/2021] [Indexed: 02/04/2023]
Abstract
Pancreatic fibrosis (PF) is an essential component of the pathobiology of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC). Activated pancreatic myofibroblasts (PMFs) are crucial for the deposition of the extracellular matrix, and fibrotic reaction in response to sustained signaling. Consequently, understanding of the molecular mechanisms of PMF activation is not only critical for understanding CP and PDAC biology but is also a fertile area of research for the development of novel therapeutic strategies for pancreatic pathologies. This review analyzes the key signaling events that drive PMF activation including, initiating signals from transforming growth factor-β1, platelet derived growth factor, as well as other microenvironmental cues, like hypoxia and extracellular matrix rigidity. Further, we discussed the intracellular signal events contributing to PMF activation, and crosstalk with different components of tumor microenvironment. Additionally, association of epidemiologically established risk factors for CP and PDAC, like alcohol intake, tobacco exposure, and metabolic factors with PMF activation, is discussed to comprehend the role of lifestyle factors on pancreatic pathologies. Overall, this analysis provides insight into the biology of PMF activation and highlights salient features of this process, which offer promising therapeutic targets.
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Affiliation(s)
- Andrew Cannon
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Christopher Michael Thompson
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Rakesh Bhatia
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | | | - Joyce Christopher Solheim
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA,Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Sushil Kumar
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Surinder Kumar Batra
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA,Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA,Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA
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Magni L, Bouazzi R, Heredero Olmedilla H, Petersen PSS, Tozzi M, Novak I. The P2X7 Receptor Stimulates IL-6 Release from Pancreatic Stellate Cells and Tocilizumab Prevents Activation of STAT3 in Pancreatic Cancer Cells. Cells 2021; 10:cells10081928. [PMID: 34440697 PMCID: PMC8391419 DOI: 10.3390/cells10081928] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Revised: 06/30/2021] [Accepted: 07/23/2021] [Indexed: 12/12/2022] Open
Abstract
Pancreatic stellate cells (PSCs) are important pancreatic fibrogenic cells that interact with pancreatic cancer cells to promote the progression of pancreatic ductal adenocarcinoma (PDAC). In the tumor microenvironment (TME), several factors such as cytokines and nucleotides contribute to this interplay. Our aim was to investigate whether there is an interaction between IL-6 and nucleotide signaling, in particular, that mediated by the ATP-sensing P2X7 receptor (P2X7R). Using human cell lines of PSCs and cancer cells, as well as primary PSCs from mice, we show that ATP is released from both PSCs and cancer cells in response to mechanical and metabolic cues that may occur in the TME, and thus activate the P2X7R. Functional studies using P2X7R agonists and inhibitors show that the receptor is involved in PSC proliferation, collagen secretion and IL-6 secretion and it promotes cancer cell migration in a human PSC-cancer cell co-culture. Moreover, conditioned media from P2X7R-stimulated PSCs activated the JAK/STAT3 signaling pathway in cancer cells. The monoclonal antibody inhibiting the IL-6 receptor, Tocilizumab, inhibited this signaling. In conclusion, we show an important mechanism between PSC-cancer cell interaction involving ATP and IL-6, activating P2X7 and IL-6 receptors, respectively, both potential therapeutic targets in PDAC.
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MESH Headings
- Animals
- Antibodies, Monoclonal, Humanized/pharmacology
- Antibodies, Monoclonal, Humanized/therapeutic use
- Antineoplastic Agents, Immunological/pharmacology
- Antineoplastic Agents, Immunological/therapeutic use
- Carcinoma, Pancreatic Ductal/drug therapy
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/physiopathology
- Cell Communication
- Cell Line, Tumor
- Cell Movement
- Cell Proliferation
- Humans
- Interleukin-6/metabolism
- Male
- Mice
- Pancreatic Stellate Cells/metabolism
- Pancreatic Stellate Cells/physiology
- Receptors, Purinergic P2X7/metabolism
- STAT3 Transcription Factor/metabolism
- Signal Transduction
- Tumor Microenvironment
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Orai1 Channel Regulates Human-Activated Pancreatic Stellate Cell Proliferation and TGF β1 Secretion through the AKT Signaling Pathway. Cancers (Basel) 2021; 13:cancers13102395. [PMID: 34063470 PMCID: PMC8156432 DOI: 10.3390/cancers13102395] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 05/04/2021] [Accepted: 05/12/2021] [Indexed: 12/24/2022] Open
Abstract
Simple Summary Activated pancreatic stellate cells (aPSCs), the main source of cancer-associated fibroblasts in pancreatic ductal adenocarcinoma (PDAC), are well known as the key actor of the abundant fibrotic stroma development surrounding the tumor cells. In permanent communication with the tumor cells, they enhance PDAC early spreading and limit the drug delivery. However, the understanding of PSC activation mechanisms and the associated signaling pathways is still incomplete. In this study, we aimed to evaluate the role of Ca2+, and Orai1 Ca2+ channels, in two main PSC activation processes: cell proliferation and cytokine secretion. Indeed, Ca2+ is a versatile second messenger implicated in the regulation of numerous biological processes. We believe that a better comprehension of PSC Ca2+ -dependent activation mechanisms will bring up new crucial PDAC early prognostic markers or new targeting approaches in PDAC treatment. Abstract Activated pancreatic stellate cells (aPSCs), the crucial mediator of pancreatic desmoplasia, are characterized, among others, by high proliferative potential and abundant transforming growth factor β1 (TGFβ1) secretion. Over the past years, the involvement of Ca2+ channels in PSC pathophysiology has attracted great interest in pancreatic cancer research. We, thus, aimed to investigate the role of the Orai1 Ca2+ channel in these two PSC activation processes. Using the siRNA approach, we invalided Orai1 expression and assessed the channel functionality by Ca2+ imaging, the effect on aPSC proliferation, and TGFβ1 secretion. We demonstrated the functional expression of the Orai1 channel in human aPSCs and its implication in the store-operated Ca2+ entry (SOCE). Orai1 silencing led to a decrease in aPSC proliferation, TGFβ1 secretion, and AKT activation. Interestingly, TGFβ1 induced a higher SOCE response by increasing Orai1 mRNAs and proteins and promoted both AKT phosphorylation and cell proliferation, abolished by Orai1 silencing. Together, our results highlight the role of Orai1-mediated Ca2+ entry in human aPSC pathophysiology by controlling cell proliferation and TGFβ1 secretion through the AKT signaling pathway. Moreover, we showed a TGFβ1-induced autocrine positive feedback loop by promoting the Orai1/AKT-dependent proliferation via the stimulation of Orai1 expression and function.
