1
|
Ghosh R, Bhowmik A, Biswas S, Samanta P, Sarkar R, Pakhira S, Mondal M, Hajra S, Saha P. Natural flavonoid Orientin restricts 5-Fluorouracil induced cancer stem cells mediated angiogenesis by regulating HIF1α and VEGFA in colorectal cancer. Mol Med 2025; 31:85. [PMID: 40045186 PMCID: PMC11881437 DOI: 10.1186/s10020-024-01032-1] [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: 09/20/2024] [Accepted: 12/06/2024] [Indexed: 03/09/2025] Open
Abstract
BACKGROUND Cancer stem cells are a small subpopulation of cells which are responsible for tumor metastasis, angiogenesis, drug resistance etc. 5-Fluorouracil (5FU), a common therapeutic drug used in colorectal cancer treatment is reported to enrich CSCs, tumor recurrence and induces severe organ toxicities resulting in poor clinical outcome in patients. Therefore, we introduced a natural flavonoid Orientin in combination with 5FU to mitigate the CSC mediated angiogenesis and induced toxicities. METHODS Tumorosphere generation, flow cytometry, immunofluorescence assay, and western blotting were performed by using 5FU and Orientin individually and both treated colorectal cells and CSCs. In silico study was carried out to check the interaction between HIF1α and Orientin. In ovo chorioallantoic membrane (CAM) assay and tube formation assay using HUVECs were performed to monitor CSC mediated angiogenesis. In vivo CT26 syngeneic mice model was used to validate in silico and ex vivo results. RESULTS We found that 5FU treatment significantly increased the CD44+/CD133+ CSC population. In contrast, this CSC population in CSC enriched spheres (CES) derived from HCT116 cells were decreased by combination of Orientin and 5FU. Decrease of CSC's stemness properties was also noted, as evidenced by the downregulation of NANOG, SOX2 and OCT4. This new therapeutic strategy also inhibited CSC mediated angiogenesis by downregulating 5FU induced ROS, NO and LPO in those tumorospheres. Combination of Orientin and 5FU significantly reduced CSC mediated angiogenesis in HUVEC and CAM. Additionally, in silico study predicted that Orientin can bind to the PAS domain of HIF1α, a crucial factor for promoting angiogenesis. Expression of HIF1α and VEGFA were also decreased when the CESs were exposed to the combinatorial treatment. Additionally, we found that treatment with 5FU alone resulted reduction in tumor volume but it enriched CSCs and produced nephrotoxicity and hepatotoxicity in vivo. Combined treatment also considerably reduced the CD44+/CD133+ CSC population and hindered angiogenesis in a therapeutic in vivo model in BALB/c mice. CONCLUSIONS This novel treatment strategy of "Orientin with 5FU" is likely to improve the efficiency of conventional chemotherapy and may suppress disease recurrence in colorectal cancer by limiting CSC mediated angiogenesis.
Collapse
Affiliation(s)
- Rituparna Ghosh
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Arijit Bhowmik
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India.
| | - Souradeep Biswas
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Priya Samanta
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Rupali Sarkar
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Shampa Pakhira
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Mrinmoyee Mondal
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Subhadip Hajra
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India
| | - Prosenjit Saha
- Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute (CNCI), 37, Shyama Prasad Mukherjee Rd, Bakul Bagan, Bhowanipore, Kolkata, West Bengal, 700026, India.
| |
Collapse
|
2
|
Lu J, Zhang Y, Yan C, Liu J, Qi D, Zhou Y, Wang Q, Yang J, Jiang J, Wu B, Yang M, Zhang W, Zhang X, Shi X, Zhang Y, Liu K, Liang Y, Wang C, Yang H, Gao Y, Sun Y, Ke R, Huang JH, Wu M, Wang H, Li C, Zhou S, Guo B, Wu E, Zhang G. TClC effectively suppresses the growth and metastasis of NSCLC via polypharmacology. Bioact Mater 2025; 45:567-583. [PMID: 39759535 PMCID: PMC11700266 DOI: 10.1016/j.bioactmat.2024.11.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2024] [Revised: 11/10/2024] [Accepted: 11/15/2024] [Indexed: 01/07/2025] Open
Abstract
Despite significant advances in targeted therapies and immunotherapies, non-small cell lung cancer (NSCLC) continues to present a global health challenge, with a modest five-year survival rate of 28 %, largely due to the emergence of treatment-resistant and metastatic tumors. In response, we synthesized a novel bioactive compound, ethyl 6-chlorocoumarin-3-carboxylyl L-theanine (TClC), which significantly inhibited NSCLC growth, epithelial mesenchymal transition (EMT), migration, and invasion in vitro and tumor growth and metastasis in vivo without inducing toxicity. TClC disrupts autocrine loops that promote tumor progression, particularly in stem-like CD133-positive NSCLC (CD133+ LC) cells, which are pivotal in tumor metastasis. Through targeted molecular assays, we identified direct binding targets of TClC, including Akt, NF-κB, β-catenin, EZH2, and PD-L1. This interaction not only suppresses the expression of oncogenic factors and cancer stem cell markers but also downregulates the expression of a multidrug resistance transporter, underscoring the compound's polypharmacological potential. These results position TClC as a promising candidate for NSCLC treatment, signaling a new era in the development of cancer therapies that directly target multiple critical cancer pathways.
Collapse
Affiliation(s)
- Jing Lu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
| | - Ying Zhang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
- Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, 58105, USA
| | - Chunyan Yan
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Department of Pharmacy, Yantai Yuhuangding Hospital, Yantai, Shandong, 264000, China
| | - Jingwen Liu
- Department of Pharmacological & Pharmaceutical Sciences, University of Houston, Houston, TX, 77204, USA
| | - Dan Qi
- Neuroscience Institute and Department of Neurosurgery, Baylor Scott & White Health, Temple, TX, 76502, USA
- Department of Neurosurgery, Baylor College of Medicine, Temple, TX, 76502, USA
| | - Yue Zhou
- Department of Statistics, North Dakota State University, Fargo, ND, 58102, USA
| | - Qinwen Wang
- The Center of Non-Traumatic Treatment and Diagnosis of Tumor, Binzhou Medical College affiliated The PLA 107 Hospital, Yantai, Shandong, 264002, China
- Outpatient Department, No. 26 Rest Center for Retired Cadres, Haidian district, Beijing, 100036, China
| | - Juechen Yang
- Department of Biomedical Informatics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
- Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Jing Jiang
- RemeGen, Ltd, Yantai, 264000, Shandong, China
- Department of Pharmacology, Binzhou Medical University, Yantai, 264003, Shandong, China
| | - Benhao Wu
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
| | - Meiling Yang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
| | - Weiwei Zhang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
| | - Xin Zhang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
| | - Xiaoyu Shi
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
| | - Yan Zhang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
- Shandong Yingdong Yinghao Biotechnology Inc., Yantai, Shandong, 264670, China
| | - Kun Liu
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
| | - Yongcai Liang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
| | - Chaoyang Wang
- Department of Thoracic Surgery, Yantai Yuhuangding Hospital, Yantai, Shandong, 264000, China
| | - Hanyu Yang
- Shiyao Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., LTD., State Key Laboratory of New Pharmaceutical Preparations and Excipients, Shijiazhuang, 050035, China
| | - Yuqing Gao
- Shiyao Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., LTD., State Key Laboratory of New Pharmaceutical Preparations and Excipients, Shijiazhuang, 050035, China
| | - Yuping Sun
- Phase I Clinical Trial Center, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, 250013, China
| | - Ronghu Ke
- Neuroscience Institute and Department of Neurosurgery, Baylor Scott & White Health, Temple, TX, 76502, USA
| | - Jason H. Huang
- Neuroscience Institute and Department of Neurosurgery, Baylor Scott & White Health, Temple, TX, 76502, USA
- College of Medicine, Texas A&M University, College Station, TX, 77843, USA
- Department of Neurosurgery, Baylor College of Medicine, Temple, TX, 76502, USA
| | - Min Wu
- Drug Discovery Center, Wenzhou Institute University of Chinese Academy of Sciences, Wenzhou, 325001, China
- Department of Critical Care Medicine, Frontiers Science Center for Disease-related Molecular Network, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 646000, China
| | - Hongbo Wang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
| | - Chunlei Li
- Shiyao Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., LTD., State Key Laboratory of New Pharmaceutical Preparations and Excipients, Shijiazhuang, 050035, China
| | - Shuang Zhou
- Department of Pharmacological & Pharmaceutical Sciences, University of Houston, Houston, TX, 77204, USA
| | - Bin Guo
- Department of Pharmacological & Pharmaceutical Sciences, University of Houston, Houston, TX, 77204, USA
| | - Erxi Wu
- Neuroscience Institute and Department of Neurosurgery, Baylor Scott & White Health, Temple, TX, 76502, USA
- College of Medicine, Texas A&M University, College Station, TX, 77843, USA
- College of Irma Lerma Rangel College of Pharmacy, Texas A&M University, College Station, TX, 77843, USA
- Department of Neurosurgery, Baylor College of Medicine, Temple, TX, 76502, USA
- LIVESTRONG Cancer Institutes and Department of Oncology, Dell Medical School, the University of Texas at Austin, Austin, TX, 78712, USA
| | - Guoying Zhang
- School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, Shandong, 264005, China
| |
Collapse
|
3
|
Khiabani NA, Doustvandi MA, Story D, Nobari SA, Hajizadeh M, Petersen R, Dunbar G, Rossignol J. Glioblastoma therapy: State of the field and future prospects. Life Sci 2024; 359:123227. [PMID: 39537100 DOI: 10.1016/j.lfs.2024.123227] [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: 06/25/2024] [Revised: 09/03/2024] [Accepted: 11/05/2024] [Indexed: 11/16/2024]
Abstract
Glioblastoma (GB) is a cancerous brain tumor that originates from glial cells and leads to thousands of deaths each year and a five-year survival of only 6.8 %. Treatments for GB include surgery, chemotherapy, radiation, and immunotherapy. GB is an incurable fatal disease, necessitating the development of innovative strategies to find a developing effective therapy. Genetic therapies may be crucial in treating GB by identifying the mutations and amplifications of multiple genes, which drive its proliferation and spread. Use of small interfering RNAs (siRNAs) provides a novel technology used to suppress the genes associated with disease, which forms a basis for targeted therapy in GB and its stem cell population, which are recognized for their ability to develop resistance to chemotherapy and tumorigenic capabilities. This review examines the use of siRNAs in GB, emphasizing their effectiveness in suppressing key oncogenes and signaling pathways associated with tumor development, invasion, stemness, and resistance to standard treatments. siRNA-based gene silencing is a promising approach for developing targeted therapeutics against GB and associated stem cell populations, potentially enhancing patient outcomes and survival rates in this devastating disease.
