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Zhu Q, He X, Liu J, Wang H, Shan X, Song G, Zhang L, Zhao Y, Yin X. DNA origami assembled spheroid for evaluating cytotoxicity and infiltration of chimeric antigen receptor macrophage (CAR-M). Commun Biol 2024; 7:1302. [PMID: 39390143 PMCID: PMC11467189 DOI: 10.1038/s42003-024-07009-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Accepted: 10/03/2024] [Indexed: 10/12/2024] Open
Abstract
Chimeric antigen receptor (CAR) T-cell therapies have shown remarkable results in patients with hematological malignancies. However, their success in treating solid tumors has been limited. As an alternative candidate for the CAR therapy, CAR-macrophages (CAR-M) have demonstrated activation and phagocytosis directed by tumor-associated antigen (TAA), showing promise in the treatment of solid tumors. Nevertheless, the mechanisms by which CARs direct tumor chemotaxis and invasion of CAR-M remain poorly understood. In this study, we aim to investigate the role of CARs in CAR-M attachment and infiltration using 3D tumor spheroids, which were created by utilizing a novel self-assembling nucleic acid nanostructure decorated living cells (NAC). Our results demonstrated that CAR-M exhibited higher invasion and killing capacity in 2D model and 3D tumor spheroids. In summary, the 3D NAC assembled tumor spheroid model provides a suitable platform for target screening and pharmacodynamic evaluation of CAR-M.
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Affiliation(s)
- Qinyao Zhu
- RocRock Biotechnology Co. Ltd, Suzhou, China
| | - Xiaofang He
- Joint Laboratory of Biomaterials and Translational Medicine, Puheng Technology Co., Ltd, Suzhou, China
| | - Junhua Liu
- RocRock Biotechnology Co. Ltd, Suzhou, China
| | - Heming Wang
- Joint Laboratory of Biomaterials and Translational Medicine, Puheng Technology Co., Ltd, Suzhou, China
| | | | - Guangqi Song
- Joint Laboratory of Biomaterials and Translational Medicine, Puheng Technology Co., Ltd, Suzhou, China.
| | - Luo Zhang
- Research Center of Bioengineering, Medical Innovation Research Division of Chinese PLA General Hospital, Beijing, China.
| | - Yicheng Zhao
- China-Japan Union Hospital of Jilin University, Changchun, China.
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, China.
| | - Xiushan Yin
- RocRock Biotechnology Co. Ltd, Suzhou, China.
- NHC Key Laboratory of Biotechnology Drugs (Shandong Academy of Medical Sciences), Biomedical Sciences College, Shandong First Medical University, Jinan, China.
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Nguyen H, Kim SH, Juang U, Gwon S, Jung W, Huang Q, Lee S, Lee B, Kwon SH, Park J. Overview of carboxyl‑terminal modulator protein 1 and its importance in various metabolic regulations (Review). Mol Med Rep 2024; 30:158. [PMID: 38994770 PMCID: PMC11258604 DOI: 10.3892/mmr.2024.13282] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Accepted: 05/21/2024] [Indexed: 07/13/2024] Open
Abstract
Acyl‑coenzyme A thioesterases (ACOTs) are crucial in mediating lipid metabolic functions, including energy expenditure, hepatic gluconeogenesis and neuronal function. The two distinct types are type I and II ACOTs, the latter of which are 'hotdog' fold superfamily members. Type II ACOTs include carboxyl‑terminal modulator protein 1 (CTMP1), also termed thioesterase superfamily member 4 (THEM4), and CTMP2, also termed THEM5. Due to their similar structural features and distinct sequence homology, CTMP1 and CTMP2 stand out from other type II ACOTs. CTMP1 was initially known as a protein kinase B (PKB) inhibitor that attenuates PKB phosphorylation. PKB is the central regulator of various cellular functions, including survival, proliferation, growth and metabolism. Therefore, by inhibiting PKB, CTMP1 can affect various cellular processes. Various other functions of CTMP1 have been revealed, including functions in cancer, brain injury, mitochondrial function and lipid metabolism. CTMP2 is a paralog of CTMP1 and was first identified as a cardiolipin remodeling factor involved in the development of fatty liver. As the functions of CTMP1 and CTMP2 were discovered separately, a review to summarize and connect these findings is essential. The current review delineates the intricate complexity of CTMP regulation across different metabolic pathways and encapsulates the principal discoveries concerning CTMP until the present day.
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Affiliation(s)
- Huonggiang Nguyen
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Seon-Hwan Kim
- Department of Neurosurgery, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Uijin Juang
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Suhwan Gwon
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Woohyeong Jung
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Qingzhi Huang
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Soohyeon Lee
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - Beomwoo Lee
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
| | - So Hee Kwon
- College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon 21983, Republic of Korea
| | - Jongsun Park
- Department of Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
- Department of Medical Science, Metabolic Syndrome and Cell Signaling Laboratory, Institute for Cancer Research, College of Medicine, Chungnam National University, Daejeon 35015, Republic of Korea
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Nakamura H, Watanabe M, Takada K, Sato T, Hikage F, Umetsu A, Muramatsu J, Furuhashi M, Ohguro H. Modulation of Epithelial-Mesenchymal Transition Is a Possible Underlying Mechanism for Inducing Chemoresistance in MIA PaCa-2 Cells against Gemcitabine and Paclitaxel. Biomedicines 2024; 12:1011. [PMID: 38790973 PMCID: PMC11118094 DOI: 10.3390/biomedicines12051011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2024] [Revised: 04/23/2024] [Accepted: 04/28/2024] [Indexed: 05/26/2024] Open
Abstract
To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial -mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than -6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.
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Affiliation(s)
- Hajime Nakamura
- Departments of Medical Oncology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (H.N.); (K.T.); (J.M.)
| | - Megumi Watanabe
- Departments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (M.W.); (F.H.); (A.U.)
| | - Kohichi Takada
- Departments of Medical Oncology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (H.N.); (K.T.); (J.M.)
| | - Tatsuya Sato
- Departments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (T.S.); (M.F.)
- Departments of Cellular Physiology and Signal Transduction, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan
| | - Fumihito Hikage
- Departments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (M.W.); (F.H.); (A.U.)
| | - Araya Umetsu
- Departments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (M.W.); (F.H.); (A.U.)
| | - Joji Muramatsu
- Departments of Medical Oncology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (H.N.); (K.T.); (J.M.)
| | - Masato Furuhashi
- Departments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (T.S.); (M.F.)
| | - Hiroshi Ohguro
- Departments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan; (M.W.); (F.H.); (A.U.)
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Dinter J, Friedrich RP, Yang H, Pilarsky C, Mangge H, Pöttler M, Janko C, Alexiou C, Lyer S. Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids. MATERIALS (BASEL, SWITZERLAND) 2023; 16:2906. [PMID: 37049199 PMCID: PMC10096321 DOI: 10.3390/ma16072906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/17/2023] [Revised: 03/20/2023] [Accepted: 03/27/2023] [Indexed: 06/19/2023]
Abstract
Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (-/-) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (-/-) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.