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30
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Hupfer A, Brichkina A, Adhikary T, Lauth M. The mammalian Hedgehog pathway is modulated by ANP32 proteins. Biochem Biophys Res Commun 2021; 553:78-84. [PMID: 33761414 DOI: 10.1016/j.bbrc.2021.03.027] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Accepted: 03/05/2021] [Indexed: 12/29/2022]
Abstract
Medulloblastoma (MB) is the most common malignant brain tumor in children. Transcriptional profiling has so far delineated four major MB subgroups of which one is driven by uncontrolled Hedgehog (Hh) signaling (SHH-MB). This pathway is amenable to drug targeting, yet clinically approved compounds exclusively target the transmembrane component Smoothened (SMO). Unfortunately, drug resistance against SMO inhibitors is encountered frequently, making the identification of novel Hh pathway components mandatory, which could serve as novel drug targets in the future. Here, we have used MB as a tool to delineate novel modulators of Hh signaling and have identified the Acidic Nuclear Phosphoprotein 32 (ANP32) family of proteins as novel regulators. The expression of all three family members (ANP32A, ANP32B, ANP32E) is increased in Hh-induced MB and their expression level is negatively associated with overall survival in SHH-MB patients. Mechanistically, we could find that ANP32 proteins function as positive modulators of mammalian Hh signaling upstream of GLI transcription factors. These findings add hitherto unknown regulators to the mammalian Hh signaling cascade and might spur future translational efforts to combat Hh-driven malignancies.
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Affiliation(s)
- Anna Hupfer
- Philipps University Marburg, Center for Tumor- and Immune Biology (ZTI), Clinics of Gastroenterology, Endocrinology, Metabolism and Infectiology, Germany
| | - Anna Brichkina
- Philipps University Marburg, Center for Tumor- and Immune Biology (ZTI), Clinics of Gastroenterology, Endocrinology, Metabolism and Infectiology, Germany
| | - Till Adhikary
- Philipps University Marburg, Center for Tumor Biology and Immunology (ZTI), Institute of Medical Bioinformatics and Biostatistics, Institute of Molecular Biology and Tumor Research, Germany
| | - Matthias Lauth
- Philipps University Marburg, Center for Tumor- and Immune Biology (ZTI), Clinics of Gastroenterology, Endocrinology, Metabolism and Infectiology, Germany.
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31
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Koustoulidou S, Hoorens MWH, Dalm SU, Mahajan S, Debets R, Seimbille Y, de Jong M. Cancer-Associated Fibroblasts as Players in Cancer Development and Progression and Their Role in Targeted Radionuclide Imaging and Therapy. Cancers (Basel) 2021; 13:1100. [PMID: 33806468 PMCID: PMC7961537 DOI: 10.3390/cancers13051100] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 02/19/2021] [Accepted: 02/20/2021] [Indexed: 12/12/2022] Open
Abstract
Cancer Associated Fibroblasts (CAFs) form a major component of the tumour microenvironment, they have a complex origin and execute diverse functions in tumour development and progression. As such, CAFs constitute an attractive target for novel therapeutic interventions that will aid both diagnosis and treatment of various cancers. There are, however, a few limitations in reaching successful translation of CAF targeted interventions from bench to bedside. Several approaches targeting CAFs have been investigated so far and a few CAF-targeting tracers have successfully been developed and applied. This includes tracers targeting Fibroblast Activation Protein (FAP) on CAFs. A number of FAP-targeting tracers have shown great promise in the clinic. In this review, we summarize our current knowledge of the functional heterogeneity and biology of CAFs in cancer. Moreover, we highlight the latest developments towards theranostic applications that will help tumour characterization, radioligand therapy and staging in cancers with a distinct CAF population.
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Affiliation(s)
- Sofia Koustoulidou
- Department of Radiology and Nuclear Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (M.W.H.H.); (S.U.D.); (Y.S.); (M.d.J.)
| | - Mark W. H. Hoorens
- Department of Radiology and Nuclear Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (M.W.H.H.); (S.U.D.); (Y.S.); (M.d.J.)
| | - Simone U. Dalm
- Department of Radiology and Nuclear Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (M.W.H.H.); (S.U.D.); (Y.S.); (M.d.J.)
| | - Shweta Mahajan
- Department of Medical Oncology, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (R.D.); (S.M.)
| | - Reno Debets
- Department of Medical Oncology, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (R.D.); (S.M.)
| | - Yann Seimbille
- Department of Radiology and Nuclear Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (M.W.H.H.); (S.U.D.); (Y.S.); (M.d.J.)
| | - Marion de Jong
- Department of Radiology and Nuclear Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; (M.W.H.H.); (S.U.D.); (Y.S.); (M.d.J.)
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Abdel Mouti M, Pauklin S. TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment. Mol Ther 2021; 29:920-936. [PMID: 33429081 PMCID: PMC7934636 DOI: 10.1016/j.ymthe.2021.01.002] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 10/17/2020] [Accepted: 01/02/2021] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer remains a grueling disease that is projected to become the second-deadliest cancer in the next decade. Standard treatment of pancreatic cancer is chemotherapy, which mainly targets the differentiated population of tumor cells; however, it paradoxically sets the roots of tumor relapse by the selective enrichment of intrinsically chemoresistant pancreatic cancer stem cells that are equipped with an indefinite capacity for self-renewal and differentiation, resulting in tumor regeneration and an overall anemic response to chemotherapy. Crosstalk between pancreatic tumor cells and the surrounding stromal microenvironment is also involved in the development of chemoresistance by creating a supportive niche, which enhances the stemness features and tumorigenicity of pancreatic cancer cells. In addition, the desmoplastic nature of the tumor-associated stroma acts as a physical barrier, which limits the intratumoral delivery of chemotherapeutics. In this review, we mainly focus on the transforming growth factor beta 1 (TGFB1)/inhibin subunit beta A (INHBA) homodimer/Nodal-SMAD2/3 signaling network in pancreatic cancer as a pivotal central node that regulates multiple key mechanisms involved in the development of chemoresistance, including enhancement of the stem cell-like properties and tumorigenicity of pancreatic cancer cells, mediating cooperative interactions between pancreatic cancer cells and the surrounding stroma, as well as regulating the deposition of extracellular matrix proteins within the tumor microenvironment.
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Affiliation(s)
- Mai Abdel Mouti
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Headington, University of Oxford, Oxford OX3 7LD, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Headington, University of Oxford, Oxford OX3 7LD, UK.