Collapse
Affiliation(s)
- Nadia Allahyarzadeh Khiabani
- Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA; Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA; College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
| | | | - Darren Story
- Department of Psychology, Saginaw Valley State University, University Center, MI 48710, USA
| | | | | | - Robert Petersen
- College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
| | - Gary Dunbar
- Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA; Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA; Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
| | - Julien Rossignol
- Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA; Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA; College of Medicine, Central Michigan University, Mount Pleasant, MI, USA.
| |
Collapse
|
4
|
Volk-Draper L, Athaiya S, Espinosa Gonzalez M, Bhattarai N, Wilber A, Ran S. Tumor microenvironment restricts IL-10 induced multipotent progenitors to myeloid-lymphatic phenotype. PLoS One 2024; 19:e0298465. [PMID: 38640116 PMCID: PMC11029653 DOI: 10.1371/journal.pone.0298465] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Accepted: 01/24/2024] [Indexed: 04/21/2024] Open
Abstract
Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.
Collapse
Affiliation(s)
- Lisa Volk-Draper
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States of America
| | - Shaswati Athaiya
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States of America
| | - Maria Espinosa Gonzalez
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States of America
| | - Nihit Bhattarai
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States of America
| | - Andrew Wilber
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States of America
- Simmons Cancer Institute, Southern Illinois University School of Medicine, Springfield, IL, United States of America
| | - Sophia Ran
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, United States of America
- Simmons Cancer Institute, Southern Illinois University School of Medicine, Springfield, IL, United States of America
| |
Collapse
|
5
|
Lemmens TP, Bröker V, Rijpkema M, Hughes CCW, Schurgers LJ, Cosemans JMEM. Fundamental considerations for designing endothelialized in vitro models of thrombosis. Thromb Res 2024; 236:179-190. [PMID: 38460307 DOI: 10.1016/j.thromres.2024.03.004] [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: 11/18/2023] [Revised: 02/19/2024] [Accepted: 03/04/2024] [Indexed: 03/11/2024]
Abstract
Endothelialized in vitro models for cardiovascular disease have contributed greatly to our current understanding of the complex molecular mechanisms underlying thrombosis. To further elucidate these mechanisms, it is important to consider which fundamental aspects to incorporate into an in vitro model. In this review, we will focus on the design of in vitro endothelialized models of thrombosis. Expanding our understanding of the relation and interplay between the different pathways involved will rely in part on complex models that incorporate endothelial cells, blood, the extracellular matrix, and flow. Importantly, the use of tissue-specific endothelial cells will help in understanding the heterogeneity in thrombotic responses between different vascular beds. The dynamic and complex responses of endothelial cells to different shear rates underlines the importance of incorporating appropriate shear in in vitro models. Alterations in vascular extracellular matrix composition, availability of bioactive molecules, and gradients in concentration and composition of these molecules can all regulate the function of both endothelial cells and perivascular cells. Factors modulating these elements in in vitro models should therefore be considered carefully depending on the research question at hand. As the complexity of in vitro models increases, so can the variability. A bottom-up approach to designing such models will remain an important tool for researchers studying thrombosis. As new techniques are continuously being developed and new pathways are brought to light, research question-dependent considerations will have to be made regarding what aspects of thrombosis to include in in vitro models.
Collapse
Affiliation(s)
- Titus P Lemmens
- Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
| | - Vanessa Bröker
- Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
| | - Minke Rijpkema
- Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
| | - Christopher C W Hughes
- Department of Molecular Biology and Biochemistry, and Department of Biomedical Engineering, University of California, Irvine, USA
| | - Leon J Schurgers
- Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
| | - Judith M E M Cosemans
- Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands.
| |
Collapse
|
6
|
Pleskač P, Fargeas CA, Veselska R, Corbeil D, Skoda J. Emerging roles of prominin-1 (CD133) in the dynamics of plasma membrane architecture and cell signaling pathways in health and disease. Cell Mol Biol Lett 2024; 29:41. [PMID: 38532366 DOI: 10.1186/s11658-024-00554-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2023] [Accepted: 02/22/2024] [Indexed: 03/28/2024] Open
Abstract
Prominin-1 (CD133) is a cholesterol-binding membrane glycoprotein selectively associated with highly curved and prominent membrane structures. It is widely recognized as an antigenic marker of stem cells and cancer stem cells and is frequently used to isolate them from biological and clinical samples. Recent progress in understanding various aspects of CD133 biology in different cell types has revealed the involvement of CD133 in the architecture and dynamics of plasma membrane protrusions, such as microvilli and cilia, including the release of extracellular vesicles, as well as in various signaling pathways, which may be regulated in part by posttranslational modifications of CD133 and its interactions with a variety of proteins and lipids. Hence, CD133 appears to be a master regulator of cell signaling as its engagement in PI3K/Akt, Src-FAK, Wnt/β-catenin, TGF-β/Smad and MAPK/ERK pathways may explain its broad action in many cellular processes, including cell proliferation, differentiation, and migration or intercellular communication. Here, we summarize early studies on CD133, as they are essential to grasp its novel features, and describe recent evidence demonstrating that this unique molecule is involved in membrane dynamics and molecular signaling that affects various facets of tissue homeostasis and cancer development. We hope this review will provide an informative resource for future efforts to elucidate the details of CD133's molecular function in health and disease.
Collapse
Affiliation(s)
- Petr Pleskač
- Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
| | - Christine A Fargeas
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Tatzberg 47/49, 01307, Dresden, Germany
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Dresden, Germany
| | - Renata Veselska
- Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic
| | - Denis Corbeil
- Biotechnology Center (BIOTEC) and Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Tatzberg 47/49, 01307, Dresden, Germany.
- Tissue Engineering Laboratories, Medizinische Fakultät der Technischen Universität Dresden, Dresden, Germany.
| | - Jan Skoda
- Laboratory of Tumor Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
- International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic.
| |
Collapse
|
7
|
Laowpanitchakorn P, Zeng J, Piantino M, Uchida K, Katsuyama M, Matsusaki M. Biofabrication of engineered blood vessels for biomedical applications. SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS 2024; 25:2330339. [PMID: 38633881 PMCID: PMC11022926 DOI: 10.1080/14686996.2024.2330339] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Accepted: 03/10/2024] [Indexed: 04/19/2024]
Abstract
To successfully engineer large-sized tissues, establishing vascular structures is essential for providing oxygen, nutrients, growth factors and cells to prevent necrosis at the core of the tissue. The diameter scale of the biofabricated vasculatures should range from 100 to 1,000 µm to support the mm-size tissue while being controllably aligned and spaced within the diffusion limit of oxygen. In this review, insights regarding biofabrication considerations and techniques for engineered blood vessels will be presented. Initially, polymers of natural and synthetic origins can be selected, modified, and combined with each other to support maturation of vascular tissue while also being biocompatible. After they are shaped into scaffold structures by different fabrication techniques, surface properties such as physical topography, stiffness, and surface chemistry play a major role in the endothelialization process after transplantation. Furthermore, biological cues such as growth factors (GFs) and endothelial cells (ECs) can be incorporated into the fabricated structures. As variously reported, fabrication techniques, especially 3D printing by extrusion and 3D printing by photopolymerization, allow the construction of vessels at a high resolution with diameters in the desired range. Strategies to fabricate of stable tubular structures with defined channels will also be discussed. This paper provides an overview of the many advances in blood vessel engineering and combinations of different fabrication techniques up to the present time.
Collapse
Affiliation(s)
| | - Jinfeng Zeng
- Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
| | - Marie Piantino
- Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
- The Consortium for Future Innovation by Cultured Meat, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
| | - Kentaro Uchida
- Materials Solution Department, Product Analysis Center, Panasonic Holdings Corporation, Kadoma, Osaka, Japan
| | - Misa Katsuyama
- Materials Solution Department, Product Analysis Center, Panasonic Holdings Corporation, Kadoma, Osaka, Japan
| | - Michiya Matsusaki
- Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
- The Consortium for Future Innovation by Cultured Meat, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
| |
Collapse
|
8
|
Moreno-Londoño AP, Robles-Flores M. Functional Roles of CD133: More than Stemness Associated Factor Regulated by the Microenvironment. Stem Cell Rev Rep 2024; 20:25-51. [PMID: 37922108 PMCID: PMC10799829 DOI: 10.1007/s12015-023-10647-6] [Citation(s) in RCA: 16] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/19/2023] [Indexed: 11/05/2023]
Abstract
CD133 protein has been one of the most used surface markers to select and identify cancer cells with stem-like features. However, its expression is not restricted to tumoral cells; it is also expressed in differentiated cells and stem/progenitor cells in various normal tissues. CD133 participates in several cellular processes, in part orchestrating signal transduction of essential pathways that frequently are dysregulated in cancer, such as PI3K/Akt signaling and the Wnt/β-catenin pathway. CD133 expression correlates with enhanced cell self-renewal, migration, invasion, and survival under stress conditions in cancer. Aside from the intrinsic cell mechanisms that regulate CD133 expression in each cellular type, extrinsic factors from the surrounding niche can also impact CD33 levels. The enhanced CD133 expression in cells can confer adaptive advantages by amplifying the activation of a specific signaling pathway in a context-dependent manner. In this review, we do not only describe the CD133 physiological functions known so far, but importantly, we analyze how the microenvironment changes impact the regulation of CD133 functions emphasizing its value as a marker of cell adaptability beyond a cancer-stem cell marker.