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Affiliation(s)
- Jonas Dinter
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
- Medical Faculty, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany
| | - Ralf P. Friedrich
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
| | - Hai Yang
- Department of Surgery, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
| | - Christian Pilarsky
- Department of Surgery, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
| | - Harald Mangge
- Clinical Institute of Medical and Chemical Laboratory Diagnosis, Medical University of Graz, 8036 Graz, Austria
| | - Marina Pöttler
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
| | - Christina Janko
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Professorship for AI-Controlled Nanomaterials, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
| | - Christoph Alexiou
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
| | - Stefan Lyer
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung Professorship, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
- Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Professorship for AI-Controlled Nanomaterials, Universitätsklinikum Erlangen, 91054 Erlangen, Germany
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Lundy J, Harris M, Zalcberg J, Zimet A, Goldstein D, Gebski V, Borsaru A, Desmond C, Swan M, Jenkins BJ, Croagh D. EUS-FNA Biopsies to Guide Precision Medicine in Pancreatic Cancer: Results of a Pilot Study to Identify KRAS Wild-Type Tumours for Targeted Therapy. Front Oncol 2021; 11:770022. [PMID: 34956889 PMCID: PMC8696205 DOI: 10.3389/fonc.2021.770022] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Accepted: 11/18/2021] [Indexed: 12/27/2022] Open
Abstract
Background Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death and lacks effective treatment options. Diagnostic endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) biopsies represent an appealing source of material for molecular analysis to inform targeted therapy, as they are often the only available tissue for patients presenting with PDAC irrespective of disease stage. However, EUS-FNA biopsies are typically not used to screen for precision medicine studies due to concerns about low tissue yield and quality. Epidermal growth factor receptor (EGFR) inhibition has shown promise in clinical trials of unselected patients with advanced pancreatic cancer, but has not been prospectively tested in KRAS wild-type patients. Here, we examine the clinical utility of EUS-FNA biopsies for molecular screening of KRAS wild-type PDAC patients for targeted anti-EGFR therapy to assess the feasibility of this approach. Patients and Methods Fresh frozen EUS-FNA or surgical biopsies from PDAC patient tumours were used to screen for KRAS mutations. Eligible patients with recurrent, locally advanced, or metastatic KRAS wild-type status who had received at least one prior line of chemotherapy were enrolled in a pilot study (ACTRN12617000540314) and treated with panitumumab at 6mg/kg intravenously every 2 weeks until progression or unacceptable toxicity. The primary endpoint was 4-month progression-free survival (PFS). Results 275 patient biopsies were screened for KRAS mutations, which were detected in 88.3% of patient samples. 8 eligible KRAS wild-type patients were enrolled onto the interventional study between November 2017 and December 2020 and treated with panitumumab. 4-month PFS was 14.3% with no objective tumour responses observed. The only grade 3/4 treatment related toxicity observed was hypomagnesaemia. Conclusions This study demonstrates proof-of-principle feasibility to molecularly screen patients with pancreatic cancer for targeted therapies, and confirms diagnostic EUS-FNA biopsies as a reliable source of tumour material for molecular analysis. Single agent panitumumab was safe and tolerable but led to no objective tumour responses in this population.
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Affiliation(s)
- Joanne Lundy
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Molecular and Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia.,Department of Surgery, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
| | - Marion Harris
- Department of Oncology, Faculty of Medicine, Nursing and Health Sciences and School of Clinical Sciences, Monash University, Clayton, VIC, Australia
| | - John Zalcberg
- Department of Medical Oncology, Alfred Health, Melbourne, VIC, Australia.,Public Health and Preventative Medicine, Monash University, Melbourne, VIC, Australia
| | - Allan Zimet
- Department of Medical Oncology, Epworth Hospital, Melbourne, VIC, Australia
| | - David Goldstein
- Prince of Wales Clinical School, University of New South Wales, Sydney, NSW, Australia.,Department of Medical Oncology, Prince of Wales Hospital, Randwick, NSW, Australia
| | - Val Gebski
- National Health and Medical Research Council Clinical Trials Centre, University of Sydney, Camperdown, NSW, Australia
| | - Adina Borsaru
- Diagnostic Imaging, Monash Health, Melbourne, VIC, Australia
| | | | - Michael Swan
- Department of Gastroenterology, Monash Health, Melbourne, VIC, Australia
| | - Brendan J Jenkins
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Molecular and Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
| | - Daniel Croagh
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Surgery, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia.,Department of Surgery, Epworth Healthcare, Melbourne, VIC, Australia
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Kaur G, Evans DM, Teicher BA, Coussens NP. Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine. SLAS DISCOVERY : ADVANCING LIFE SCIENCES R & D 2021; 26:1298-1314. [PMID: 34772287 DOI: 10.1177/24725552211038362] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.
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Affiliation(s)
- Gurmeet Kaur
- Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA
| | | | - Beverly A Teicher
- Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA
| | - Nathan P Coussens
- Molecular Pharmacology Laboratories, Applied and Developmental Research Directorate, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
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Doctor A, Seifert V, Ullrich M, Hauser S, Pietzsch J. Three-Dimensional Cell Culture Systems in Radiopharmaceutical Cancer Research. Cancers (Basel) 2020; 12:cancers12102765. [PMID: 32993034 PMCID: PMC7600608 DOI: 10.3390/cancers12102765] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Revised: 09/14/2020] [Accepted: 09/16/2020] [Indexed: 12/12/2022] Open
Abstract
In preclinical cancer research, three-dimensional (3D) cell culture systems such as multicellular spheroids and organoids are becoming increasingly important. They provide valuable information before studies on animal models begin and, in some cases, are even suitable for reducing or replacing animal experiments. Furthermore, they recapitulate microtumors, metastases, and the tumor microenvironment much better than monolayer culture systems could. Three-dimensional models show higher structural complexity and diverse cell interactions while reflecting (patho)physiological phenomena such as oxygen and nutrient gradients in the course of their growth or development. These interactions and properties are of great importance for understanding the pathophysiological importance of stromal cells and the extracellular matrix for tumor progression, treatment response, or resistance mechanisms of solid tumors. Special emphasis is placed on co-cultivation with tumor-associated cells, which further increases the predictive value of 3D models, e.g., for drug development. The aim of this overview is to shed light on selected 3D models and their advantages and disadvantages, especially from the radiopharmacist's point of view with focus on the suitability of 3D models for the radiopharmacological characterization of novel radiotracers and radiotherapeutics. Special attention is paid to pancreatic ductal adenocarcinoma (PDAC) as a predestined target for the development of new radionuclide-based theranostics.
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Affiliation(s)
- Alina Doctor
- Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; (A.D.); (V.S.); (M.U.); (S.H.)
- School of Science, Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, 01069 Dresden, Germany
| | - Verena Seifert
- Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; (A.D.); (V.S.); (M.U.); (S.H.)
- School of Science, Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, 01069 Dresden, Germany
| | - Martin Ullrich
- Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; (A.D.); (V.S.); (M.U.); (S.H.)
| | - Sandra Hauser
- Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; (A.D.); (V.S.); (M.U.); (S.H.)
| | - Jens Pietzsch
- Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; (A.D.); (V.S.); (M.U.); (S.H.)