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Feldmann K, Maurer C, Peschke K, Teller S, Schuck K, Steiger K, Engleitner T, Öllinger R, Nomura A, Wirges N, Papargyriou A, Jahan Sarker RS, Ranjan RA, Dantes Z, Weichert W, Rustgi AK, Schmid RM, Rad R, Schneider G, Saur D, Reichert M. Mesenchymal Plasticity Regulated by Prrx1 Drives Aggressive Pancreatic Cancer Biology. Gastroenterology 2021; 160:346-361.e24. [PMID: 33007300 DOI: 10.1053/j.gastro.2020.09.010] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Revised: 08/11/2020] [Accepted: 09/06/2020] [Indexed: 12/18/2022]
Abstract
BACKGROUND & AIMS Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology. METHODS To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis. RESULTS Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC. CONCLUSIONS Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
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Affiliation(s)
- Karin Feldmann
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Carlo Maurer
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Katja Peschke
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Steffen Teller
- Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Kathleen Schuck
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Katja Steiger
- Institute of Pathology, Technical University of Munich, Munich, Germany; Comparative Experimental Pathology, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Thomas Engleitner
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Rupert Öllinger
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Alice Nomura
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Nils Wirges
- Institute of Pathology, Technical University of Munich, Munich, Germany; Comparative Experimental Pathology, Technical University of Munich, Munich, Germany
| | - Aristeidis Papargyriou
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; Institute of Stem Cell Research, Helmholtz Center for Health and Environmental Research Munich, Neuherberg, Germany
| | - Rim Sabrina Jahan Sarker
- Institute of Pathology, Technical University of Munich, Munich, Germany; Comparative Experimental Pathology, Technical University of Munich, Munich, Germany
| | - Raphela Aranie Ranjan
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Zahra Dantes
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany
| | - Wilko Weichert
- Institute of Pathology, Technical University of Munich, Munich, Germany; Comparative Experimental Pathology, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Anil K Rustgi
- Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, Columbia University, New York, New York
| | - Roland M Schmid
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Roland Rad
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Günter Schneider
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Dieter Saur
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany
| | - Maximilian Reichert
- Klinik und Poliklinik für Innere Medizin II, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany; German Cancer Consortium, Partner Site Munich, Germany.
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Choi SR, Yang Y, Huang KY, Kong HJ, Flick MJ, Han B. Engineering of biomaterials for tumor modeling. MATERIALS TODAY. ADVANCES 2020; 8:100117. [PMID: 34541484 PMCID: PMC8448271 DOI: 10.1016/j.mtadv.2020.100117] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Development of biomaterials mimicking tumor and its microenvironment has recently emerged for the use of drug discovery, precision medicine, and cancer biology. These biomimetic models have developed by reconstituting tumor and stroma cells within the 3D extracellular matrix. The models are recently extended to recapitulate the in vivo tumor microenvironment, including biological, chemical, and mechanical conditions tailored for specific cancer type and its microenvironment. In spite of the recent emergence of various innovative engineered tumor models, many of these models are still early stage to be adapted for cancer research. In this article, we review the current status of biomaterials engineering for tumor models considering three main aspects - cellular engineering, matrix engineering, and engineering for microenvironmental conditions. Considering cancer-specific variability in these aspects, our discussion is focused on pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC). In addition, we further discussed the current challenges and future opportunities to create reliable and relevant tumor models.
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Affiliation(s)
- Sae Rome Choi
- School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
| | - Yi Yang
- Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Blood Research Center, University of North Carolina, Chapel Hill, NC, USA
| | - Kai-Yu Huang
- Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Hyun Joon Kong
- Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Matthew J. Flick
- Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Blood Research Center, University of North Carolina, Chapel Hill, NC, USA
| | - Bumsoo Han
- School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
- Weldon School of Biomedical Engineering and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA
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Huber M, Brehm CU, Gress TM, Buchholz M, Alashkar Alhamwe B, Pogge von Strandmann E, Slater EP, Bartsch JW, Bauer C, Lauth M. The Immune Microenvironment in Pancreatic Cancer. Int J Mol Sci 2020; 21:E7307. [PMID: 33022971 PMCID: PMC7583843 DOI: 10.3390/ijms21197307] [Citation(s) in RCA: 154] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Revised: 09/28/2020] [Accepted: 09/29/2020] [Indexed: 02/07/2023] Open
Abstract
The biology of solid tumors is strongly determined by the interactions of cancer cells with their surrounding microenvironment. In this regard, pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) represents a paradigmatic example for the multitude of possible tumor-stroma interactions. PDAC has proven particularly refractory to novel immunotherapies, which is a fact that is mediated by a unique assemblage of various immune cells creating a strongly immunosuppressive environment in which this cancer type thrives. In this review, we outline currently available knowledge on the cross-talk between tumor cells and the cellular immune microenvironment, highlighting the physiological and pathological cellular interactions, as well as the resulting therapeutic approaches derived thereof. Hopefully a better understanding of the complex tumor-stroma interactions will one day lead to a significant advancement in patient care.
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Affiliation(s)
- Magdalena Huber
- Institute for Medical Microbiology and Hospital Hygiene, Philipps University Marburg, 35043 Marburg, Germany;
| | - Corinna U. Brehm
- Institute of Pathology, University Hospital Giessen-Marburg, 35043 Marburg, Germany;
| | - Thomas M. Gress
- Department of Gastroenterology, Endocrinology, Metabolism and Infectiology, Center for Tumor- and Immunology (ZTI), Philipps University Marburg, 35043 Marburg, Germany; (T.M.G.); (M.B.); (C.B.)
| | - Malte Buchholz
- Department of Gastroenterology, Endocrinology, Metabolism and Infectiology, Center for Tumor- and Immunology (ZTI), Philipps University Marburg, 35043 Marburg, Germany; (T.M.G.); (M.B.); (C.B.)
| | - Bilal Alashkar Alhamwe
- Institute for Tumor Immunology, Clinic for Hematology, Oncology and Immunology, Center for Tumor Biology and Immunology (ZTI), Philipps University Marburg, 35043 Marburg, Germany; (E.P.v.S.); (B.A.A.)
| | - Elke Pogge von Strandmann
- Institute for Tumor Immunology, Clinic for Hematology, Oncology and Immunology, Center for Tumor Biology and Immunology (ZTI), Philipps University Marburg, 35043 Marburg, Germany; (E.P.v.S.); (B.A.A.)
| | - Emily P. Slater
- Department of Visceral-, Thoracic- and Vascular Surgery, Philipps University Marburg, Baldingerstrasse, 35043 Marburg, Germany;
| | - Jörg W. Bartsch
- Department of Neurosurgery, Philipps University Marburg, Baldingerstrasse, 35043 Marburg, Germany;
| | - Christian Bauer
- Department of Gastroenterology, Endocrinology, Metabolism and Infectiology, Center for Tumor- and Immunology (ZTI), Philipps University Marburg, 35043 Marburg, Germany; (T.M.G.); (M.B.); (C.B.)
| | - Matthias Lauth
- Department of Gastroenterology, Endocrinology, Metabolism and Infectiology, Center for Tumor- and Immunology (ZTI), Philipps University Marburg, 35043 Marburg, Germany; (T.M.G.); (M.B.); (C.B.)