Collapse
Affiliation(s)
- Angela Patricia Moreno-Londoño
- Department of Biochemistry, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), 04510, Mexico City, Mexico
| | - Martha Robles-Flores
- Department of Biochemistry, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), 04510, Mexico City, Mexico.
| |
Collapse
|
9
|
Romeo HE, Barreiro Arcos ML. Clinical relevance of stem cells in lung cancer. World J Stem Cells 2023; 15:576-588. [PMID: 37424954 PMCID: PMC10324501 DOI: 10.4252/wjsc.v15.i6.576] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 03/18/2023] [Accepted: 05/08/2023] [Indexed: 06/26/2023] Open
Abstract
Lung cancer is the major cause of cancer-related deaths worldwide, it has one of the lowest 5-year survival rate, mainly because it is diagnosed in the late stage of the disease. Lung cancer is classified into two groups, small cell lung cancer (SCLC) and non-SCLC (NSCLC). In turn, NSCLC is categorized into three distinct cell subtypes: Adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. NSCLC is the most common lung cancer, accounting for 85% of all lung cancers. Treatment for lung cancer is linked to the cell type and stage of the disease, involving chemotherapy, radiation therapy, and surgery. Despite improvements in therapeutic treatments, lung cancer patients show high rates of recurrence, metastasis, and resistance to chemotherapy. Lung stem cells (SCs) are undifferentiated cells capable of self-renewal and proliferation, are resistant to chemotherapy and radiotherapy and, due to their properties, could be involved in the development and progression of lung cancer. The presence of SCs in the lung tissue could be the reason why lung cancer is difficult to treat. The identification of lung cancer stem cells biomarkers is of interest for precision medicine using new therapeutic agents directed against these cell populations. In this review, we present the current knowledge on lung SCs and discuss their functional role in the initiation and progression of lung cancer, as well as their role in tumor resistance to chemotherapy.
Collapse
Affiliation(s)
- Horacio Eduardo Romeo
- School of Engineering and Agrarian Sciences, Pontifical Catholic University of Argentina, Institute of Biomedical Research (BIOMED-UCA-CONICET), CABA C1107AAZ, Buenos Aires, Argentina
| | - María Laura Barreiro Arcos
- School of Engineering and Agrarian Sciences, Pontifical Catholic University of Argentina, Institute of Biomedical Research (BIOMED-UCA-CONICET), CABA C1107AAZ, Buenos Aires, Argentina
| |
Collapse
|
10
|
Siurana A, Cánovas A, Casellas J, Calsamiglia S. Transcriptome Profile in Dairy Cows Resistant or Sensitive to Milk Fat Depression. Animals (Basel) 2023; 13:ani13071199. [PMID: 37048455 PMCID: PMC10093643 DOI: 10.3390/ani13071199] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2023] [Revised: 03/26/2023] [Accepted: 03/27/2023] [Indexed: 04/01/2023] Open
Abstract
Feeding linseed to dairy cows results in milk fat depression (MFD), but there is a wide range of sensitivity among cows. The objectives of this study were to identify target genes containing SNP that may play a key role in the regulation of milk fat synthesis in cows resistant or sensitive to MFD. Four cows were selected from a dairy farm after a switch from a control diet to a linseed-rich diet; two were resistant to MFD with a high milk fat content in the control (4.06%) and linseed-rich (3.90%) diets; and two were sensitive to MFD with the milk fat content decreasing after the change from the control (3.87%) to linseed-rich (2.52%) diets. Transcriptome and SNP discovery analyses were performed using RNA-sequencing technology. There was a large number of differentially expressed genes in the control (n = 1316) and linseed-rich (n = 1888) diets. Of these, 15 genes were detected as key gene regulators and harboring SNP in the linseed-rich diet. The selected genes MTOR, PDPK1, EREG, NOTCH1, ZNF217 and TGFB3 may form a network with a principal axis PI3K/Akt/MTOR/SREBP1 involved in milk fat synthesis and in the response to diets that induced MFD. These 15 genes are novel candidate genes to be involved in the resistance or sensitivity of dairy cows to milk fat depression.
Collapse
|
11
|
Chen MY, Hsu CH, Setiawan SA, Tzeng DTW, Ma HP, Ong JR, Chu YC, Hsieh MS, Wu ATH, Tzeng YM, Yeh CT. Ovatodiolide and antrocin synergistically inhibit the stemness and metastatic potential of hepatocellular carcinoma via impairing ribosome biogenesis and modulating ERK/Akt-mTOR signaling axis. PHYTOMEDICINE : INTERNATIONAL JOURNAL OF PHYTOTHERAPY AND PHYTOPHARMACOLOGY 2023; 108:154478. [PMID: 36265255 DOI: 10.1016/j.phymed.2022.154478] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/01/2022] [Revised: 08/28/2022] [Accepted: 09/25/2022] [Indexed: 06/16/2023]
Abstract
Activation of mitogen-activated protein kinase (MAPK) and PI3K signaling confers resistance against sorafenib, a mainstay treatment for advanced hepatocellular carcinoma (HCC). Antrocin and ovatodiolide constitute as the most potent secondary metabolites isolated from Antrodia camphorata and Anisomeles indica, respectively. Both natural compounds have recently gained a lot of attention due to their putative inhibition of MAPK and PI3K signaling in various solid cancers. However, whether their combination is effective in HCC remains unknown. Here, we investigated their effect, alone or in various combinations, on MAPK and PI3K signaling pathways in HCC cells. An array of in vitro study were used to investigate anticancer and stemness effects to treat HCC, such as cytotoxicity, drug combination index, migration, invasion, colony formation, and tumor sphere formation. Drug effect in vivo was evaluated using mouse xenograft models. In this study, antrocin and ovatodiolide synergistically inhibited the SNU387, Hep3B, Mahlavu, and Huh7 cell lines. Sequential combination treatment of Huh7 and Mahlavu with ovatodiolide followed by antrocin resulted stronger cytotoxic effect than did treatment with antrocin followed by ovatodiolide, their simultaneous administration, antrocin alone, or ovatodiolide alone. In the Huh7 and Mahlavu cell lines, ovatodiolide→antrocin significantly suppressed colony formation and proliferation as well as markedly downregulated ERK1/2, Akt, and mTOR expression. Inhibition of ERK1/2 and Akt/mTOR signaling by ovatodiolide→antrocin suppressed ribosomal biogenesis, autophagy, and cancer stem cell-like phenotypes and promoted apoptosis in Huh7 and Mahlavu cells. The sorafenib-resistant clone of Huh7 was effectively inhibited by synergistic combination of both compound in vitro. Eventually, the ovatodiolide→antrocin combination synergistically suppressed the growth of HCC xenografts. Taken together, our findings suggested that ovatodiolide→antrocin combination may represent potential therapeutic approach for patients with advanced HCC.
Collapse
Affiliation(s)
- Ming-Yao Chen
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei City 110, Taiwan; Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University-Shuang Ho Hospital, New Taipei City 235, Taiwan
| | - Chia-Hung Hsu
- Department of Emergency Medicine, Taipei Medical University-Shuang Ho Hospital, New Taipei City 235, Taiwan; Graduate Institute of Injury Prevention and Control, College of Public Health, Taipei Medical University, Taipei City 110, Taiwan; Department of Emergency Medicine, School of Medicine, Taipei Medical University, Taipei City 110, Taiwan
| | - Syahru Agung Setiawan
- International Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei City 110, Taiwan; Department of Medical Research & Education, Taipei Medical University - Shuang-Ho Hospital, New Taipei City 235, Taiwan
| | - David T W Tzeng
- School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China; Lifebit, Mindspace Shoreditch, London, England, EC2A 2AP, UK
| | - Hon-Ping Ma
- Department of Emergency Medicine, Taipei Medical University-Shuang Ho Hospital, New Taipei City 235, Taiwan; Graduate Institute of Injury Prevention and Control, College of Public Health, Taipei Medical University, Taipei City 110, Taiwan; Department of Emergency Medicine, School of Medicine, Taipei Medical University, Taipei City 110, Taiwan
| | - Jiann Ruey Ong
- Department of Emergency Medicine, Taipei Medical University-Shuang Ho Hospital, New Taipei City 235, Taiwan; Graduate Institute of Injury Prevention and Control, College of Public Health, Taipei Medical University, Taipei City 110, Taiwan; Department of Emergency Medicine, School of Medicine, Taipei Medical University, Taipei City 110, Taiwan
| | - Yi Cheng Chu
- Department of Medicine, St. George's University School of Medicine, St. George, Grenada
| | - Ming-Shou Hsieh
- Department of Medical Research & Education, Taipei Medical University - Shuang-Ho Hospital, New Taipei City 235, Taiwan
| | - Alexander T H Wu
- Department of Medical Research & Education, Taipei Medical University - Shuang-Ho Hospital, New Taipei City 235, Taiwan
| | - Yew-Min Tzeng
- Department of Applied Science, National Taitung University, Taitung 95092, Taiwan.
| | - Chi-Tai Yeh
- Department of Medical Research & Education, Taipei Medical University - Shuang-Ho Hospital, New Taipei City 235, Taiwan; Continuing Education Program of Food Biotechnology Applications, College of Science and Engineering, National Taitung University, Taitung 95092, Taiwan.
| |
Collapse
|
12
|
Cao W, Zhang H, Zhou N, Zhou R, Zhang X, Yin J, Deng J, Ao X, Shi C. Functional recovery of myocardial infarction by specific EBP-PR1P peptides bridging injectable cardiac extracellular matrix and vascular endothelial growth factor. J Biomed Mater Res A 2022; 111:995-1005. [PMID: 36579729 DOI: 10.1002/jbm.a.37483] [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: 09/11/2022] [Revised: 11/26/2022] [Accepted: 12/08/2022] [Indexed: 12/30/2022]
Abstract
Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor and plays an important role in therapy of myocardial infarction (MI). Currently, how to retain regional concentration and decrease rapid diffusion is critical for its clinical application of VEGF. In recent years, the application of targeting peptides has been developed rapidly and provides new strategies for the sustained release of VEGF. In present study, a bi-functional EBP-PR1P peptide was designed and bridged VEGF to injectable cardiac extracellular matrix (c-ECM). Through EBP-PR1P peptides, VEGF could specifically bind with c-ECM to realize the sustained release, without impacting the bioactivity of VEGF. Then VEGF/EBP-PR1P/c-ECM scaffolds were constructed and administrated into rats with MI. The results showed VEGF/EBP-PR1P/c-ECM could promote angiogenesis, protect cardiomyocytes survival against apoptosis, and improve the recovery of cardiac function. In addition, the mechanism of EBP-PR1P/VEGF was also investigated which canonical downstream of VEGF-Akt signaling pathway was activated. These results showed specific VEGF/EBP-PR1P/c-ECM scaffolds served as promising delivery system for VEGF that facilitated the functional recovery of MI.