- School of Science, Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, 01069 Dresden, Germany
- Correspondence: ; Tel.: +49-351-260-2622
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Puppi D, Chiellini F. Biodegradable Polymers for Biomedical Additive Manufacturing. APPLIED MATERIALS TODAY 2020; 20:100700. [DOI: 10.1016/j.apmt.2020.100700] [Citation(s) in RCA: 60] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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Tawfik D, Zaccagnino A, Bernt A, Szczepanowski M, Klapper W, Schwab A, Kalthoff H, Trauzold A. The A818-6 system as an in-vitro model for studying the role of the transportome in pancreatic cancer. BMC Cancer 2020; 20:264. [PMID: 32228510 PMCID: PMC7106758 DOI: 10.1186/s12885-020-06773-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Accepted: 03/23/2020] [Indexed: 02/08/2023] Open
Abstract
Background The human pancreatic cancer cell line A818–6 can be grown in vitro either as a highly malignant, undifferentiated monolayer (ML) or as three-dimensional (3D) single layer hollow spheres (HS) simulating a benign, highly differentiated, duct-like pancreatic epithelial structure. This characteristic allowing A818–6 cells to switch from one phenotype to another makes these cells a unique system to characterize the cellular and molecular modifications during differentiation on one hand and malignant transformation on the other hand. Ion channels and transport proteins (transportome) have been implicated in malignant transformation. Therefore, the current study aimed to analyse the transportome gene expression profile in the A818–6 cells growing as a monolayer or as hollow spheres. Methods & Results The study identified the differentially expressed transportome genes in both cellular states of A818–6 using Agilent and Nanostring arrays and some targets were validated via immunoblotting. Additionally, these results were compared to a tissue Affymetrix microarray analysis of pancreatic adenocarcinoma patients’ tissues. The overall transcriptional profile of the ML and HS cells confirmed the formerly described mesenchymal features of ML and epithelial nature of HS which was further verified via high expression of E-cadherin and low expression of vimentin found in HS in comparison to ML. Among the predicted features between HS and ML was the involvement of miRNA-9 in this switch. Importantly, the bioinformatics analysis also revealed substantial number (n = 126) of altered transportome genes. Interestingly, three genes upregulated in PDAC tissue samples (GJB2, GJB5 and SLC38A6) were found to be also upregulated in ML and 3 down-regulated transportome genes (KCNQ1, TRPV6 and SLC4A) were also reduced in ML. Conclusion This reversible HS/ML in vitro system might help in understanding the pathophysiological impact of the transportome in the dedifferentiation process in pancreatic carcinogenesis. Furthermore, the HS/ML model represents a novel system for studying the role of the transportome during the switch from a more benign, differentiated (HS) to a highly malignant, undifferentiated (ML) phenotype.
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Affiliation(s)
- Doaa Tawfik
- Institute for Experimental Cancer Research, Christian-Albrechts-University of Kiel, Arnold-Heller Str. 3, 24105, Kiel, Germany
| | - Angela Zaccagnino
- Institute for Experimental Cancer Research, Christian-Albrechts-University of Kiel, Arnold-Heller Str. 3, 24105, Kiel, Germany
| | - Alexander Bernt
- Institute for Experimental Cancer Research, Christian-Albrechts-University of Kiel, Arnold-Heller Str. 3, 24105, Kiel, Germany
| | - Monika Szczepanowski
- Clinic for Internal Medicine II, Christian-Albrechts-University of Kiel, UKSH, Kiel, Germany
| | - Wolfram Klapper
- Institute of Pathology, Hematopathology Section and Lymph Node Registry, Christian-Albrechts-University of Kiel, UKSH, Kiel, Germany
| | - Albrecht Schwab
- Institute of Physiology II, Westfälische Wilhelms-Universität, Münster, Germany
| | - Holger Kalthoff
- Institute for Experimental Cancer Research, Christian-Albrechts-University of Kiel, Arnold-Heller Str. 3, 24105, Kiel, Germany
| | - Anna Trauzold
- Institute for Experimental Cancer Research, Christian-Albrechts-University of Kiel, Arnold-Heller Str. 3, 24105, Kiel, Germany.
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10
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Fukuchi H, Hayashida Y, Inoue K, Sadamura Y. Forkhead box B2 inhibits the malignant characteristics of the pancreatic cancer cell line Panc-1 in vitro. Genes Cells 2019; 24:674-681. [PMID: 31433897 DOI: 10.1111/gtc.12717] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2019] [Revised: 08/09/2019] [Accepted: 08/19/2019] [Indexed: 01/10/2023]
Abstract
Forkhead box (FOX) proteins constitute a family of transcription factors that are evolutionarily conserved in various species ranging from yeast to humans. These proteins have functions during development as well as in adulthood. To date, many reports have described the functions of FOX family genes in cancer cells, but the role of FOXB2 is not well understood. In one of the pancreas ductal adenocarcinoma cell lines, Panc-1 cells, we showed here that FOXB2 expression is barely detectable and that CpG islands in the 5' regions of the FOXB2 are highly methylated. These findings led us to hypothesize that FOXB2 acts as a tumor suppressor. To clarify our hypotheses, we investigated the effects of FOXB2 over-expression in Panc-1 cells. We obtained FOXB2 stable transfectants, and these clones exhibited reduced spheroid formation ability. Expression of β-catenin, which is reported to be over-expressed in various cancer cells, was highly suppressed in FOXB2 stable transfectants. Moreover, side population (SP) cell fractions, which have a high tumorigenesis and metastatic potential, as well as anchorage-independent growth ability, were reduced. These results suggest that FOXB2 has the ability to inhibit the malignant characteristics of Panc-1 in vitro.
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Affiliation(s)
- Hiroki Fukuchi
- Life Science Research Laboratories, FUJIFILM Wako Pure Chemical Corporation, Amagasaki, Japan
- Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan
| | - Yukinobu Hayashida
- Life Science Research Laboratories, FUJIFILM Wako Pure Chemical Corporation, Amagasaki, Japan
| | - Kunio Inoue
- Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan
| | - Yoshifusa Sadamura
- Life Science Research Laboratories, FUJIFILM Wako Pure Chemical Corporation, Amagasaki, Japan
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11
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Tataranni T, Agriesti F, Pacelli C, Ruggieri V, Laurenzana I, Mazzoccoli C, Sala GD, Panebianco C, Pazienza V, Capitanio N, Piccoli C. Dichloroacetate Affects Mitochondrial Function and Stemness-Associated Properties in Pancreatic Cancer Cell Lines. Cells 2019; 8:cells8050478. [PMID: 31109089 PMCID: PMC6562462 DOI: 10.3390/cells8050478] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Revised: 05/14/2019] [Accepted: 05/15/2019] [Indexed: 12/14/2022] Open
Abstract
Targeting metabolism represents a possible successful approach to treat cancer. Dichloroacetate (DCA) is a drug known to divert metabolism from anaerobic glycolysis to mitochondrial oxidative phosphorylation by stimulation of PDH. In this study, we investigated the response of two pancreatic cancer cell lines to DCA, in two-dimensional and three-dimension cell cultures, as well as in a mouse model. PANC-1 and BXPC-3 treated with DCA showed a marked decrease in cell proliferation and migration which did not correlate with enhanced apoptosis indicating a cytostatic rather than a cytotoxic effect. Despite PDH activation, DCA treatment resulted in reduced mitochondrial oxygen consumption without affecting glycolysis. Moreover, DCA caused enhancement of ROS production, mtDNA, and of the mitophagy-marker LC3B-II in both cell lines but reduced mitochondrial fusion markers only in BXPC-3. Notably, DCA downregulated the expression of the cancer stem cells markers CD24/CD44/EPCAM only in PANC-1 but inhibited spheroid formation/viability in both cell lines. In a xenograft pancreatic cancer mouse-model DCA treatment resulted in retarding cancer progression. Collectively, our results clearly indicate that the efficacy of DCA in inhibiting cancer growth mechanistically depends on the cell phenotype and on multiple off-target pathways. In this context, the novelty that DCA might affect the cancer stem cell compartment is therapeutically relevant.