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Verloy R, Privat-Maldonado A, Smits E, Bogaerts A. Cold Atmospheric Plasma Treatment for Pancreatic Cancer-The Importance of Pancreatic Stellate Cells. Cancers (Basel) 2020; 12:cancers12102782. [PMID: 32998311 PMCID: PMC7601057 DOI: 10.3390/cancers12102782] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2020] [Revised: 09/17/2020] [Accepted: 09/26/2020] [Indexed: 01/18/2023] Open
Abstract
Simple Summary This review aims to highlight the potential of cold plasma, the fourth state of matter, as anti-cancer treatment for pancreatic cancer, and the importance of pancreatic stellate cells in the response to this treatment. Currently, a significant lack of basic research on cold plasma considering both pancreatic cancer and stellate cells exists. However, co-cultures of these populations can be advantageous, as they resemble the cell-to-cell interactions occurring in a tumor in response to therapy. Even more, these studies should be performed prior to clinical trials of cold plasma to avoid unforeseen responses to treatment. This review article provides a framework for future research of cold plasma therapies for pancreatic cancer, considering the critical role of pancreatic stellate cells in the disease and treatment outcome. Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with low five-year survival rates of 8% by conventional treatment methods, e.g., chemotherapy, radiotherapy, and surgery. PDAC shows high resistance towards chemo- and radiotherapy and only 15–20% of all patients can have surgery. This disease is predicted to become the third global leading cause of cancer death due to its significant rise in incidence. Therefore, the development of an alternative or combinational method is necessary to improve current approaches. Cold atmospheric plasma (CAP) treatments could offer multiple advantages to this emerging situation. The plasma-derived reactive species can induce oxidative damage and a cascade of intracellular signaling pathways, which could lead to cell death. Previous reports have shown that CAP treatment also influences cells in the tumor microenvironment, such as the pancreatic stellate cells (PSCs). These PSCs, when activated, play a crucial role in the propagation, growth and survival of PDAC tumors. However, the effect of CAP on PSCs is not yet fully understood. This review focuses on the application of CAP for PDAC treatment and the importance of PSCs in the response to treatment.
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Affiliation(s)
- Ruben Verloy
- Plasma Lab for Applications in Sustainability and Medicine-ANTwerp, University of Antwerp, 2610 Wilrijk, Belgium;
- Center for Oncological Research, University of Antwerp, 2610 Wilrijk, Belgium;
- Correspondence: (R.V.); (A.P.-M.); Tel.: +32-3265-2343 (R.V. & A.P.-M.)
| | - Angela Privat-Maldonado
- Plasma Lab for Applications in Sustainability and Medicine-ANTwerp, University of Antwerp, 2610 Wilrijk, Belgium;
- Center for Oncological Research, University of Antwerp, 2610 Wilrijk, Belgium;
- Correspondence: (R.V.); (A.P.-M.); Tel.: +32-3265-2343 (R.V. & A.P.-M.)
| | - Evelien Smits
- Center for Oncological Research, University of Antwerp, 2610 Wilrijk, Belgium;
| | - Annemie Bogaerts
- Plasma Lab for Applications in Sustainability and Medicine-ANTwerp, University of Antwerp, 2610 Wilrijk, Belgium;
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Buchholz SM, Goetze RG, Singh SK, Ammer-Herrmenau C, Richards FM, Jodrell DI, Buchholz M, Michl P, Ellenrieder V, Hessmann E, Neesse A. Depletion of Macrophages Improves Therapeutic Response to Gemcitabine in Murine Pancreas Cancer. Cancers (Basel) 2020; 12:E1978. [PMID: 32698524 PMCID: PMC7409345 DOI: 10.3390/cancers12071978] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Revised: 07/09/2020] [Accepted: 07/16/2020] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND The tumor microenvironment (TME) is composed of fibro-inflammatory cells and extracellular matrix (ECM) components. However, the exact contribution of the various TME compartments towards therapeutic response is unknown. Here, we aim to dissect the specific contribution of tumor-associated macrophages (TAMs) towards drug delivery and response in pancreatic ductal adenocarcinoma (PDAC). METHODS The effect of gemcitabine was assessed in human and murine macrophages, human pancreatic stellate cells (hPSCs), and tumor cells (L3.6pl, BxPC3 and KPC) in vitro. The drug metabolism of gemcitabine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Preclinical studies were conducted using KrasG12D;p48-Cre and KrasG12D;p53172H;Pdx-Cre mice to investigate gemcitabine delivery at different stages of tumor progression and upon pharmacological TAM depletion. RESULTS Gemcitabine accumulation was significantly increased in murine PDAC tissue compared to pancreatic intraepithelial neoplasia (PanIN) lesions and healthy control pancreas tissue. In vitro, macrophages accumulated and rapidly metabolized gemcitabine resulting in a significant drug scavenging effect for gemcitabine. Finally, pharmacological TAM depletion enhanced therapeutic response to gemcitabine in tumor-bearing KPC mice. CONCLUSION Macrophages rapidly metabolize gemcitabine in vitro, and pharmacological depletion improves the therapeutic response to gemcitabine in vivo. Our study supports the notion that TAMs might be a promising therapeutic target in PDAC.
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Affiliation(s)
- Soeren M. Buchholz
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
| | - Robert G. Goetze
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
| | - Shiv K. Singh
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
| | - Christoph Ammer-Herrmenau
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
| | - Frances M. Richards
- Cancer Research UK Cambridge Institute, The University of Cambridge, Li Ka Shing Centre, Cambridge CB2 1TN, UK; (F.M.R.); (D.I.J.)
| | - Duncan I. Jodrell
- Cancer Research UK Cambridge Institute, The University of Cambridge, Li Ka Shing Centre, Cambridge CB2 1TN, UK; (F.M.R.); (D.I.J.)
| | - Malte Buchholz
- Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps University Marburg, 35037 Marburg, Germany;
| | - Patrick Michl
- Department of Internal Medicine I, Martin-Luther-University of Halle-Wittenberg, 06120 Halle, Germany;
| | - Volker Ellenrieder
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
| | - Elisabeth Hessmann
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
| | - Albrecht Neesse
- Department of Gastroenterology, Gastrointestinal Oncology and Endocrinology, University Medical Center Göttingen, 37075 Göttingen, Germany; (S.M.B.); (R.G.G.); (S.K.S.); (C.A.-H.); (V.E.); (E.H.)