Collapse
Affiliation(s)
- Wenxuan Cao
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Hong Zhang
- Department of Cardiac Surgery, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Ning Zhou
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Runxue Zhou
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Xiaojing Zhang
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Jia Yin
- Department of Neurology, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Jin Deng
- Department of Neurology, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Xiang Ao
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Chunying Shi
- School of Basic Medicine, Qingdao University, Qingdao, China
| |
Collapse
|
13
|
Hajimoradi Javarsiani M, Sajedianfard J, Haghjooy Javanmard S. The effects of metformin on the hippo pathway in the proliferation of melanoma cancer cells: a preclinical study. Arch Physiol Biochem 2022; 128:1150-1155. [PMID: 32407182 DOI: 10.1080/13813455.2020.1760304] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
YAP and TAZ, two closely related transcriptional regulators, have crucial roles in tissue repair upon injury, organ size control, and cancer treatment. Some drugs, such as metformin, that alter cell metabolism can play a role in the regulation of the Hippo pathway. The cells were treated with various concentrations of metformin, dacarbazine (IC50), and both of them. The evaluation of the biomarker and proteins was performed by FACS and immunoblotting, respectively. Cell viability was reduced by 50% after 24 h. Data showed that metformin treatment down-regulated YAP and TAZ (p = .002) expressions and enhanced YAP phosphorylation (p < .001). Metformin, alone and in combination, inhibited the growth and viability of melanoma cells in vitro. The increase in the phosphorylation of YAP renders it a potential target in the development of anticancer drugs. This study showed the effects of metformin on the inhibition of oncogenic YAP and TAZ in the proliferation of melanoma cells.
Collapse
Affiliation(s)
| | - Javad Sajedianfard
- Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
| | - Shagayegh Haghjooy Javanmard
- Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
| |
Collapse
|
14
|
Zheng Y, Wang L, Yin L, Yao Z, Tong R, Xue J, Lu Y. Lung Cancer Stem Cell Markers as Therapeutic Targets: An Update on Signaling Pathways and Therapies. Front Oncol 2022; 12:873994. [PMID: 35719973 PMCID: PMC9204354 DOI: 10.3389/fonc.2022.873994] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Accepted: 04/25/2022] [Indexed: 02/05/2023] Open
Abstract
Cancer stem cells, a relatively small group of self-renewing cancer cells, were first isolated from acute myeloid leukemia. These cells can play a crucial role in tumor metastasis, relapse, and therapy resistance. The cancer stem cell theory may be applied to lung cancer and explain the inefficiency of traditional treatments and eventual recurrence. However, because of the unclear accuracy and illusive biological function of cancer stem cells, some researchers remain cautious about this theory. Despite the ongoing controversy, cancer stem cells are still being investigated, and their biomarkers are being discovered for application in cancer diagnosis, targeted therapy, and prognosis prediction. Potential lung cancer stem cell markers mainly include surface biomarkers such as CD44, CD133, epithelial cell adhesion molecule, and ATP-binding cassette subfamily G member 2, along with intracellular biomarkers such as aldehyde dehydrogenase, sex-determining region Y-box 2, NANOG, and octamer-binding transcription factor 4. These markers have different structures and functions but are closely associated with the stem potential and uncontrollable proliferation of tumor cells. The aberrant activation of major signaling pathways, such as Notch, Hedgehog, and Wnt, may be associated with the expression and regulation of certain lung cancer stem cell markers, thus leading to lung cancer stem cell maintenance, chemotherapy resistance, and cancer promotion. Treatments targeting lung cancer stem cell markers, including antibody drugs, nanoparticle drugs, chimeric antigen receptor T-cell therapy, and other natural or synthetic specific inhibitors, may provide new hope for patients who are resistant to conventional lung cancer therapies. This review provides comprehensive and updated data on lung cancer stem cell markers with regard to their structures, functions, signaling pathways, and promising therapeutic target approaches, aiming to elucidate potential new therapies for lung cancer.
Collapse
Affiliation(s)
- Yue Zheng
- Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Laduona Wang
- Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Limei Yin
- Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Zhuoran Yao
- Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China
| | - Ruizhan Tong
- Laboratory of Clinical Cell Therapy, West China Hospital, Sichuan University, Chengdu, China
| | - Jianxin Xue
- Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.,Laboratory of Clinical Cell Therapy, West China Hospital, Sichuan University, Chengdu, China
| | - You Lu
- Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.,Laboratory of Clinical Cell Therapy, West China Hospital, Sichuan University, Chengdu, China
| |
Collapse
|
15
|
Yuan Z, Sheng D, Jiang L, Shafiq M, Khan AUR, Hashim R, Chen Y, Li B, Xie X, Chen J, Morsi Y, Mo X, Chen S. Vascular Endothelial Growth Factor-Capturing Aligned Electrospun Polycaprolactone/Gelatin Nanofibers Promote Patellar Ligament Regeneration. Acta Biomater 2022; 140:233-246. [PMID: 34852300 DOI: 10.1016/j.actbio.2021.11.040] [Citation(s) in RCA: 45] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2021] [Revised: 11/21/2021] [Accepted: 11/24/2021] [Indexed: 01/11/2023]
Abstract
Ligament injuries are common in sports and other rigorous activities. It is a great challenge to achieve ligament regeneration after an injury due the avascular structure and low self-renewal capability. Herein, we developed vascular endothelial growth factor (VEGF)-binding aligned electrospun poly(caprolactone)/gelatin (PCL/Gel) scaffolds by incorporating prominin-1-binding peptide (BP) sequence and exploited them for patellar ligament regeneration. The adsorption of BP onto scaffolds was discerned by various techniques, such as Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and confocal laser scanning microscope. The accumulation of VEGF onto scaffolds correlated with the concentration of the peptide in vitro. BP-anchored PCL/Gel scaffolds (BP@PCL/Gel) promoted the tubular formation of human umbilical vein endothelial cells (HUVECs) and wound healing in vitro. Besides, BP containing scaffolds exhibited higher content of CD31+ cells than that of the control scaffolds at 1 week after implantation in vivo. Moreover, BP containing scaffolds improved biomechanical properties and facilitated the regeneration of matured collagen in patellar ligament 4 weeks after implantation in mice. Overall, this strategy of peptide-mediated orchestration of VEGF provides an enticing platform for the ligament regeneration, which may also have broad implications for tissue repair applications. STATEMENT OF SIGNIFICANCE: Ligament injuries are central to sports and other rigorous activities. Given to the avascular nature and poor self-healing capability of injured ligament tissues, it is a burgeoning challenge to fabricate tissue-engineered scaffolds for ligament reconstruction. Vascular endothelial growth factor (VEGF) is pivotal to the neo-vessel formation. However, the high molecular weight of VEGF as well as its short half-life in vitro and in vivo limits its therapeutic potential. To circumvent these limitations, herein, we functionalized aligned electrospun polycaprolactone/gelatin (PCL/Gel)-based scaffolds with VEGF-binding peptide (BP) and assessed their biocompatibility and performance in vitro and in vivo. BP-modified scaffolds accumulated VEGF, improved tube formation of HUVECs, and induced wound healing in vitro, which may have broad implications for regenerative medicine and tissue engineering.
Collapse
Affiliation(s)
- Zhengchao Yuan
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China
| | - Dandan Sheng
- Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China; State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, China
| | - Liping Jiang
- State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, China.
| | - Muhammad Shafiq
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China; Department of Biotechnology, Faculty of Life Sciences, University of Central Punjab (UCP), Lahore 54000, Pakistan.
| | - Atta Ur Rehman Khan
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China
| | - Rashida Hashim
- Department of Chemistry, Quaid-i-Azam University (QAU), Islamabad 45320, Pakistan
| | - Yujie Chen
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China
| | - Baojie Li
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China
| | - Xianrui Xie
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China
| | - Jun Chen
- Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China
| | - Yosry Morsi
- Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Boroondara, VIC 3122, Australia
| | - Xiumei Mo
- State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China.
| | - Shiyi Chen
- Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China.
| |
Collapse
|
16
|
Schumacher M, Habibović P, van Rijt S. Peptide-Modified Nano-Bioactive Glass for Targeted Immobilization of Native VEGF. ACS APPLIED MATERIALS & INTERFACES 2022; 14:4959-4968. [PMID: 35041377 PMCID: PMC8815037 DOI: 10.1021/acsami.1c21378] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
A limiting factor in large bone defect regeneration is the slow and disorganized formation of a functional vascular network in the defect area, often resulting in delayed healing or implant failure. To overcome this, strategies that induce angiogenic processes should be combined with potent bone graft substitutes in new bone regeneration approaches. To this end, we describe a unique approach to immobilize the pro-angiogenic growth factor VEGF165 in its native state on the surface of nanosized bioactive glass particles (nBGs) via a binding peptide (PR1P). We demonstrate that covalent coupling of the peptide to amine functional groups grafted on the nBG surface allows immobilization of VEGF with high efficiency and specificity. The amount of coupled peptide could be controlled by varying amine density, which eventually allows tailoring the amount of bound VEGF within a physiologically effective range. In vitro analysis of endothelial cell tube formation in response to VEGF-carrying nBG confirmed that the biological activity of VEGF is not compromised by the immobilization. Instead, comparable angiogenic stimulation was found for lower doses of immobilized VEGF compared to exogenously added VEGF. The described system, for the first time, employs a binding peptide for growth factor immobilization on bioactive glass nanoparticles and represents a promising strategy to overcome the problem of insufficient neovascularization in large bone defect regeneration.