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Affiliation(s)
- Tiziana Tataranni
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
| | - Francesca Agriesti
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
| | - Consiglia Pacelli
- Department of Clinical and Experimental Medicine, University of Foggia, 71100 Foggia, Italy.
| | - Vitalba Ruggieri
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
| | - Ilaria Laurenzana
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
| | - Carmela Mazzoccoli
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
| | - Gerardo Della Sala
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
| | - Concetta Panebianco
- Division of Gastroenterology, IRCCS "Casa Sollievo della Sofferenza" Hospital, 71013 San Giovanni Rotondo, Italy.
| | - Valerio Pazienza
- Division of Gastroenterology, IRCCS "Casa Sollievo della Sofferenza" Hospital, 71013 San Giovanni Rotondo, Italy.
| | - Nazzareno Capitanio
- Department of Clinical and Experimental Medicine, University of Foggia, 71100 Foggia, Italy.
| | - Claudia Piccoli
- Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, 85028 Rionero in Vulture (Pz), Italy.
- Department of Clinical and Experimental Medicine, University of Foggia, 71100 Foggia, Italy.
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12
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Chen M, Shah MP, Shelper TB, Nazareth L, Barker M, Tello Velasquez J, Ekberg JAK, Vial ML, St John JA. Naked Liquid Marbles: A Robust Three-Dimensional Low-Volume Cell-Culturing System. ACS APPLIED MATERIALS & INTERFACES 2019; 11:9814-9823. [PMID: 30724549 DOI: 10.1021/acsami.8b22036] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Three-dimensional (3D) multicellular structures allow cells to behave and interact with each other in a manner that mimics the in vivo environment. In recent years, many 3D cell culture methods have been developed with the goal of producing the most in vivo-like structures possible. Whilst strongly preferable to conventional cell culture, these approaches are often poorly reproducible, time-consuming, expensive, and labor-intensive and require specialized equipment. Here, we describe a novel 3D culture platform, which we have termed the naked liquid marble (NLM). Cells are cultured in a liquid drop (the NLM) in superhydrophobic-coated plates, which causes the cells to naturally form 3D structures. Inside the NLMs, cells are free to interact with each other, forming multiple 3D spheroids that are uniform in size and shape in less than 24 h. We showed that this system is highly reproducible, suitable for cell coculture, compound screening, and also compatible with laboratory automation systems. The low cost of production, small volume of each NLM, and production via automated liquid handling make this 3D cell-culturing system particularly suitable for high-throughput screening assays such as drug testing as well as numerous other cell-based research applications.
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Affiliation(s)
- Mo Chen
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
| | - Megha P Shah
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
| | - Todd B Shelper
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
| | - Lynn Nazareth
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
| | | | | | - Jenny A K Ekberg
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
| | - Marie-Laure Vial
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
| | - James A St John
- Menzies Health Institute Queensland , Griffith University , Southport , 4222 Queensland , Australia
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13
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Cruz-Acuña R, García AJ. Engineered materials to model human intestinal development and cancer using organoids. Exp Cell Res 2019; 377:109-114. [PMID: 30794801 DOI: 10.1016/j.yexcr.2019.02.017] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2018] [Revised: 01/27/2019] [Accepted: 02/18/2019] [Indexed: 02/06/2023]
Abstract
Human organoids provide constructive in vitro models of human development and disease, as these recapitulate important morphogenetic and functional features of the tissue and species of origin. However, organoid culture technologies often involve the use of biologically-derived materials (e.g. Matrigel™) that do not allow dissection of the independent contributions of the biochemical and biophysical matrix properties to organoid development. Additionally, their inherent lot-to-lot variability and, in the case of Matrigel™, tumor-derived nature limits their applicability as platforms for drug and tissue transplantation therapies. Here, we highlight recent studies that overcome these limitations through engineering of novel biomaterial platforms that (1) allow to study the independent contributions of physicochemical matrix properties to organoid development and their potential for translational therapies, and (2) better recreate the tumor microenvironment for high-throughput, pre-clinical drug development. These studies illustrate how innovative biomaterial constructs can contribute to the modeling of human development and disease using organoids, and as platforms for development of organoid-based therapies. Finally, we discuss the current limitations of the organoid field and how they can potentially be addressed using engineered biomaterials.
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Affiliation(s)
- Ricardo Cruz-Acuña
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, United States; Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332, United States; George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, United States
| | - Andrés J García
- Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332, United States; George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, United States.
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14
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Lin CC, Korc M. Designer hydrogels: Shedding light on the physical chemistry of the pancreatic cancer microenvironment. Cancer Lett 2018; 436:22-27. [PMID: 30118843 PMCID: PMC6557435 DOI: 10.1016/j.canlet.2018.08.008] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Revised: 07/13/2018] [Accepted: 08/09/2018] [Indexed: 01/18/2023]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer mortality in the United States, with a 5-year survival of ∼8%. PDAC is characterized by a dense and hypo-vascularized stroma consisting of proliferating cancer cells, cancer-associated fibroblasts, macrophages and immune cells, as well as excess matrices including collagens, fibronectin, and hyaluronic acid. In addition, PDAC has increased interstitial pressures and a hypoxic/acidic tumor microenvironment (TME) that impedes drug delivery and blocks cancer-directed immune mechanisms. In spite of increasing options in targeted therapy, PDAC has mostly remained treatment recalcitrant. Owing to its critical roles on governing PDAC progression and treatment outcome, TME and its interplay with the cancer cells are increasingly studied. In particular, three-dimensional (3D) hydrogels derived from or inspired by components in the TME are progressively developed. When properly designed, these hydrogels (e.g., Matrigel, collagen gel, hyaluronic acid-based, and semi-synthetic hydrogels) can provide pathophysiologically relevant compositions, conditions, and contexts for supporting PDAC cell fate processes. This review summarizes recent efforts in using 3D hydrogels for fundamental studies on cell-matrix or cell-cell interactions in PDAC.