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Norberg KJ, Liu X, Fernández Moro C, Strell C, Nania S, Blümel M, Balboni A, Bozóky B, Heuchel RL, Löhr JM. A novel pancreatic tumour and stellate cell 3D co-culture spheroid model. BMC Cancer 2020; 20:475. [PMID: 32460715 PMCID: PMC7251727 DOI: 10.1186/s12885-020-06867-5] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Accepted: 04/14/2020] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells. METHOD A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting. RESULTS We found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids. CONCLUSIONS We developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.
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Affiliation(s)
- K J Norberg
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden
| | - X Liu
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden
| | - C Fernández Moro
- Department of Laboratory Medicine (LabMed), Division of Pathology, Karolinska Institutet, Stockholm, Sweden
- Department of Clinical Pathology/Cytology, Karolinska University Hospital, Stockholm, Sweden
| | - C Strell
- Department of Cancer, Division of Upper GI, Karolinska University Hospital, Stockholm, Sweden
| | - S Nania
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden
| | - M Blümel
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden
| | - A Balboni
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden
| | - B Bozóky
- Department of Laboratory Medicine (LabMed), Division of Pathology, Karolinska Institutet, Stockholm, Sweden
- Department of Clinical Pathology/Cytology, Karolinska University Hospital, Stockholm, Sweden
| | - R L Heuchel
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden.
| | - J M Löhr
- Pancreas Cancer Research Lab, Department of Clinical Intervention and Technology (CLINTEC), Karolinska Institutet, Novum, floor 6, room 613, SE-141 86, Stockholm, Sweden
- Department of Cancer, Division of Upper GI, Karolinska University Hospital, Stockholm, Sweden
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Hartung F, Krüwel T, Shi X, Pfizenmaier K, Kontermann R, Chames P, Alves F, Pardo LA. A Novel Anti-Kv10.1 Nanobody Fused to Single-Chain TRAIL Enhances Apoptosis Induction in Cancer Cells. Front Pharmacol 2020; 11:686. [PMID: 32528281 PMCID: PMC7246340 DOI: 10.3389/fphar.2020.00686] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Accepted: 04/27/2020] [Indexed: 01/11/2023] Open
Abstract
Antibody-based therapies hold promise for a safe and efficient treatment of cancer. The identification of target tumor cells through a specific antigen enriched on their surface and the subsequent delivery of the therapeutic agent only to those cells requires, besides the efficacy of the therapeutic agent itself, the identification of an antigen enriched on the surface of tumor cells, the generation of high affinity antibodies against that antigen. We have generated single-domain antibodies (nanobodies) against the voltage-gated potassium channel Kv10.1, which outside of the brain is detectable almost exclusively in tumor cells. The nanobody with highest affinity was fused to an improved form of the tumor necrosis factor-related apoptosis inducing ligand TRAIL, to target this cytokine to the surface of tumor cells. The resulting construct, VHH-D9-scTRAIL, shows rapid and strong apoptosis induction in different tumor models in cell culture. The construct combines two sources of specificity, the expression of the antigen restricted to tumor cells and the tumor selectivity of TRAIL. Such specificity combined with the high affinity obtained through nanobodies make the novel agent a promising concept for cancer therapy.
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Affiliation(s)
- Franziska Hartung
- Oncophysiology Group, Max Planck, Institute of Experimental Medicine, Göttingen, Germany
| | - Thomas Krüwel
- Institute of Diagnostic and Interventional Radiology, University Medical Center Göttingen, Göttingen, Germany
| | - Xiaoyi Shi
- Oncophysiology Group, Max Planck, Institute of Experimental Medicine, Göttingen, Germany
| | - Klaus Pfizenmaier
- Institut für Zellbiologie und Immunologie, Universität Stuttgart, Stuttgart, Germany
| | - Roland Kontermann
- Institut für Zellbiologie und Immunologie, Universität Stuttgart, Stuttgart, Germany
| | - Patrick Chames
- Aix Marseille Univ, CNRS, Inserm, Institut Paoli-Calmettes, CRCM, Marseille, France
| | - Frauke Alves
- Institute of Diagnostic and Interventional Radiology, University Medical Center Göttingen, Göttingen, Germany.,Translational Molecular Imaging Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany
| | - Luis A Pardo
- Oncophysiology Group, Max Planck, Institute of Experimental Medicine, Göttingen, Germany
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Proton Pump Inhibitors Reduce Pancreatic Adenocarcinoma Progression by Selectively Targeting H +, K +-ATPases in Pancreatic Cancer and Stellate Cells. Cancers (Basel) 2020; 12:cancers12030640. [PMID: 32164284 PMCID: PMC7139746 DOI: 10.3390/cancers12030640] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Revised: 02/28/2020] [Accepted: 03/05/2020] [Indexed: 02/06/2023] Open
Abstract
Pancreatic duct cells are equipped with acid/base transporters important for exocrine secretion. Pancreatic ductal adenocarcinoma (PDAC) cells may utilize such transporters to acidify extracellular tumor microenvironment, creating a niche favoring cell proliferation, fibrosis and resistance to chemotherapy-all contributing to the notoriously bad prognosis of this disease. Here, we report that gastric and non-gastric H+, K+-ATPases (coded by ATP4A and ATP12A) are overexpressed in human and murine pancreatic cancer and that we can target them specifically with proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs) in in vitro models of PDAC. Focusing on pantoprazole, we show that it significantly reduced human cancer cell proliferation by inhibiting cellular H+ extrusion, increasing K+ conductance and promoting cyclin D1-dependent cell cycle arrest and preventing STAT3 activation. Pantoprazole also decreased collagen secretion from pancreatic stellate cells. Importantly, in vivo studies show that pantoprazole treatment of tumor-bearing mice reduced tumor size, fibrosis and expression of angiogenic markers. This work provides the first evidence that H+, K+-ATPases contribute to PDAC progression and that these can be targeted by inhibitors of these pumps, thus proving a promising therapeutic strategy.