Collapse
|
17
|
Adini A, Wu H, Dao DT, Ko VH, Yu LJ, Pan A, Puder M, Mitiku SZ, Potla R, Chen H, Rice JM, Matthews BD. PR1P Stabilizes VEGF and Upregulates Its Signaling to Reduce Elastase-induced Murine Emphysema. Am J Respir Cell Mol Biol 2020; 63:452-463. [PMID: 32663413 PMCID: PMC7528927 DOI: 10.1165/rcmb.2019-0434oc] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2019] [Accepted: 06/04/2020] [Indexed: 12/21/2022] Open
Abstract
Emphysema is a progressive and fatal lung disease with no cure that is characterized by thinning, enlargement, and destruction of alveoli, leading to impaired gas exchange. Disease progression is due in part to dysregulation of VEGF (vascular endothelial growth factor) signaling in the lungs and increased lung-cell apoptosis. Here we asked whether PR1P (Prominin-1-derived peptide), a novel short peptide we designed that increases VEGF binding to endothelial cells, could be used to improve outcome in in vitro and in vivo models of emphysema. We used computer simulation and in vitro and in vivo studies to show that PR1P upregulated endogenous VEGF receptor-2 signaling by binding VEGF and preventing its proteolytic degradation. In so doing, PR1P mitigated toxin-induced lung-cell apoptosis, including from cigarette-smoke extract in vitro and from LPS in vivo in mice. Remarkably, inhaled PR1P led to significantly increased VEGF concentrations in murine lungs within 30 minutes that remained greater than twofold above that of control animals 24 hours later. Finally, inhaled PR1P reduced acute lung injury in 4- and 21-day elastase-induced murine emphysema models. Taken together, these results highlight the potential of PR1P as a novel therapeutic agent for the treatment of emphysema or other lung diseases characterized by VEGF signaling dysregulation.
Collapse
Affiliation(s)
- Avner Adini
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
- Department of Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
| | - Hao Wu
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
| | - Duy T. Dao
- Vascular Biology Program
- Department of Surgery, and
| | | | - Lumeng J. Yu
- Vascular Biology Program
- Department of Surgery, and
| | - Amy Pan
- Vascular Biology Program
- Department of Surgery, and
| | - Mark Puder
- Vascular Biology Program
- Department of Surgery, and
| | - Selome Z. Mitiku
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
- Department of Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
| | - Ratnakar Potla
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
| | - Hong Chen
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
| | - James M. Rice
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
| | - Benjamin D. Matthews
- Vascular Biology Program
- Department of Pathology
- Department of Surgery, and
- Department of Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts
| |
Collapse
|
18
|
Rossi E, Poirault-Chassac S, Bieche I, Chocron R, Schnitzler A, Lokajczyk A, Bourdoncle P, Dizier B, Bacha NC, Gendron N, Blandinieres A, Guerin CL, Gaussem P, Smadja DM. Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties. Stem Cell Rev Rep 2020; 15:590-600. [PMID: 30879244 DOI: 10.1007/s12015-019-09881-8] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.
Collapse
Affiliation(s)
- Elisa Rossi
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France
| | - Sonia Poirault-Chassac
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France
| | - Ivan Bieche
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Department of genetics, Pharmacogenomics Unit, Institut Curie, Paris, France
| | - Richard Chocron
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S970, Paris, France.,AP-HP, Emergency Medicine Department, Hôpital Européen Georges Pompidou, Paris, France
| | - Anne Schnitzler
- Department of genetics, Pharmacogenomics Unit, Institut Curie, Paris, France
| | - Anna Lokajczyk
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France
| | - Pierre Bourdoncle
- Plate-forme IMAG'IC Institut Cochin Inserm U1016-CNRS UMR8104, Université Paris Descartes, Paris, France
| | - Blandine Dizier
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France
| | - Nour C Bacha
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France
| | - Nicolas Gendron
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France.,AP-HP, Hematology Department, Hôpital Européen Georges Pompidou, Paris, France
| | - Adeline Blandinieres
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France.,AP-HP, Hematology Department, Hôpital Européen Georges Pompidou, Paris, France
| | - Coralie L Guerin
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France.,Cytometry Unit, Institut Curie, Paris, France
| | - Pascale Gaussem
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France.,Inserm UMR-S1140, Paris, France.,AP-HP, Hematology Department, Hôpital Européen Georges Pompidou, Paris, France
| | - David M Smadja
- Sorbonne Paris Cité, Université Paris Descartes, Paris, France. .,Inserm UMR-S1140, Paris, France. .,AP-HP, Hematology Department, Hôpital Européen Georges Pompidou, Paris, France. .,Laboratory of Biosurgical Research, Carpentier Foundation, Hôpital Européen Georges Pompidou, Paris, France.
| |
Collapse
|
19
|
Lee J, Shin JE, Lee B, Kim H, Jeon Y, Ahn SH, Chi SW, Cho Y. The stem cell marker Prom1 promotes axon regeneration by down-regulating cholesterol synthesis via Smad signaling. Proc Natl Acad Sci U S A 2020; 117:15955-15966. [PMID: 32554499 PMCID: PMC7355016 DOI: 10.1073/pnas.1920829117] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Axon regeneration is regulated by a neuron-intrinsic transcriptional program that is suppressed during development but that can be reactivated following peripheral nerve injury. Here we identify Prom1, which encodes the stem cell marker prominin-1, as a regulator of the axon regeneration program. Prom1 expression is developmentally down-regulated, and the genetic deletion of Prom1 in mice inhibits axon regeneration in dorsal root ganglion (DRG) cultures and in the sciatic nerve, revealing the neuronal role of Prom1 in injury-induced regeneration. Elevating prominin-1 levels in cultured DRG neurons or in mice via adeno-associated virus-mediated gene delivery enhances axon regeneration in vitro and in vivo, allowing outgrowth on an inhibitory substrate. Prom1 overexpression induces the consistent down-regulation of cholesterol metabolism-associated genes and a reduction in cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axonal regrowth. We find that prominin-1 interacts with the type I TGF-β receptor ALK4, and that they synergistically induce phosphorylation of Smad2. These results suggest that Prom1 and cholesterol metabolism pathways are possible therapeutic targets for the promotion of neural recovery after injury.
Collapse
Affiliation(s)
- Jinyoung Lee
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea
| | - Jung Eun Shin
- Department of Molecular Neuroscience, Dong-A University College of Medicine, 49201 Busan, Republic of Korea
| | - Bohm Lee
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea
| | - Hyemin Kim
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea
| | - Yewon Jeon
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea
| | - Seung Hyun Ahn
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea
| | - Sung Wook Chi
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea
| | - Yongcheol Cho
- Department of Life Sciences, Korea University, 02841 Seoul, Republic of Korea;
| |
Collapse
|
20
|
Wang H, Gong P, Li J, Fu Y, Zhou Z, Liu L. Role of CD133 in human embryonic stem cell proliferation and teratoma formation. Stem Cell Res Ther 2020; 11:208. [PMID: 32460847 PMCID: PMC7251672 DOI: 10.1186/s13287-020-01729-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 04/29/2020] [Accepted: 05/13/2020] [Indexed: 01/16/2023] Open
Abstract
Background Pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs), hold great potential for regenerative medicine and cell therapy. One of the major hurdles hindering the clinical development of PSC-based therapy is the potential risk of tumorigenesis. CD133 (Prominin 1, PROM1) is a transmembrane protein whose mRNA and glycosylated forms are highly expressed in many human cancer cell types. CD133 also serves as a cancer stem cell (CSC) marker associated with cancer progression and patient outcome. Interestingly, CD133 is highly expressed in hESCs as well as in human preimplantation embryos, but its function in hESCs has remained largely unknown. Methods CD133 knockout hESC WA26 cell line was generated with CRISPR/Cas9. CD133 knockout and wide type hESC lines were subjected to pluripotency, proliferation, telomere biology, and teratoma tests; the related global changes and underlying mechanisms were further systemically analyzed by RNA-seq. Results CD133 deficiency did not affect hESC pluripotency or in vivo differentiation into three germ layers but significantly decreased cell proliferation. RNA-seq revealed that CD133 deficiency dysregulated the p53, PI3K-Akt, AMPK, and Wnt signaling pathways. Alterations in these pathways have been implicated in tumor proliferation and apoptotic escape. Conclusions Our data imply that CD133 could be an additional target and used as a selective marker to sort and eliminate undifferentiated cells in reducing potential teratoma formation risk of hESCs in regenerative medicine.
Collapse
Affiliation(s)
- Hua Wang
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.,Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China
| | - Peng Gong
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.,Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China
| | - Jie Li
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.,Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China
| | - Yudong Fu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.,Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China
| | - Zhongcheng Zhou
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.,Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China
| | - Lin Liu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China. .,Department of Cell Biology and Genetics, College of Life Sciences, Nankai University, Tianjin, 300071, China.
| |
Collapse
|
21
|
Imaging VEGF Receptors and α vβ 3 Integrins in a Mouse Hindlimb Ischemia Model of Peripheral Arterial Disease. Mol Imaging Biol 2019; 20:963-972. [PMID: 29687324 DOI: 10.1007/s11307-018-1191-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
PURPOSE To compare targeted imaging of vascular endothelial growth factor (VEGF) receptors vs. αvβ3 integrins in a mouse hindlimb ischemia model of peripheral artery disease. PROCEDURES Male wild-type (WT) C57BL/6 mice (8- to 10-week old) (n = 24) underwent left femoral artery ligation. The right leg served as control. Five days later, mice were injected with either VEGF receptor targeting [99mTc]DOTA-PEG-scVEGF ([99mTc]scV) (n = 8) or with αvβ3-targeting tracer [99mTc]HYNIC-cycloRGD ([99mTc]RGD) (n = 8) and underwent single photon emission computed tomography (SPECT) x-ray computed tomography imaging. To assess non-specific [99mTc]scV uptake, six additional mice received a mixture of [99mTc]scV and 30-fold excess of targeting protein, scVEGF. Tracer uptake as %ID was measured using volumetric regions encompassing the hindlimb muscles and as %ID/g from harvested limb muscles. Double and triple immunofluorescent analysis on tissue sections established localization of αvβ3, VEGFR-1, VEGFR-2, as well as certain cell lineage markers. RESULTS Tracer uptake, as %ID/g, was higher in ligated limbs of mice injected with [99mTc]scV compared to ligated hindlimbs in mice injected with [99mTc]RGD (p = 0.02). The ratio of tracer uptake for ligated/control hindlimb was borderline higher for [99mTc]scV than for [99mTc]RGD (p = 0.06). Immunofluorescent analysis showed higher prevalence of VEGFR-1, VEGFR-2, and αvβ3, in damaged vs. undamaged hindlimb tissue, but with little co-localization of these markers. Double immunofluorescent staining showed partial co-localization of VEGFR-1, VEGFR-2, and αvβ3, with endothelial cell marker FVIII, but not with CD31. Immunostaining for VEGFR-1 and VEGFR-2 additionally co-localized with lineage markers for endothelial progenitor cell and monocytes/macrophages, with a more diverse pattern of co-localization for VEGFR-2. CONCLUSION In a mouse hindlimb ischemia model of peripheral artery disease, [99mTc]scV SPECT tracer-targeting VEGF receptors showed a more robust signal than [99mTc]RGD tracer-targeting αvβ3. Immunofluorescent analysis suggests that uptake of [99mTc]scV and [99mTc]RGD in damaged tissue is due to non-overlapping cell populations and reflects different dynamic processes and that enhanced uptake of [99mTc]scV may be due to the presence of VEGF receptors on additional cell types.