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Affiliation(s)
- Chien-Chi Lin
- Department of Biomedical Engineering, Purdue School of Engineering & Technology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202, USA; Indiana University Melvin and Bren Simon Cancer Center and the Pancreatic Cancer Signature Center, Indianapolis, IN, 46202, USA.
| | - Murray Korc
- Indiana University Melvin and Bren Simon Cancer Center and the Pancreatic Cancer Signature Center, Indianapolis, IN, 46202, USA; Departments of Medicine and Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
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15
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Multi-functional nanoparticles as theranostic agents for the treatment & imaging of pancreatic cancer. Acta Biomater 2017; 49:422-433. [PMID: 27890622 DOI: 10.1016/j.actbio.2016.11.053] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Revised: 11/02/2016] [Accepted: 11/22/2016] [Indexed: 01/02/2023]
Abstract
Theranostics has received considerable attention since both therapy and imaging modalities can be integrated into a single nanocarrier. In this study, fluorescent iron oxide (FIO) nanoparticles and gemcitabine (G) encapsulated poly(lactide-co-glycolide) (PLGA) nanospheres (PGFIO) conjugated with human epidermal growth factor receptor 2, (HER-2) antibody (HER-PGFIO) were prepared and characterized. HER-PGFIO showed the magnetic moment of 10emu/g, relaxivity (r2) of 773mM-1s-1 and specific absorption rate (SAR) of 183W/g. HER-PGFIO showed a sustained release of gemcitabine for 11days in PBS (pH 7.4). In vitro cytotoxicity evaluation of HER-PGFIO in 3D MIAPaCa-2 cultures showed 50% inhibitory concentration (IC50) of 0.11mg/mL. Subcutaneous tumor xenografts of MIAPaCa-2 in SCID mice were developed and the tumor regression study at the end of 30days showed significant tumor regression (86±3%) in the HER-PGFIO with magnetic hyperthermia (MHT) treatment group compared to control group. In vivo MRI imaging showed the enhanced contrast in HER-PGFIO+MHT treated group compared to control. HER-PGFIO showed significant tumor regression and enhanced MRI in treatment groups, which could be an effective nanocarrier system for the treatment of pancreatic cancer. STATEMENT OF SIGNIFICANCE Combination therapies are best suitable to treat pancreatic cancer. Theranostics are the next generation therapeutics with both imaging and treatment agents encapsulated in a single nanocarrier. The novelty of the present work is the development of targeted nanocarrier that provides chemotherapy, thermotherapy and MRI imaging properties. The present work is the next step in developing the nanocarriers for pancreatic cancer treatment. Different treatment modalities embedding into a single nanocarrier is the biggest challenge that was achieved without compromising the functionality of each other. The surface modification of polymeric nanocarriers for antibody binding and their multifunctional abilities will appeal to wider audience.
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16
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Abstract
Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy for which new treatment and diagnostic approaches are urgently needed. In order for such breakthroughs to be discovered, researchers require systems that accurately model the development and biology of PDA. While cell lines, genetically engineered murine models, and xenografts have all led to valuable clinical insights, organotypic culture models have emerged as tractable systems to recapitulate the complex three-dimensional organization of PDA. Recently, multiple methods for modeling PDA using organoids have been reported. This review aims to summarize these organoid methods in the context of other PDA models. While each model system has unique benefits and drawbacks, ultimately, organoids hold special promise for the development of personalized medicine approaches.
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Affiliation(s)
- Lindsey A. Baker
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
- Lustgarten Pancreatic Cancer Research Laboratory, Cold Spring Harbor, NY 11724, USA
| | - Hervé Tiriac
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
- Lustgarten Pancreatic Cancer Research Laboratory, Cold Spring Harbor, NY 11724, USA
| | - Hans Clevers
- Hubrecht Institute and University Medical Centre Utrecht, Utrecht, The Netherlands
| | - David A. Tuveson
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
- Lustgarten Pancreatic Cancer Research Laboratory, Cold Spring Harbor, NY 11724, USA
- Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
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17
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Chiellini F, Puppi D, Piras AM, Morelli A, Bartoli C, Migone C. Modelling of pancreatic ductal adenocarcinoma in vitro with three-dimensional microstructured hydrogels. RSC Adv 2016. [DOI: 10.1039/c6ra08420f] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Development of a novelin vitro3D model of pancreas cancer based on microstructured polyelectrolyte complex (mPEC) hydrogel.
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Affiliation(s)
- F. Chiellini
- BIOLab Research Group
- Department of Chemistry and Industrial Chemistry
- University of Pisa
- UdR INSTM Pisa
- Pisa
| | - D. Puppi
- BIOLab Research Group
- Department of Chemistry and Industrial Chemistry
- University of Pisa
- UdR INSTM Pisa
- Pisa
| | - A. M. Piras
- BIOLab Research Group
- Department of Chemistry and Industrial Chemistry
- University of Pisa
- UdR INSTM Pisa
- Pisa
| | - A. Morelli
- BIOLab Research Group
- Department of Chemistry and Industrial Chemistry
- University of Pisa
- UdR INSTM Pisa
- Pisa
| | - C. Bartoli
- BIOLab Research Group
- Department of Chemistry and Industrial Chemistry
- University of Pisa
- UdR INSTM Pisa
- Pisa
| | - C. Migone
- BIOLab Research Group
- Department of Chemistry and Industrial Chemistry
- University of Pisa
- UdR INSTM Pisa
- Pisa
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18
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Lovitt CJ, Shelper TB, Avery VM. Miniaturized three-dimensional cancer model for drug evaluation. Assay Drug Dev Technol 2015; 11:435-48. [PMID: 25310845 DOI: 10.1089/adt.2012.483] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
A more relevant in vitro cell culture model that closely mimics tumor biology and provides better predictive information on anticancer therapies has been the focus of much attention in recent years. We have developed a three-dimensional (3D) human tumor cell culture model that attempts to recreate the in vivo microenvironment and tumor biology in a miniaturized 384-well plate format. This model aims to exploit the potential of 3D cell culture as a screening tool for novel therapeutics for discovery programs. Here we have evaluated a Matrigel™ based induction of 3D tumor formation using standard labware and plate reading equipment. We have demonstrated that with an optimized protocol, reproducible proliferation, and cell viability data can be obtained across a range of cell lines and reagent batches. A panel of reference drugs was used to validate the suitability of the assays for a high throughput drug discovery program. Indicators of assay reproducibility, such as Z'-factor and coefficient of variation, as well as dose response curves confirmed the robustness of the assays. Several methods of drug activity determination were examined, including metabolic and imaging based assays. These data demonstrate this model as a robust tool for drug discovery bridging the gap between monolayer cell culture and animal models, providing insights into drug efficacy at an earlier time point, ultimately reducing costs and high attrition rates.