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Sun L, Qu L, Brigstock DR, Li H, Li Y, Gao R. Biological and Proteomic Characteristics of an Immortalized Human Pancreatic Stellate Cell Line. Int J Med Sci 2020; 17:137-144. [PMID: 31929747 PMCID: PMC6945563 DOI: 10.7150/ijms.36337] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/05/2019] [Accepted: 12/12/2019] [Indexed: 02/07/2023] Open
Abstract
Human pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during chronic pancreatitis (CP). However, primary PSCs have a short lifespan in vitro, which seriously affects their use in various applications. We have established a stable immortalized human PSC line (HP-1) by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated human PSCs. HP-1 cells express cytoskeleton proteins including glial fibrillary acidic protein (GFAP), α-smooth muscle actin (α-SMA), vimentin and desmin, and are typical of PSCs, which are high transfeciability and viable in 0.5% serum. The cells express receptors such as TGFβR2, PDGFR, TGF-β pseudoreceptor Bambi and PPRPγ that are commonly found in PSCs. HP-1 cells are similar to activated human PSCs in that they have enhanced expression of α-SMA, CTGF, Col1 and TIMP-2 mRNAs or proteins, as well as decreased expression of MMP-1/2 mRNAs or proteins in response to TGF-β1 stimulation. Comparative proteomics revealed 4,537 shared proteins between HP-1 cells and PSCs and no single protein in HP-1 cells versus PSCs. Statistical analysis reveals no significantly difference between HP-1 cells and PSCs in their expression of proteins associated with matrix and matrix remodeling. The similarity between HP-1 cell and PSC is further shown by the finding that only 9 proteins are differentially up-regulated > ± 2-fold in HP-1 cells and 13 proteins are up-regulated > ± 2-fold in PSCs and none of these proteins include ECM proteins, cytokines, growth factors or matrix remodeling regulatory proteins. Therefore, HP-1 cells can be used as an effective tool for the study of PSC-mediated pancreatic fibrosis.
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Affiliation(s)
- Li Sun
- Department of Hepatic Biliary Pancreatic Medicine, First Hospital of Jilin University, Changchun, 130021, China
| | - Limei Qu
- Department of Hepatic Biliary Pancreatic Medicine, First Hospital of Jilin University, Changchun, 130021, China
| | - David R Brigstock
- Research Institute at Nationwide Children's Hospital, Columbus, 43205, United States
| | - Hongyan Li
- Department of Hepatic Biliary Pancreatic Medicine, First Hospital of Jilin University, Changchun, 130021, China
| | - Yanyan Li
- Department of Hepatic Biliary Pancreatic Medicine, First Hospital of Jilin University, Changchun, 130021, China
| | - Runping Gao
- Department of Hepatic Biliary Pancreatic Medicine, First Hospital of Jilin University, Changchun, 130021, China
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Al Alawi R, Alhamdani MSS, Hoheisel JD, Baqi Y. Antifibrotic and tumor microenvironment modulating effect of date palm fruit (Phoenix dactylifera L.) extracts in pancreatic cancer. Biomed Pharmacother 2020; 121:109522. [DOI: 10.1016/j.biopha.2019.109522] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Revised: 09/22/2019] [Accepted: 10/01/2019] [Indexed: 12/16/2022] Open
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Cold Atmospheric Plasma-Treated PBS Eliminates Immunosuppressive Pancreatic Stellate Cells and Induces Immunogenic Cell Death of Pancreatic Cancer Cells. Cancers (Basel) 2019; 11:cancers11101597. [PMID: 31635070 PMCID: PMC6826654 DOI: 10.3390/cancers11101597] [Citation(s) in RCA: 68] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Revised: 10/16/2019] [Accepted: 10/17/2019] [Indexed: 02/08/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a low response to treatment and a five-year survival rate below 5%. The ineffectiveness of treatment is partly because of an immunosuppressive tumor microenvironment, which comprises tumor-supportive pancreatic stellate cells (PSCs). Therefore, new therapeutic strategies are needed to tackle both the immunosuppressive PSC and pancreatic cancer cells (PCCs). Recently, physical cold atmospheric plasma consisting of reactive oxygen and nitrogen species has emerged as a novel treatment option for cancer. In this study, we investigated the cytotoxicity of plasma-treated phosphate-buffered saline (pPBS) using three PSC lines and four PCC lines and examined the immunogenicity of the induced cell death. We observed a decrease in the viability of PSC and PCC after pPBS treatment, with a higher efficacy in the latter. Two PCC lines expressed and released damage-associated molecular patterns characteristic of the induction of immunogenic cell death (ICD). In addition, pPBS-treated PCC were highly phagocytosed by dendritic cells (DCs), resulting in the maturation of DC. This indicates the high potential of pPBS to trigger ICD. In contrast, pPBS induced no ICD in PSC. In general, pPBS treatment of PCCs and PSCs created a more immunostimulatory secretion profile (higher TNF-α and IFN-γ, lower TGF-β) in coculture with DC. Altogether, these data show that plasma treatment via pPBS has the potential to induce ICD in PCCs and to reduce the immunosuppressive tumor microenvironment created by PSCs. Therefore, these data provide a strong experimental basis for further in vivo validation, which might potentially open the way for more successful combination strategies with immunotherapy for PDAC.
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Ramu I, Buchholz SM, Patzak MS, Goetze RG, Singh SK, Richards FM, Jodrell DI, Sipos B, Ströbel P, Ellenrieder V, Hessmann E, Neesse A. SPARC dependent collagen deposition and gemcitabine delivery in a genetically engineered mouse model of pancreas cancer. EBioMedicine 2019; 48:161-168. [PMID: 31597597 PMCID: PMC6838446 DOI: 10.1016/j.ebiom.2019.09.024] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Revised: 09/07/2019] [Accepted: 09/13/2019] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) is characterised by extensive matrix deposition that has been implicated in impaired drug delivery and therapeutic resistance. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates collagen deposition and is highly upregulated in the activated stroma subtype with poor prognosis in PDAC patients. METHODS KrasG12D;p48-Cre;SPARC-/- (KC-SPARC-/-) and KrasG12D;p48-Cre;SPARCWT (KC-SPARCWT) were generated and analysed at different stages of carcinogenesis by histological grading, immunohistochemistry for epithelial and stromal markers, survival and preclinical analysis. Pharmacokinetic and pharmacodynamic studies were conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunohistochemistry following gemcitabine treatment (100 mg/kg) in vivo. FINDINGS Global genetic ablation of SPARC in a KrasG12D driven mouse model resulted in significantly reduced overall and mature collagen deposition around early and advanced pancreatic intraepithelial neoplasia (PanIN) lesions and in invasive PDAC (p < .001). However, detailed pathological scoring and molecular analysis showed no effects on PanIN to PDAC progression, vessel density (CD31), tumour incidence, grading or metastatic frequency. Despite comparable tumour kinetics, ablation of SPARC resulted in a significantly shortened survival in KC-SPARC-/- mice (280 days versus 485 days, p < .03, log-rank-test). Using LC-MS/MS, we show that SPARC dependent collagen deposition does not affect intratumoural gemcitabine accumulation or immediate therapeutic response in tumour bearing KC-SPARCWT and KC-SPARC-/-mice. INTERPRETATION Global SPARC ablation reduces the collagen-rich microenvironment in murine PDAC. Moreover, global SPARC depletion did not affect tumour growth kinetics, grading or metastatic frequency. Notably, the dense-collagen matrix did not restrict access of gemcitabine to the tumour. These findings may have direct translational implications in clinical trial design.