Collapse
|
22
|
Chi ZL, Adini A, Birsner AE, Bazinet L, Akula JD, D'Amato RJ. PR1P ameliorates neurodegeneration through activation of VEGF signaling pathway and remodeling of the extracellular environment. Neuropharmacology 2018; 148:96-106. [PMID: 30594697 DOI: 10.1016/j.neuropharm.2018.12.029] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2018] [Revised: 11/26/2018] [Accepted: 12/26/2018] [Indexed: 12/13/2022]
Abstract
Neurodegenerative diseases affect millions of people worldwide. Optic neuropathies are the most commonly occurring neurodegenerative diseases, characterized by progressive retinal ganglion cell (RGC) degeneration. We recently reported that Prominin-1, a protein found on the surface of stem cells, interacts with VEGF and enhances its activity. VEGF is known to have various protective roles in the nervous system. Subsequently, we have developed a 12-mer peptide derived from Prominin-1, named PR1P, and investigated its effects on neuronal survival of damaged RGCs in a rat model of optic nerve crush (ONC). PR1P prevented RGC apoptosis resulting in improvement of retinal function in the rat ONC model. PR1P treatment significantly increased phosphorylation of ERK and AKT and expression its downstream proteins c-fos and Egr-1 in the retina. Additionally, PR1P beneficially increased the MMP-9/TIMP-1 ratio and promoted glial activation in the retina of ONC rats. Thus, PR1P displayed neuroprotective effects through enhanced VEGF-driven neuronal survival and reconstruction of the extracellular environment in ONC model. Our data indicate that PR1P may be a promising new clinical candidate for the treatment of neurodegenerative diseases.
Collapse
Affiliation(s)
- Zai-Long Chi
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, The Eye Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China; Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Boston, MA, 02115, USA.
| | - Avner Adini
- Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Boston, MA, 02115, USA
| | - Amy E Birsner
- Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Boston, MA, 02115, USA
| | - Lauren Bazinet
- Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Boston, MA, 02115, USA
| | - James D Akula
- Department of Ophthalmology, Boston Children's Hospital, Boston, 02115, MA, USA
| | - Robert J D'Amato
- Vascular Biology Program, Department of Surgery, Boston Children's Hospital, Boston, MA, 02115, USA; Department of Ophthalmology, Harvard Medical School, Boston, 02115, MA, USA.
| |
Collapse
|
23
|
Nikitakis NG, Gkouveris I, Aseervatham J, Barahona K, Ogbureke KUE. DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells. Cell Mol Biol Lett 2018; 23:30. [PMID: 30002682 PMCID: PMC6040065 DOI: 10.1186/s11658-018-0096-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Accepted: 06/19/2018] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. METHODS The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. RESULTS DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 μM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). CONCLUSIONS We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.
Collapse
Affiliation(s)
- Nikolaos G. Nikitakis
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Sciences Center at Houston School of Dentistry, 7500 Cambridge Street, Houston, TX 77054 USA
- Department of Oral Pathology and Medicine, School of Dentistry, University of Athens, Athens, Greece
| | - Ioannis Gkouveris
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Sciences Center at Houston School of Dentistry, 7500 Cambridge Street, Houston, TX 77054 USA
| | - Jaya Aseervatham
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Sciences Center at Houston School of Dentistry, 7500 Cambridge Street, Houston, TX 77054 USA
| | - Kelvin Barahona
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Sciences Center at Houston School of Dentistry, 7500 Cambridge Street, Houston, TX 77054 USA
| | - Kalu U. E. Ogbureke
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Sciences Center at Houston School of Dentistry, 7500 Cambridge Street, Houston, TX 77054 USA
| |
Collapse
|
24
|
Chaurasiya S, Chen NG, Warner SG. Oncolytic Virotherapy versus Cancer Stem Cells: A Review of Approaches and Mechanisms. Cancers (Basel) 2018; 10:E124. [PMID: 29671772 PMCID: PMC5923379 DOI: 10.3390/cancers10040124] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2018] [Revised: 04/11/2018] [Accepted: 04/14/2018] [Indexed: 12/26/2022] Open
Abstract
A growing body of evidence suggests that a subset of cells within tumors are resistant to conventional treatment modalities and may be responsible for disease recurrence. These cells are called cancer stem cells (CSC), which share properties with normal stem cells including self-renewal, pluripotency, drug resistance, and the ability to maintain quiescence. While most conventional therapies can efficiently destroy rapidly dividing cancer cells comprising the bulk of a tumor, they often fail to kill the less abundant and quiescent CSCs. Furthermore, killing of only differentiated cells in the tumor may actually allow for enrichment of CSCs and thereby portend a bad prognosis. Therefore, targeting of CSCs is important to achieve long-term success in cancer therapy. Oncolytic viruses represent a completely different class of therapeutics that can kill cancer cells in a variety of ways, which differ from those of conventional therapies. Hence, CSCs that are inherently resistant to conventional therapies may be susceptible to oncolytic virus-mediated killing. Recent studies have shown that oncolytic viruses can efficiently kill CSCs in many types of cancer. Here, we discuss the mechanism through which CSCs can escape conventional therapies and how they may still be susceptible to different classes of oncolytic viruses. Furthermore, we provide a summary of recent studies that have tested oncolytic viruses on CSCs of different origins and discuss possible future directions for this fascinating subset of oncolytic virus research.
Collapse
Affiliation(s)
- Shyambabu Chaurasiya
- Department of Surgery, Division of Surgical Oncology, City of Hope National Medical Center, Duarte, CA 91010, USA.
| | - Nanhai G Chen
- Department of Surgery, Division of Surgical Oncology, City of Hope National Medical Center, Duarte, CA 91010, USA.
- Center for Gene Therapy, Department of Hematologic and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
- Gene Editing and Viral Vector Core, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
| | - Susanne G Warner
- Department of Surgery, Division of Surgical Oncology, City of Hope National Medical Center, Duarte, CA 91010, USA.
| |
Collapse
|
25
|
Impact of Blood Vessel Quantity and Vascular Expression of CD133 and ICAM-1 on Survival of Glioblastoma Patients. NEUROSCIENCE JOURNAL 2017; 2017:5629563. [PMID: 29250531 PMCID: PMC5698821 DOI: 10.1155/2017/5629563] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/12/2017] [Revised: 08/07/2017] [Accepted: 10/04/2017] [Indexed: 11/18/2022]
Abstract
Glioblastoma (GB) is the most angiogenic tumor. Nevertheless, antiangiogenic therapy has not shown significant clinical efficacy. The aim of this study was to assess blood vessel characteristics on survival of GB patients. Surgically excised GB tissues were histologically examined for overall proportion of glomeruloid microvascular proliferation (MP) and the total number of blood vessels. Also, immunohistochemical vascular staining intensities of CD133 and ICAM-1 were determined. Vessel parameters were correlated with patients' overall survival. The survival time depended on the number of blood vessels (p = 0.03) but not on the proportion of MP. Median survival times for patients with low (<median) and high (≥median) number of blood vessels were 9.0 months (95% CI: 7.5–10.5) and 12.0 months (95% CI: 9.3–14.7). Also, median survival times for patients with low (<median) and high (≥median) vascular expression level of CD133 were 9.0 months (95% CI: 8.0–10.1) and 12.0 months (95% CI: 10.3–13.7). In contrast, the staining intensity of vascular ICAM-1 did not affect survival. In multivariate analysis, the number of blood vessels emerged as an independent predictor for longer overall survival (HR: 2.4, 95% CI: 1.2–5.0, p = 0.02). For success in antiangiogenic therapy, better understanding about tumor vasculature biology is needed.
Collapse
|
26
|
Almasry SM, Habib EK, Elmansy RA, Hassan ZA. Hyperglycemia Alters the Protein Levels of Prominin-1 and VEGFA in the Retina of Albino Rats. J Histochem Cytochem 2017; 66:33-45. [PMID: 29076766 DOI: 10.1369/0022155417737484] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
In this study, we addressed the potential relationship between prominin-1 (prom1) and vascular endothelial growth factor (VEGFA) in diabetes-induced retinopathy. In total, we examined 28 retinas from 14 rats with streptozotocin-induced diabetes and 30 retinas from 15 untreated control rats. ELISA was used to measure the level of prom1 and VEGFA in retinal tissue homogenates. Immunohistochemical techniques were used with antibodies directed against prom1, VEGFA, and CASP-3. After 180 days of diabetes induction, we performed light and electron microscopy studies on rat eyes to evaluate histopathological changes and to estimate the de novo metric "Diabetic Retinopathy Histopathological Index" (DRHI). These changes were then correlated to the tissue and immunoexpression levels of prom1 and VEGFA. The data showed a significant upregulation of the tissue levels and optical densities (ODs) of VEGFA and prom1 immunoreactivity in diabetic retinas compared with controls. Both the tissue levels and OD values of prom1 and VEGFA correlated significantly with each other and to the diabetic structural changes as calculated by DRHI. Taken together, these data provide new insight into the potential role of prom1 and VEGFA in the development of diabetic retinopathy.