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Affiliation(s)
- Carrie J Lovitt
- Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University , Nathan, Australia
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19
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Coleman SJ, Watt J, Arumugam P, Solaini L, Carapuca E, Ghallab M, Grose RP, Kocher HM. Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation. World J Gastroenterol 2014; 20:8471-8481. [PMID: 25024603 PMCID: PMC4093698 DOI: 10.3748/wjg.v20.i26.8471] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/18/2013] [Revised: 01/15/2014] [Accepted: 04/01/2014] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer carries a terrible prognosis, as the fourth most common cause of cancer death in the Western world. There is clearly a need for new therapies to treat this disease. One of the reasons no effective treatment has been developed in the past decade may in part, be explained by the diverse influences exerted by the tumour microenvironment. The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate. Thus, appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer. Here we discuss the evolution of 3D organotypic models, which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma (PDAC). Organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC. A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short time-frame. This allows new therapies that can target the cancer, the stromal compartment or both to be tested in a model that mirrors the in vivo situation. A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely. We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients.
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20
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Seeberger KL, Anderson SJ, Ellis CE, Yeung TY, Korbutt GS. Identification and differentiation of PDX1 β-cell progenitors within the human pancreatic epithelium. World J Diabetes 2014; 5:59-68. [PMID: 24567802 PMCID: PMC3932428 DOI: 10.4239/wjd.v5.i1.59] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2013] [Revised: 11/27/2013] [Accepted: 12/16/2013] [Indexed: 02/05/2023] Open
Abstract
AIM: To minimize the expansion of pancreatic mesenchymal cells in vitro and confirm that β-cell progenitors reside within the pancreatic epithelium.
METHODS: Due to mesenchymal stem cell (MSC) expansion and overgrowth, progenitor cells within the pancreatic epithelium cannot be characterized in vitro, though β-cell dedifferentiation and expansion of MSC intermediates via epithelial-mesenchymal transition (EMT) may generate β-cell progenitors. Pancreatic epithelial cells from endocrine and non-endocrine tissue were expanded and differentiated in a novel pancreatic epithelial expansion medium supplemented with growth factors known to support epithelial cell growth (dexamethasone, epidermal growth factor, 3,5,3’-triiodo-l-thyronine, bovine brain extract). Cells were also infected with a single and dual lentiviral reporter prior to cell differentiation. Enhanced green fluorescent protein was controlled by the rat Insulin 1 promoter and the monomeric red fluorescent protein was controlled by the mouse PDX1 promoter. In combination with lentiviral tracing, cells expanded and differentiated in the pancreatic medium were characterized by flow cytometry (BD fluorescence activated cell sorting), immunostaining and real-time polymerase chain reaction (PCR) (7900HT Fast Realtime PCR System).
RESULTS: In the presence of 10% serum MSCs rapidly expand in vitro while the epithelial cell population declines. The percentage of vimentin+ cells increased from 22% ± 5.83% to 80.43% ± 3.24% (14 d) and 99.00% ± 0.0% (21 d), and the percentage of epithelial cells decreased from 74.71% ± 8.34% to 26.57% ± 9.75% (14 d) and 4.00% ± 1.53% (21 d), P < 0.01 for all time points. Our novel pancreatic epithelial expansion medium preserved the epithelial cell phenotype and minimized epithelial cell dedifferentiation and EMT. Cells expanded in our epithelial medium contained significantly less mesenchymal cells (vimentin+) compared to controls (44.87% ± 4.93% vs 95.67% ± 1.36%; P < 0.01). During cell differentiation lentiviral reporting demonstrated that, PDX1+ and insulin+ cells were localized within adherent epithelial cell aggregates compared to controls. Compared to starting islets differentiated cells had at least two fold higher gene expression of PDX1, insulin, PAX4 and RFX (P < 0.05).
CONCLUSION: PDX1+ cells were confined to adherent epithelial cell aggregates and not vimentin+ cells (mesenchymal), suggesting that EMT is not a mechanism for generating pancreatic progenitor cells.
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21
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Zhang S, Zhou X, Chen T, Shang Y, Lu R, Yin D, Liu J, Xu H, Mo X. Single primary fetal lung cells generate alveolar structures in vitro. In Vitro Cell Dev Biol Anim 2013; 50:87-93. [PMID: 24092015 DOI: 10.1007/s11626-013-9657-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2013] [Accepted: 06/26/2013] [Indexed: 02/05/2023]
Abstract
Organ morphogenesis, including lung morphogenesis, involves a series of cellular behaviors that are difficult to observe and document in vivo due to current limitations in imaging techniques. Therefore, in vitro models are necessary to study these cellular behaviors as well as basic developmental processes relevant to in vivo morphogenesis. Here, we describe a novel in vitro three-dimensional (3D) culture system for assessing mouse lung alveolar morphogenesis using primary fetal mouse lung cells cultured in Matrigel supplemented with fibroblast growth factor 10 and hepatocyte growth factor. In our in vitro 3D culture system, single primary mouse fetal lung cells successfully grew, developed lumen, and formed multivesicular epithelial structures, resulting in a morphology that was highly similar to that of lung alveoli. This culture system is a useful tool for investigating the cellular and molecular mechanisms involved in lung alveolar morphogenesis.
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Affiliation(s)
- Shengliang Zhang
- Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China, 610041
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22
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Lieu CH, Tan AC, Leong S, Diamond JR, Eckhardt SG. From bench to bedside: lessons learned in translating preclinical studies in cancer drug development. J Natl Cancer Inst 2013; 105:1441-56. [PMID: 24052618 PMCID: PMC3787906 DOI: 10.1093/jnci/djt209] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The development of targeted agents in oncology has rapidly expanded over the past 2 decades and has led to clinically significant improvements in the treatment of numerous cancers. Unfortunately, not all success at the bench in preclinical experiments has translated to success at the bedside. As preclinical studies shift toward defining proof of mechanism, patient selection, and rational drug combinations, it is critical to understand the lessons learned from prior translational studies to gain an understanding of prior drug development successes and failures. By learning from prior drug development, future translational studies will provide more clinically relevant data, and the underlying hope is that the clinical success rate will improve and the treatment of patients with ineffective targeted therapy will be limited.
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Affiliation(s)
- Christopher H Lieu
- Affiliation of authors: Division of Medical Oncology, University of Colorado, Aurora, CO (CHL, A-CT, SL, JRD, SGE)
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23
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Longati P, Jia X, Eimer J, Wagman A, Witt MR, Rehnmark S, Verbeke C, Toftgård R, Löhr M, Heuchel RL. 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer 2013; 13:95. [PMID: 23446043 PMCID: PMC3617005 DOI: 10.1186/1471-2407-13-95] [Citation(s) in RCA: 275] [Impact Index Per Article: 22.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2012] [Accepted: 02/24/2013] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. METHODS Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. RESULTS Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. CONCLUSIONS We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.