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Affiliation(s)
- Iswarya Ramu
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Sören M Buchholz
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Melanie S Patzak
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Robert G Goetze
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Shiv K Singh
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Frances M Richards
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, The University of Cambridge, United Kingdom
| | - Duncan I Jodrell
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, The University of Cambridge, United Kingdom
| | - Bence Sipos
- Institute of Pathology and Neuropathology, University Clinic Tübingen, Germany
| | - Philipp Ströbel
- Institute of Pathology, University Medical Centre Göttingen, Germany
| | - Volker Ellenrieder
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Elisabeth Hessmann
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany
| | - Albrecht Neesse
- Department of Gastroenterology and Gastrointestinal Oncology, University Medical Centre Göttingen, Germany.
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Chen YT, Chen FW, Chang TH, Wang TW, Hsu TP, Chi JY, Hsiao YW, Li CF, Wang JM. Hepatoma-derived growth factor supports the antiapoptosis and profibrosis of pancreatic stellate cells. Cancer Lett 2019; 457:180-190. [PMID: 31078734 DOI: 10.1016/j.canlet.2019.05.001] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Revised: 03/30/2019] [Accepted: 05/02/2019] [Indexed: 12/13/2022]
Abstract
Pancreatic cancer is refractory and is characterized by extensively surrounding and intratumor fibrotic reactions that are contributed by activated pancreatic stellate cells (PSCs). Herein, we show that CCAAT/enhancer-binding protein δ (CEBPD) responds to transforming growth factor-β1 (TGF-β1) through reciprocal loop regulation and that activated hypoxia inducible factor-1α (HIF-1α) further contributes to the upregulation of the hepatoma-derived growth factor (HDGF) gene. Secreted HDGF contributes to the antiapoptosis of PSCs and consequently leads to the synthesis and deposition of extracellular matrix proteins for stabilizing PSC/pancreatic cancer cell (PCC) tumor foci. This result agrees with the observation that severe stromal growth positively correlated with stromal HDGF and CEBPD expression in pancreatic cancer specimens. Collectively, the identification of the TGF-β1-activated CEBPD/HIF-1α/HDGF axis provides new insights into novel discoveries of HDGF in the antiapoptosis and profibrosis of PSCs and the outgrowth of PCCs.
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Affiliation(s)
- Yi-Ting Chen
- Department of Biotechnology and Bioindustry Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan; Medical Research Department, Chi Mei Medical Center, Tainan, Taiwan
| | - Feng-Wei Chen
- Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Tsung-Hao Chang
- Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Tso-Wen Wang
- Department of Biotechnology and Bioindustry Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan
| | - Teng-Po Hsu
- Department of Life Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan
| | - Jhih-Ying Chi
- Department of Biotechnology and Bioindustry Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan
| | - Yu-Wei Hsiao
- Department of Biotechnology and Bioindustry Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan
| | - Chien-Feng Li
- Medical Research Department, Chi Mei Medical Center, Tainan, Taiwan; Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.
| | - Ju-Ming Wang
- Department of Biotechnology and Bioindustry Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan; International Research Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan, Taiwan; Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
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Firuzi O, Che PP, El Hassouni B, Buijs M, Coppola S, Löhr M, Funel N, Heuchel R, Carnevale I, Schmidt T, Mantini G, Avan A, Saso L, Peters GJ, Giovannetti E. Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells. Cancers (Basel) 2019; 11:638. [PMID: 31072019 PMCID: PMC6562408 DOI: 10.3390/cancers11050638] [Citation(s) in RCA: 63] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 04/18/2019] [Accepted: 05/02/2019] [Indexed: 12/18/2022] Open
Abstract
Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids' architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for "stage specific embryonic antigen-4" (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC-PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.
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Affiliation(s)
- Omidreza Firuzi
- Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, 71348-14336 Shiraz, Iran.
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
| | - Pei Pei Che
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
| | - Btissame El Hassouni
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
| | - Mark Buijs
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
| | - Stefano Coppola
- Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, 2333 CA, Leiden, The Netherlands.
| | - Matthias Löhr
- Division of Surgery, CLINTEC, Karolinska Institutet, SE-171, Stockholm, Sweden.
| | - Niccola Funel
- Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa, 56124 Pisa, Italy.
| | - Rainer Heuchel
- Division of Surgery, CLINTEC, Karolinska Institutet, SE-171, Stockholm, Sweden.
| | - Ilaria Carnevale
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
- Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa, 56124 Pisa, Italy.
| | - Thomas Schmidt
- Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden University, 2333 CA, Leiden, The Netherlands.
| | - Giulia Mantini
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
| | - Amir Avan
- Metabolic syndrome Research center, Mashhad University of Medical Sciences, 91778-99191 Mashhad, Iran.
| | - Luciano Saso
- Department of Physiology and Pharmacology "Vittorio Erspamer", Sapienza University, 00185, Rome, Italy.
| | - Godefridus J Peters
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
| | - Elisa Giovannetti
- Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, VU University Medical Center (VUmc), 1081 HV, Amsterdam, The Netherlands.
- Cancer Pharmacology Lab, AIRC Start Up Unit, University of Pisa, 56124 Pisa, Italy.
- Fondazione Pisana per la Scienza, 56017, Pisa, Italy.
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Dooling LJ, Discher DE. Inhibiting Tumor Fibrosis and Actomyosin through GPCR activation. Trends Cancer 2019; 5:197-199. [PMID: 30961827 DOI: 10.1016/j.trecan.2019.02.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2019] [Accepted: 02/07/2019] [Indexed: 12/14/2022]
Abstract
Myofibroblasts produce desmoplastic stroma around tumors and have emerged as therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) and other cancers. Differentiation of pancreatic stellate cells (PSCs) into myofibroblasts is inhibited by the estrogen-receptor modulator, tamoxifen, which activates a G-protein-coupled receptor (GPCR) for estrogen (GPER). This negatively regulates actomyosin contractility and downstream mechanosensitive signaling to profoundly alter the tumor microenvironment, which appears less fibrotic, less immunosuppressive, and more vascularized.
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Affiliation(s)
- Lawrence J Dooling
- Biophysical Engineering Labs University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Dennis E Discher
- Biophysical Engineering Labs University of Pennsylvania, Philadelphia, PA 19104, USA.