Collapse
Affiliation(s)
- Shaima M Almasry
- Department of Anatomy, Al-Mansoura University, Mansoura, Egypt.,Department of Anatomy, Taibah University, Medina, Saudi Arabia
| | - Eman K Habib
- Department of Anatomy, Ain Shams University, Cairo, Egypt
| | | | - Zeinab A Hassan
- Department of Anatomy, Taibah University, Medina, Saudi Arabia.,Department of Histology and Cell Biology, Zagazig University, Zagazig, Egypt
| |
Collapse
|
27
|
A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P. Angiogenesis 2017; 20:399-408. [PMID: 28397127 DOI: 10.1007/s10456-017-9556-7] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2017] [Accepted: 04/04/2017] [Indexed: 12/21/2022]
Abstract
Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.
Collapse
|
28
|
Argaw-Denboba A, Balestrieri E, Serafino A, Cipriani C, Bucci I, Sorrentino R, Sciamanna I, Gambacurta A, Sinibaldi-Vallebona P, Matteucci C. HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features. J Exp Clin Cancer Res 2017; 36:20. [PMID: 28125999 PMCID: PMC5270369 DOI: 10.1186/s13046-016-0485-x] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2016] [Accepted: 12/27/2016] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications. METHODS TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression. RESULTS Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells. CONCLUSIONS HERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.
Collapse
Affiliation(s)
- Ayele Argaw-Denboba
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Emanuela Balestrieri
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Annalucia Serafino
- Institute of Translational Pharmacology, National Research Council, Via Fosso del Cavaliere 100, 00133 Rome, Italy
| | - Chiara Cipriani
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Ilaria Bucci
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Roberta Sorrentino
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Ilaria Sciamanna
- S.B.G.S.A. Istituto Superiore di Sanità (Italian National Institute of Health), Viale Regina Elena 299, 00161 Rome, Italy
| | - Alessandra Gambacurta
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| | - Paola Sinibaldi-Vallebona
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
- Institute of Translational Pharmacology, National Research Council, Via Fosso del Cavaliere 100, 00133 Rome, Italy
| | - Claudia Matteucci
- Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata”, Via Montpellier 1, 00133 Rome, Italy
| |
Collapse
|
29
|
Weng L, Hu X, Kumar B, Garcia M, Todorov I, Jung X, Marcucci G, Forman SJ, Chen CC. Identification of a CD133-CD55- population functions as a fetal common skeletal progenitor. Sci Rep 2016; 6:38632. [PMID: 27929130 PMCID: PMC5144148 DOI: 10.1038/srep38632] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Accepted: 11/10/2016] [Indexed: 01/19/2023] Open
Abstract
In this study, we identified a CD105+CD90.1−CD133−CD55− (CD133−CD55−) population in the fetal skeletal element that can generate bone and bone marrow. Besides osteoblasts and chondrocytes, the CD133−CD55− common progenitors can give rise to marrow reticular stromal cells and perivascular mesenchymal progenitors suggesting they function as the fetal common skeletal progenitor. Suppression of CXCL12 and Kitl expression in CD133−CD55− common progenitors severely disrupted the BM niche formation but not bone generation. Thus, CD133−CD55− common progenitors are the main source of CXCL12 and Kitl producing cells in the developing marrow.
Collapse
Affiliation(s)
- Lihong Weng
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA.,Departments of Cancer Immunotherapeutic and Immunology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA
| | - Xingbin Hu
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Department of Transfusion Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an 7100032, P.R. China
| | - Bijender Kumar
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA
| | - Mayra Garcia
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA
| | - Ivan Todorov
- Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA
| | - Xiaoman Jung
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA
| | - Guido Marcucci
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA
| | - Stephen J Forman
- Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA.,Departments of Cancer Immunotherapeutic and Immunology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Irell &Manella Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010, USA
| | - Ching-Cheng Chen
- Divison of Hematopoietic Stem Cell and Leukemia Research, Gehr Family Center for Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.,Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, CA 91010, USA.,Irell &Manella Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010, USA
| |
Collapse
|
30
|
CD133, Selectively Targeting the Root of Cancer. Toxins (Basel) 2016; 8:toxins8060165. [PMID: 27240402 PMCID: PMC4926132 DOI: 10.3390/toxins8060165] [Citation(s) in RCA: 72] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Revised: 05/08/2016] [Accepted: 05/10/2016] [Indexed: 12/20/2022] Open
Abstract
Cancer stem cells (CSC) are capable of promoting tumor initiation and self-renewal, two important hallmarks of carcinoma formation. This population comprises a small percentage of the tumor mass and is highly resistant to chemotherapy, causing the most difficult problem in the field of cancer research, drug refractory relapse. Many CSC markers have been reported. One of the most promising and perhaps least ubiquitous is CD133, a membrane-bound pentaspan glycoprotein that is frequently expressed on CSC. There is evidence that directly targeting CD133 with biological drugs might be the most effective way to eliminate CSC. We have investigated two entirely unrelated, but highly effective approaches for selectively targeting CD133. The first involves using a special anti-CD133 single chain variable fragment (scFv) to deliver a catalytic toxin. The second utilizes this same scFv to deliver components of the immune system. In this review, we discuss the development and current status of these CD133 associated biological agents. Together, they show exceptional promise by specific and efficient CSC elimination.
Collapse
|
31
|
Liu C, Li Y, Xing Y, Cao B, Yang F, Yang T, Ai Z, Wei Y, Jiang J. The Interaction between Cancer Stem Cell Marker CD133 and Src Protein Promotes Focal Adhesion Kinase (FAK) Phosphorylation and Cell Migration. J Biol Chem 2016; 291:15540-50. [PMID: 27226554 DOI: 10.1074/jbc.m115.712976] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2015] [Indexed: 12/28/2022] Open
Abstract
CD133, a widely known cancer stem cell marker, has been proved to promote tumor metastasis. However, the mechanism by which CD133 regulates metastasis remains largely unknown. Here, we report that CD133 knockdown inhibits cancer cell migration, and CD133 overexpression promotes cell migration. CD133 expression is beneficial to activate the Src-focal adhesion kinase (FAK) signaling pathway. Further studies show that CD133 could interact with Src, and the region between amino acids 845 and 857 in the CD133 C-terminal domain is indispensable for its interaction with Src. The interaction activates Src to phosphorylate its substrate FAK and to promote cell migration. Likewise, a Src binding-deficient CD133 mutant loses the abilities to increase Src and FAK phosphorylation and to promote cell migration. Inhibition of Src activity by PP2, a known Src activity inhibitor, could block the activation of FAK phosphorylation and cell migration induced by CD133. In summary, our data suggest that activation of FAK by the interaction between CD133 and Src promotes cell migration, providing clues to understand the migratory mechanism of CD133(+) tumor cells.
Collapse
Affiliation(s)
- Chanjuan Liu
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Yinan Li
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Yang Xing
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Benjin Cao
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Fan Yang
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Tianxiao Yang
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Zhilong Ai
- Division of General Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China
| | - Yuanyan Wei
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| | - Jianhai Jiang
- From the Key Laboratory of Glycoconjugates Research, Ministry of Public Health, Department of Biochemistry and Molecular Biology, Shanghai Medical College of Fudan University, Shanghai 200032, China and
| |
Collapse
|
32
|
Wylot B, Konarzewska K, Bugajski L, Piwocka K, Zawadzka M. Isolation of vascular endothelial cells from intact and injured murine brain cortex-technical issues and pitfalls in FACS analysis of the nervous tissue. Cytometry A 2015; 87:908-20. [PMID: 25892199 DOI: 10.1002/cyto.a.22677] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Revised: 03/11/2015] [Accepted: 04/01/2015] [Indexed: 12/15/2022]
Abstract
CNS endothelial cells (CNS-ECs), one of the main non-neural CNS cell populations, play a vital role in physiology, pathology, and regeneration of the nervous system. Therefore, there is an urgent need to enhance our knowledge on their biology to elucidate mechanisms responsible for the blood brain barrier function in normal and pathological conditions, interaction between brain endothelium and neural stem cells in the neurogenic niche, the paracrine processes in the brain and spinal cord, etc. Here, we described a novel, simple, and efficient protocol for isolation of endothelial, vessel-forming cells from the murine CNS, which is based on Sca-1 expression. Using this newly described protocol we were able to detect and sort viable, highly pure CNS-ECs with minimal contamination by cells of non-endothelial origin. This method will increase the availability of CNS-ECs for in vitro research. Moreover, we compared phenotype of CNS-ECs isolated from neonatal mice and adult intact and injured brain looking for the cells of endothelial precursor characteristic, such as those found in the bone marrow and circulating in the bloodstream after organ injuries. We have found that neonatal brain capillaries contain proportion of endothelial precursor cells (Sca-1(+) , CD45(-) , c-Kit(+) ). Such precursors were also found in adult brain cortex, both in intact and injured brain. Finally, we discuss several crucial technical issues concerning CNS tissue preparation for flow cytometry analysis and cell sorting as well as nonspecific antibody binding caused by inflammatory microglia/macrophages which should be avoided in order to reliable isolation of pure CNS cells for downstream procedures including cell transplantation-based translational studies.