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Affiliation(s)
- Paola Longati
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
| | - Xiaohui Jia
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
| | - Johannes Eimer
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
| | - Annika Wagman
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
| | | | - Stefan Rehnmark
- Axcentua Pharmaceuticals AB, Nobels Allé 10, Stockholm, 14157, Sweden
| | - Caroline Verbeke
- Department of Pathology, Karolinska Institutet, Stockholm, 14186, Sweden
| | - Rune Toftgård
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
| | - Matthias Löhr
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
| | - Rainer L Heuchel
- CLINTEC, Karolinska Institutet, Stockholm 14186, Sweden
- Center of Biosciences, Karolinska Institutet, Stockholm 14186, Sweden
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Sakurai A, Matsuda M, Kiyokawa E. Activated Ras protein accelerates cell cycle progression to perturb Madin-Darby canine kidney cystogenesis. J Biol Chem 2012; 287:31703-11. [PMID: 22829590 DOI: 10.1074/jbc.m112.377804] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
In a number of human cancer cells, K-RAS is frequently mutated and activated constitutively, culminating in the induction of continuous cell growth, a hallmark of cancer cells. It is still unclear, however, how the mutated K-RAS induces morphological abnormalities in cancerous tissues. To investigate the mechanism underlying the K-RAS-induced morphological changes, we utilized an auxin-dependent protein expression system, which enabled us to rapidly induce and evaluate constitutively active K-Ras in MDCK (Madin-Darby canine kidney) cysts, a model for polarized epithelial structure. Cells carrying the constitutively active KRasV12 gene were morphologically indistinguishable from normal cells in two-dimensional culture. However, in a gel of extracellular matrix, KRasV12-expressing cells failed to form a spherical cyst. When KRasV12 induction was delayed until after cyst formation, some cells in the cyst wall lost polarity and were extruded into and accumulated in the luminal space. With effector-specific mutants of KRasV12 and inhibitors for MEK and PI3-kinase, we found that both the Raf-MEK-ERK and PI3-kinase axes are necessary and sufficient for this phenotype. Live cell imaging with cell cycle indicators showed that KRasV12 expression promoted cell cycle progression, which was prevented by either MEK or PI3-kinase inhibitors. From these results, we provide a model wherein active-Ras induces cell cycle progression leading to apical cell extrusion through Raf and PI3-kinase in a cooperative manner. The system developed here can be applied to drug screening for various cancers originating from epithelial cells.
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Affiliation(s)
- Atsuro Sakurai
- Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Kyoto 606-8501, Japan
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Winterhoff BJN, Arlt A, Duttmann A, Ungefroren H, Schäfer H, Kalthoff H, Kruse ML. Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system. Differentiation 2011; 83:148-57. [PMID: 22364882 DOI: 10.1016/j.diff.2011.11.013] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2010] [Revised: 09/27/2011] [Accepted: 11/23/2011] [Indexed: 01/15/2023]
Abstract
The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation-dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a "snowball effect" of apoptotic events.
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Affiliation(s)
- Boris J N Winterhoff
- Institute for Experimental Cancer Research, Division Molecular Oncology, University Hospital Schleswig-Holstein Campus Kiel, Germany
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Sempere LF, Gunn JR, Korc M. A novel 3-dimensional culture system uncovers growth stimulatory actions by TGFβ in pancreatic cancer cells. Cancer Biol Ther 2011; 12:198-207. [PMID: 21613822 DOI: 10.4161/cbt.12.3.15979] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Transforming Growth Factor-β (TGF-β) exerts cell type-specific and context-dependent effects. Understanding the intrinsic effects of TGF-β on cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a prerequisite for rationalized clinical implementation of TGF-β targeting therapies. Since the tumor microenvironment can affect how cancer cell respond to TGF-β, we employed a novel three-dimensional (3D) culturing system to recapitulate stromal and extracellular matrix interactions. We show here that TGF-β stimulates growth of human and murine pancreatic cancer cell lines (PCCs) when embedded in a 3% collagen IV/laminin-rich gelatinous medium (Matrigel™) over a solidified layer of soft agar. Moreover, in this novel 3D model, concomitant treatment with TGF-β1 and epidermal growth factor (EGF) enhanced PCC growth to a greater extent than either growth factor alone, and conferred increased chemoresistance to cytotoxic compounds. These cooperative growth-stimulatory effects were blocked by pharmacological inhibition of TGF-β type I receptor with SB431542 or the EGF receptor with erlotinib. Co-incubation with SB431542 and erlotinib enhanced the efficacy of gemcitabine and cisplatin in PCCs and in primary cell cultures established from pancreata of genetically-engineered mouse models of PDAC. These findings suggest that concomitant inhibition of TGF-β and EGF signaling may represent an effective therapeutic strategy in PDAC, and that this 3D culturing system could be utilized to test ex vivo the therapeutic response of pancreatic tumor biopsies from PDAC patients, thereby providing a functional assay to facilitate personalized targeted therapies.
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Affiliation(s)
- Lorenzo F Sempere
- Department of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA.
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Foglieni C, Cavarelli M, Piscopiello M, Fulgenzi A, Ferrero ME. Mn bioavailability by polarized Caco-2 cells: comparison between Mn gluconate and Mn oxyprolinate. Nutr J 2011; 10:77. [PMID: 21781350 PMCID: PMC3171306 DOI: 10.1186/1475-2891-10-77] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2010] [Accepted: 07/25/2011] [Indexed: 12/20/2022] Open
Abstract
Background Micronutrient inadequate intake is responsible of pathological deficiencies and there is a need of assessing the effectiveness of metal supplementation, frequently proposed to rebalance poor diets. Manganese (Mn) is present in many enzymatic intracellular systems crucial for the regulation of cell metabolism, and is contained in commercially available metal supplements. Methods We compared the effects of two different commercial Mn forms, gluconate (MnGluc) and oxyprolinate (MnOxP). For this purpose we used the polarized Caco-2 cells cultured on transwell filters, an established in vitro model of intestinal epithelium. Since micronutrient deficiency may accelerate mitochondrial efficiency, the mitochondrial response of these cells, in the presence of MnGluc and MnOxP, by microscopy methods and by ATP luminescence assay was used. Results In the presence of both MnOxP and MnGluc a sustained mitochondrial activity was shown by mitoTraker labeling (indicative of mitochondrial respiration), but ATP intracellular content remained comparable to untreated cells only in the presence of MnOxP. In addition MnOxP transiently up-regulated the antioxidant enzyme Mn superoxide dismutase more efficiently than MnGluc. Both metal treatments preserved NADH and βNADPH diaphorase oxidative activity, avoided mitochondrial dysfunction, as assessed by the absence of a sustained phosphoERK activation, and were able to maintain cell viability. Conclusions Collectively, our data indicate that MnOxP and MnGluc, and primarily the former, produce a moderate and safe modification of Caco-2 cell metabolism, by activating positive enzymatic mechanisms, thus could contribute to long-term maintenance of cell homeostasis.