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Cortes E, Lachowski D, Rice A, Chronopoulos A, Robinson B, Thorpe S, Lee DA, Possamai LA, Wang H, Pinato DJ, Del Río Hernández AE. Retinoic Acid Receptor-β Is Downregulated in Hepatocellular Carcinoma and Cirrhosis and Its Expression Inhibits Myosin-Driven Activation and Durotaxis in Hepatic Stellate Cells. Hepatology 2019; 69:785-802. [PMID: 30055117 DOI: 10.1002/hep.30193] [Citation(s) in RCA: 44] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/05/2018] [Accepted: 07/25/2018] [Indexed: 01/17/2023]
Abstract
Hepatic stellate cells (HSCs) are essential perisinusoidal cells in both healthy and diseased liver. HSCs modulate extracellular matrix (ECM) homeostasis when quiescent, but in liver fibrosis, HSCs become activated and promote excess deposition of ECM molecules and tissue stiffening via force generation and mechanosensing. In hepatocellular carcinoma (HCC), activated HSCs infiltrate the stroma and migrate to the tumor core to facilitate paracrine signaling with cancer cells. Because the function of HSCs is known to be modulated by retinoids, we investigated the expression profile of retinoic acid receptor beta (RAR-β) in patients with cirrhosis and HCC, as well as the effects of RAR-β activation in HSCs. We found that RAR-β expression is significantly reduced in cirrhotic and HCC tissues. Using a comprehensive set of biophysical methods combined with cellular and molecular biology, we have elucidated the biomechanical mechanism by which all trans-retinoic acid promotes HSC deactivation via RAR-β-dependent transcriptional downregulation of myosin light chain 2 expression. Furthermore, this also abrogated mechanically driven migration toward stiffer substrates. Conclusion: Targeting mechanotransduction in HSCs at the transcriptional level may offer therapeutic options for a range of liver diseases.
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Affiliation(s)
- Ernesto Cortes
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, United Kingdom
| | - Dariusz Lachowski
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, United Kingdom
| | - Alistair Rice
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, United Kingdom
| | - Antonios Chronopoulos
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, United Kingdom
| | - Benjamin Robinson
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, United Kingdom
| | - Stephen Thorpe
- School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom
| | - David A Lee
- School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom
| | - Lucia A Possamai
- Division of Integrative Systems Medicine and Digestive Disease, Imperial College London, London, United Kingdom
| | - Haiyun Wang
- School of Life Sciences and Technology, Tongji University, Shanghai, China
| | - David J Pinato
- Department of Surgery and Cancer, Hammersmith Hospital Campus, Imperial College London, London, United Kingdom
| | - Armando E Del Río Hernández
- Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, United Kingdom
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Lenggenhager D, Amrutkar M, Sántha P, Aasrum M, Löhr JM, Gladhaug IP, Verbeke CS. Commonly Used Pancreatic Stellate Cell Cultures Differ Phenotypically and in Their Interactions with Pancreatic Cancer Cells. Cells 2019; 8:23. [PMID: 30621293 PMCID: PMC6356867 DOI: 10.3390/cells8010023] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2018] [Revised: 12/24/2018] [Accepted: 12/28/2018] [Indexed: 02/07/2023] Open
Abstract
Activated pancreatic stellate cells (PSCs) play a central role in the tumor stroma of pancreatic ductal adenocarcinoma (PDAC). Given the limited availability of patient-derived PSCs from PDAC, immortalized PSC cell lines of murine and human origin have been established; however, it is not elucidated whether differences in species, organ disease status, donor age, and immortalization alter the PSC phenotype and behavior compared to that of patient-derived primary PSC cultures. Therefore, a panel of commonly used PSC cultures was examined for important phenotypical and functional features: three primary cultures from human PDAC, one primary from normal human pancreas, and three immortalized (one from human, two from murine pancreas). Growth rate was considerably lower in primary PSCs from human PDAC. Basal collagen synthesis varied between the PSC cultures, and TGF-β stimulation increased collagen synthesis only in non-immortalized cultures. Differences in secretome composition were observed along with a divergence in the DNA synthesis, migration, and response to gemcitabine of PDAC cell lines that were grown in conditioned medium from the various PSC cultures. The findings reveal considerable differences in features and functions that are key to PSCs and in the interactions with PDAC. These observations may be relevant to researchers when selecting the most appropriate PSC culture for their experiments.
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Affiliation(s)
- Daniela Lenggenhager
- Department of Pathology, Institute of Clinical Medicine, University of Oslo, Blindern, 0316 Oslo, Norway.
- Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Blindern, 0316 Oslo, Norway.
- Department of Pathology and Molecular Pathology, University Hospital Zürich, University of Zürich, Schmelzbergstrasse 12, 8091 Zürich, Switzerland.
| | - Manoj Amrutkar
- Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Blindern, 0316 Oslo, Norway.
- Department of Hepato-Pancreato-Biliary Surgery, Institute of Clinical Medicine, University of Oslo, P.O. Box 1171 Blindern, 0318 Oslo, Norway.
| | - Petra Sántha
- Department of Pathology, Oslo University Hospital Rikshospitalet, Nydalen, 0424 Oslo, Norway.
| | - Monica Aasrum
- Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Blindern, 0316 Oslo, Norway.
| | - Johannes-Matthias Löhr
- Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K 53, 141 86 Stockholm, Sweden.
| | - Ivar P Gladhaug
- Department of Hepato-Pancreato-Biliary Surgery, Institute of Clinical Medicine, University of Oslo, P.O. Box 1171 Blindern, 0318 Oslo, Norway.
- Department of Hepato-Pancreato-Biliary Surgery, Oslo University Hospital Rikshospitalet, Nydalen, 0424 Oslo, Norway.
| | - Caroline S Verbeke
- Department of Pathology, Institute of Clinical Medicine, University of Oslo, Blindern, 0316 Oslo, Norway.
- Department of Pathology, Oslo University Hospital Rikshospitalet, Nydalen, 0424 Oslo, Norway.
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Abstract
Primary cultures of pancreatic stellate cells (PSCs) remain an important basis for in vitro study. However, effective methods for isolating abundant PSCs are currently lacking. This purpose of this chapter is to report our novel approach to isolating PSCs from normal rat pancreas and human pancreatic ductal adenocarcinoma (PDAC) tissue. Normal PSCs were isolated with enzyme digestion and ladder centrifugation with Nycodenz solution. Isolated PSCs were cultured in DMEM/F12 containing 10% fetal bovine serum. Cancer-associated PSCs were obtained by an outgrow method from fresh human PDAC tissues. Isolated activated PSCs were cultured in DMEM/F12 containing 20% fetal bovine serum. With our modification, normal pancreas tissue yields an adequate number of PSCs (approximately 0.5-5 million/g pancreas) for in vitro studies, and the cell viability was about 90%. And a modified outgrowth method made tissue blocks attached more tightly and significantly shortened the outgrowth time of the activated cells. Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period, thus facilitating future PSC-related research.
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