Collapse
Affiliation(s)
- Bartosz Wylot
- Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland
| | - Katarzyna Konarzewska
- Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland
| | - Lukasz Bugajski
- Laboratory of Cytometry, Nencki Institute of Experimental Biology, Warsaw, Poland
| | - Katarzyna Piwocka
- Laboratory of Cytometry, Nencki Institute of Experimental Biology, Warsaw, Poland
| | - Malgorzata Zawadzka
- Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland
| |
Collapse
|
33
|
Bongiovanni D, Bassetti B, Gambini E, Gaipa G, Frati G, Achilli F, Scacciatella P, Carbucicchio C, Pompilio G. The CD133+Cell as Advanced Medicinal Product for Myocardial and Limb Ischemia. Stem Cells Dev 2014; 23:2403-21. [DOI: 10.1089/scd.2014.0111] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Affiliation(s)
- Dario Bongiovanni
- Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino-IRCCS, Milan, Italy
- Cardiovascular and Thoracic Diseases Department, Azienda Ospedaliera Città della Salute e della Scienza di Torino, Turin, Italy
| | - Beatrice Bassetti
- Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino-IRCCS, Milan, Italy
| | - Elisa Gambini
- Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino-IRCCS, Milan, Italy
| | - Giuseppe Gaipa
- Laboratorio Interdipartimentale di Terapia Cellulare Stefano Verri, Azienda Ospedaliera San Gerardo, Monza, Milan, Italy
| | - Giacomo Frati
- Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy
- Department of AngioCardioNeurology, IRCCS NeuroMed, Pozzilli, Italy
| | - Felice Achilli
- Department of Cardiology, Azienda Ospedaliera San Gerardo, Monza, Italy
| | - Paolo Scacciatella
- Cardiovascular and Thoracic Diseases Department, Azienda Ospedaliera Città della Salute e della Scienza di Torino, Turin, Italy
| | - Corrado Carbucicchio
- Cardiac Arrhythmia Research Centre, Centro Cardiologico Monzino-IRCCS, Milan, Italy
| | - Giulio Pompilio
- Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino-IRCCS, Milan, Italy
- Department of Clinical and Community Sciences, Università degli Studi di Milano, Milano, Italy
| |
Collapse
|
34
|
Pastushenko I, Vermeulen PB, Van den Eynden GG, Rutten A, Carapeto FJ, Dirix LY, Van Laere S. Mechanisms of tumour vascularization in cutaneous malignant melanoma: clinical implications. Br J Dermatol 2014; 171:220-33. [PMID: 24641095 DOI: 10.1111/bjd.12973] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/11/2014] [Indexed: 01/02/2023]
Abstract
Malignant melanoma represents < 10% of all skin cancers but is responsible for the majority of skin-cancer-related deaths. Metastatic melanoma has historically been considered as one of the most therapeutically challenging malignancies. Fortunately, for the first time after decades of basic research and clinical investigation, new drugs have produced major clinical responses. Angiogenesis has been considered an important target for cancer treatment. Initial efforts have focused primarily on targeting endothelial and tumour-related vascular endothelial growth factor signalling. Here, we review different mechanisms of tumour vascularization described in melanoma and discuss the potential clinical implications.
Collapse
Affiliation(s)
- I Pastushenko
- Department of Dermatology, Hospital Clínico Universitario 'Lozano Blesa', Zaragoza, 50009, Spain
| | | | | | | | | | | | | |
Collapse
|
35
|
Braden BP, Taketa DA, Pierce JD, Kassmer S, Lewis DD, De Tomaso AW. Vascular regeneration in a basal chordate is due to the presence of immobile, bi-functional cells. PLoS One 2014; 9:e95460. [PMID: 24736432 PMCID: PMC3988187 DOI: 10.1371/journal.pone.0095460] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2013] [Accepted: 03/27/2014] [Indexed: 11/18/2022] Open
Abstract
The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process.
Collapse
Affiliation(s)
- Brian P. Braden
- Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - Daryl A. Taketa
- Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - James D. Pierce
- Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - Susannah Kassmer
- Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - Daniel D. Lewis
- Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America
| | - Anthony W. De Tomaso
- Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara, Santa Barbara, California, United States of America
| |
Collapse
|
36
|
Abstract
Rho GTPases are a family of small GTPases, which play an important role in the regulation of the actin cytoskeleton. Not surprisingly, Rho GTPases are crucial for cell migration and therefore highly important for cancer cell invasion and the formation of metastases. In addition, Rho GTPases are involved in growth and survival of tumor cells, in the interaction of tumor cells with their environment, and they are vital for the cancer supporting functions of the tumor stroma. Recent research has significantly improved our understanding of the regulation of Rho GTPase activity, the specificity of Rho GTPases, and their function in tumor stem cells and tumor stroma. This review summarizes these novel findings and tries to define challenging questions for future research.
Collapse
Affiliation(s)
- Hui Li
- University of Copenhagen, BRIC, BMI, 2200, Copenhagen, Denmark
| | | | | | | |
Collapse
|
37
|
Mak AB, Schnegg C, Lai CY, Ghosh S, Yang MH, Moffat J, Hsu MY. CD133-targeted niche-dependent therapy in cancer: a multipronged approach. THE AMERICAN JOURNAL OF PATHOLOGY 2014; 184:1256-62. [PMID: 24589338 DOI: 10.1016/j.ajpath.2014.01.008] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 11/27/2013] [Revised: 01/05/2014] [Accepted: 01/16/2014] [Indexed: 02/07/2023]
Abstract
Cancer treatment continues to be challenged by the development of therapeutic resistances and relapses in the clinical setting, which are largely attributed to tumor heterogeneity, particularly the existence of cancer stem cells (CSCs). Thus, targeting the CSC subpopulation may represent an effective therapeutic strategy. However, despite advances in identifying and characterizing CD133(+) CSCs in various human cancers, efforts to translate these experimental findings to clinical modalities have been slow in the making, especially in light of the growing awareness of CSC plasticity and the foreseeable pitfall of therapeutically targeting CSC base sorely on a surface marker. We, and others, have demonstrated that the CD133(+) CSCs reside in complex vascular niches, where reciprocal signaling between the CD133(+) CSCs and their microenvironment may govern niche morphogenesis and homeostasis. Herein, we discuss the multifaceted functional role of the CD133(+) cells in the context of their niche, and the potential of targeting CD133 as a niche-dependent approach in effective therapy.
Collapse
Affiliation(s)
- Anthony B Mak
- Department of Dermatology, Boston University Medical Center, Boston, Massachusetts; Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
| | - Caroline Schnegg
- Department of Dermatology, Boston University Medical Center, Boston, Massachusetts
| | - Chiou-Yan Lai
- Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts
| | - Subrata Ghosh
- Department of Dermatology, Boston University Medical Center, Boston, Massachusetts
| | - Moon Hee Yang
- Department of Dermatology, Boston University Medical Center, Boston, Massachusetts
| | - Jason Moffat
- Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
| | - Mei-Yu Hsu
- Department of Dermatology, Boston University Medical Center, Boston, Massachusetts; Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts.
| |
Collapse
|
38
|
Mund JA, Shannon H, Sinn AL, Cai S, Wang H, Pradhan KR, Pollok KE, Case J. Human proangiogenic circulating hematopoietic stem and progenitor cells promote tumor growth in an orthotopic melanoma xenograft model. Angiogenesis 2013; 16:953-62. [PMID: 23877751 DOI: 10.1007/s10456-013-9368-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2013] [Accepted: 07/13/2013] [Indexed: 12/19/2022]
Abstract
We previously identified a distinct population of human circulating hematopoietic stem and progenitor cells (CHSPCs; CD14(-)glyA(-)CD34(+)AC133(+/-)CD45(dim)CD31(+) cells) in the peripheral blood (PB) and bone marrow, and their frequency in the PB can correlate with disease state. The proangiogenic subset (pCHSPC) play a role in regulating tumor progression, for we previously demonstrated a statistically significant increase in C32 melanoma growth in NOD.Cg-Prkdc (scid) (NOD/SCID) injected with human pCHSPCs (p < 0.001). We now provide further evidence that pCHSPCs possess proangiogenic properties. In vitro bio-plex cytokine analyses and tube forming assays indicate that pCHSPCs secrete a proangiogenic profile and promote vessel formation respectively. We also developed a humanized bone marrow-melanoma orthotopic model to explore in vivo the biological significance of the pCHSPC population. Growth of melanoma xenografts increased more rapidly at 3-4 weeks post-tumor implantation in mice previously transplanted with human CD34(+) cells compared to control mice. Increases in pCHSPCs in PB correlated with increases in tumor growth. Additionally, to determine if we could prevent the appearance of pCHSPCs in the PB, mice with humanized bone marrow-melanoma xenografts were administered Interferon α-2b, which is used clinically for treatment of melanoma. The mobilization of the pCHSPCs was decreased in the mice with the humanized bone marrow-melanoma xenografts. Taken together, these data indicate that pCHSPCs play a functional role in tumor growth. The novel in vivo model described here can be utilized to further validate pCHSPCs as a biomarker of tumor progression. The model can also be used to screen and optimize anticancer/anti-angiogenic therapies in a humanized system.
Collapse
Affiliation(s)
- Julie A Mund
- Department of Pediatrics, Indiana University School of Medicine, 1044 West Walnut St, R4-470, Indianapolis, IN, 46202, USA
| | | | | | | | | | | | | | | |
Collapse
|
39
|
Peitzsch C, Kurth I, Kunz-Schughart L, Baumann M, Dubrovska A. Discovery of the cancer stem cell related determinants of radioresistance. Radiother Oncol 2013; 108:378-87. [PMID: 23830195 DOI: 10.1016/j.radonc.2013.06.003] [Citation(s) in RCA: 143] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2013] [Revised: 05/31/2013] [Accepted: 06/05/2013] [Indexed: 12/23/2022]
Abstract
Tumors are known to be heterogeneous containing a dynamic mixture of phenotypically and functionally different tumor cells. The two concepts attempting to explain the origin of intratumor heterogeneity are the cancer stem cell hypothesis and the clonal evolution model. The stochastic model argues that tumors are biologically homogenous and all cancer cells within the tumor have equal ability to propagate the tumor growth depending on continuing mutations and selective pressure. By contrast, the stem cells model suggests that cancer heterogeneity is due to the hierarchy that originates from a small population of cancer stem cells (CSCs) which are biologically distinct from the bulk tumor and possesses self-renewal, tumorigenic and multilineage potential. Although these two hypotheses have been discussed for a long time as mutually exclusive explanations of tumor heterogeneity, they are easily reconciled serving as a driving force of cancer evolution and diversity. Recent discovery of the cancer cell plasticity and heterogeneity makes the CSC population a moving target that could be hard to track and eradicate. Understanding the signaling mechanisms regulating CSCs during the course of cancer treatment can be indispensable for the optimization of current treatment strategies.
Collapse
Affiliation(s)
- Claudia Peitzsch
- OncoRay National Center for Radiation Research in Oncology, University Hospital/Medical Faculty Carl Gustav Carus, TU Dresden, Germany
| | | | | | | | | |
Collapse
|