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Affiliation(s)
- Chiara Foglieni
- Department of Human Morphology Città Studi, Università degli Studi di Milano, Milan, Italy
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Pancreatic cancer organotypic cultures. J Biotechnol 2010; 148:16-23. [PMID: 20083148 DOI: 10.1016/j.jbiotec.2010.01.008] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2009] [Revised: 12/20/2009] [Accepted: 01/12/2010] [Indexed: 01/27/2023]
Abstract
Pancreatic cancer, the fourth most common cause of cancer-related death, is a devastating disease with poor prognosis. Over the last four decades, no effective new treatments have been developed for this cancer. As a result, its prognosis has remained unchanged. Appropriate cancer models, representing all aspects of pancreatic cancer, will enhance our understanding of its biology. In this review we discuss the evolution and merit of organotypic culture models. These co-culture in vitro systems of cancer and stromal cells grown within, or on top of, reconstituted extracellular matrix gels model pancreatic cancer more realistically than 2D systems. Different methodologies are discussed which enable interrogation of various hypotheses examining the tumour-stroma cross-talk. Thus this validated organotypic culture model provides a system, which can be easily manipulated and used to test (novel) treatment options targeting the cancer, the stromal compartment or both, in a physiologically relevant environment. The big challenge for future research is to expand this model further so that its mimicry of the human tumour is more robust. This will increase our understanding of the biology of this aggressive tumour; ultimately resulting in improved therapies and, therefore, a better prognosis.
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Philip PA, Mooney M, Jaffe D, Eckhardt G, Moore M, Meropol N, Emens L, O'Reilly E, Korc M, Ellis L, Benedetti J, Rothenberg M, Willett C, Tempero M, Lowy A, Abbruzzese J, Simeone D, Hingorani S, Berlin J, Tepper J. Consensus report of the national cancer institute clinical trials planning meeting on pancreas cancer treatment. J Clin Oncol 2009; 27:5660-9. [PMID: 19858397 DOI: 10.1200/jco.2009.21.9022] [Citation(s) in RCA: 186] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality, despite significant improvements in diagnostic imaging and operative mortality rates. The 5-year survival rate remains less than 5% because of microscopic or gross metastatic disease at time of diagnosis. The Clinical Trials Planning Meeting in pancreatic cancer was convened by the National Cancer Institute's Gastrointestinal Cancer Steering Committee to discuss the integration of basic and clinical knowledge in the design of clinical trials in PDAC. Major emphasis was placed on the enhancement of research to identify and validate the relevant targets and molecular pathways in PDAC, cancer stem cells, and the microenvironment. Emphasis was also placed on developing rational combinations of targeted agents and the development of predictive biomarkers to assist selection of patient subsets. The development of preclinical tumor models that are better predictive of human PDAC must be supported with wider availability to the research community. Phase III clinical trials should be implemented only if there is a meaningful clinical signal of efficacy and safety in the phase II setting. The emphasis must therefore be on performing well-designed phase II studies with uniform sets of basic entry and evaluation criteria with survival as a primary endpoint. Patients with either metastatic or locally advanced PDAC must be studied separately.
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Affiliation(s)
- Philip A Philip
- Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA.
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Froeling FEM, Mirza TA, Feakins RM, Seedhar A, Elia G, Hart IR, Kocher HM. Organotypic culture model of pancreatic cancer demonstrates that stromal cells modulate E-cadherin, beta-catenin, and Ezrin expression in tumor cells. THE AMERICAN JOURNAL OF PATHOLOGY 2009; 175:636-48. [PMID: 19608876 PMCID: PMC2716963 DOI: 10.2353/ajpath.2009.090131] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 05/15/2009] [Indexed: 12/21/2022]
Abstract
Pancreatic cancer is characterized by an intense stromal reaction. Reproducible three-dimensional in vitro systems for exploring interactions of the stroma with pancreatic cancer cells have not previously been available, prompting us to develop such a model. Cancer cells were grown on collagen/Matrigel and embedded with or without stromal cells (hTERT-immortalized human PS-1 stellate cells or MRC-5 fibroblasts) for 7 days. Proliferation and apoptosis, as well as important cell-cell adhesion and cytoskeleton-regulating proteins, were studied. PS-1 cells were confirmed as stellate based on the expression of key cytoskeletal proteins and lipid vesicles. Capan-1, and to a lesser extent PaCa-3, cells differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and an apico-basal polarity. Presence of either stromal cell type translocated Ezrin from apical (when stromal cells were absent) to basal aspects of cancer cells, where it was associated with invasive activity. Interestingly, the presence of 'normal' (not tumor-derived) stromal cells induced total tumor cell number reduction (P < 0.005) associated with a significant decrease in E-cadherin expression (P < 0.005). Conversely, beta-catenin expression was up-regulated (P < 0.01) in the presence of stromal cells with predominant cytoplasmic expression. Moreover, patient samples confirmed that these data recapitulated the clinical situation. In conclusion, pancreatic organotypic culture offers a reproducible, bio-mimetic, three-dimensional in vitro model that allows examination of the interactions between stromal elements and pancreatic cancer cells.
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Affiliation(s)
- Fieke E M Froeling
- Centre for Tumour Biology, Institute of Cancer, Barts and the London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ
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31
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Piao L, Li Y, Yang KJ, Park KA, Byun HS, Won M, Hong J, Kim JL, Kweon GR, Hur GM, Seok JH, Cho JY, Chun T, Hess D, Sack R, Maira SM, Brazil DP, Hemmings BA, Park J. Heat shock protein 70-mediated sensitization of cells to apoptosis by Carboxyl-Terminal Modulator Protein. BMC Cell Biol 2009; 10:53. [PMID: 19604401 PMCID: PMC2729731 DOI: 10.1186/1471-2121-10-53] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2009] [Accepted: 07/15/2009] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The serine/threonine protein kinase B (PKB/Akt) is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP) has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process. RESULTS CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38) were identified as novel in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70) inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function. CONCLUSION Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria.
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Affiliation(s)
- Longzhen Piao
- Department of Pharmacology, Daejeon Regional Cancer Center, Cancer Research Institute, Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Taejeon, 301-131, Korea.
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Abstract
How do animal cells assemble into tissues and organs? A diverse array of tissue structures and shapes can be formed by organizing groups of cells into different polarized arrangements and by coordinating their polarity in space and time. Conserved design principles underlying this diversity are emerging from studies of model organisms and tissues. We discuss how conserved polarity complexes, signalling networks, transcription factors, membrane-trafficking pathways, mechanisms for forming lumens in tubes and other hollow structures, and transitions between different types of polarity, such as between epithelial and mesenchymal cells, are used in similar and iterative manners to build all tissues.
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Affiliation(s)
- David M. Bryant
- Department of Anatomy, University of California San Francisco, California 94143-2140, USA
| | - Keith E. Mostov
- Department of Anatomy, University of California San Francisco, California 94143-2140, USA
- Department of Biochemistry and Biophysics, University of California San Francisco, California 94143-2140, USA
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Pancreatic duct secretion: experimental methods, ion transport mechanisms and regulation. J Physiol Biochem 2008; 64:243-57. [PMID: 19244938 DOI: 10.1007/bf03178846] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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Franke TF. Akt-interacting proteins: attractive opposites. focus on "Carboxy-terminal modulator protein induces Akt phosphorylation and activation, thereby enhancing antiapoptotic, glycogen synthetic, and glucose uptake pathways". Am J Physiol Cell Physiol 2007; 293:C1768-70. [PMID: 17913839 DOI: 10.1152/ajpcell.00451.2007] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Thomas F Franke
- Department of Psychiatry, Milhauser Laboratories, NYU School of Medicine, New York, NY 10016, usa.
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