[en] The tumor microenvironment and its contribution to tumorigenesis has been a focal highlight in recent years. A two-way communication between the tumor and the surrounding microenvironment sustains and contributes to the growth and metastasis of tumors. Progression and metastasis of hepatocellular carcinoma (HCC) have been reported to be exceedingly influenced by diverse microenvironmental cues. In this study, we present a 3D-culture model of liver cancer to better mimic in vivo tumor settings. By creating novel 3D co-culture model that combines free-floating and scaffold-based 3D-culture techniques of liver cancer cells and fibroblasts, we aimed to establish a simple albeit reproducible ex vivo cancer microenvironment model that captures tumor-stroma interactions. The model presented herein exhibited unique gene expression and protein expression profiles when compared to 2D and 3D mono-cultures of liver cancer cells. Our results showed that in vivo like conditions cannot be mimicked by simply growing cancer cells as spheroids, but by co-culturing them with 3D fibroblast with which they were able to crosstalk. This was evident by the upregulation of several pathways involved in HCC, and the increase in secreted factors by co-cultured cancer cells, many of which are also involved in tumor-stroma interactions. Compared to the conventional 2D culture, the proposed model exhibits an increase in the expression of genes associated with development, progression, and poor prognosis of HCC. Our results correlated with an aggressive outcome that better mirrors in vivo HCC, and therefore, a more reliable platform for molecular understanding of HCC.
Disciplines :
Laboratory medicine & medical technology Human health sciences: Multidisciplinary, general & others Oncology Biotechnology Life sciences: Multidisciplinary, general & others
Author, co-author :
Al Hrout, Ala'a; Institute of Experimental Immunology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland. ; Biology Department, College of Science, UAE University, P.O. Box 15551, Al-Ain, United Arab Emirates.
Cervantes-Gracia, Karla; Institute of Experimental Immunology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
CHAHWAN, Richard ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Health, Medicine and Life Sciences (DHML) ; Institute of Experimental Immunology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland. chahwan@immunology.uzh.ch.
Amin, Amr; Biology Department, College of Science, UAE University, P.O. Box 15551, Al-Ain, United Arab Emirates. a.amin@uaeu.ac.ae. ; The University of Chicago, Chicago, IL, 60637, USA. a.amin@uaeu.ac.ae.
External co-authors :
yes
Language :
English
Title :
Modelling liver cancer microenvironment using a novel 3D culture system.
Publication date :
14 May 2022
Journal title :
Scientific Reports
eISSN :
2045-2322
Publisher :
Nature Publishing Group, London, Gb
Volume :
12
Issue :
1
Pages :
8003
Peer reviewed :
Peer Reviewed verified by ORBi
Funding number :
BB/N017773/2/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom; CRSK-3_190550/SNSF_/Swiss National Science Foundation/Switzerland
Quail, D. F. & Joyce, J. A. Microenvironmental regulation of tumor progression and metastasis. Nat. Med. 19, 1423–1437 (2013).
Catalano, V. et al. Tumor and its microenvironment: A synergistic interplay. Semin. Cancer Biol. 10.1016/j.semcancer.2013.08.007 (2013). DOI: 10.1016/j.semcancer.2013.08.007
Hanahan, D. & Weinberg, R. A. A. Hallmarks of cancer: The next generation. Cell 144, 646–674 (2011).
Hanahan, D. & Coussens, L. M. Accessories to the crime: Functions of cells recruited to the tumor microenvironment. Cancer Cell 10.1016/j.ccr.2012.02.022 (2012). DOI: 10.1016/j.ccr.2012.02.022
Bissell, M. J. & Hines, W. C. Why don’t we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nat. Med. 17, 320–329 (2011).
Öhlund, D., Elyada, E. & Tuveson, D. Fibroblast heterogeneity in the cancer wound. J. Exp. Med. 211, 1503–1523 (2014).
Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335–348 (2005).
Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6, 392–401 (2006).
Cirri, P. & Chiarugi, P. Cancer-associated-fibroblasts and tumour cells: A diabolic liaison driving cancer progression. Cancer Metastasis Rev. 31, 195–208 (2012).
Zhang, J. & Liu, J. Tumor stroma as targets for cancer therapy. Pharmacol. Ther. 137, 200–215 (2013).
Chen, X. & Song, E. Turning foes to friends: Targeting cancer-associated fibroblasts. Nat. Rev. Drug Discov. 18, 99–115 (2019).
Span, P. N. & Bussink, J. Biology of hypoxia. Semin. Nucl. Med. 45, 101–109 (2015).
Brahimi-Horn, M. C., Chiche, J. & Pouysségur, J. Hypoxia and cancer. J. Mol. Med. 85, 1301–1307 (2007).
Casazza, A. et al. Tumor stroma: A complexity dictated by the hypoxic tumor microenvironment. Oncogene 33, 1743–1754 (2014).
Gilkes, D. M., Semenza, G. L. & Wirtz, D. Hypoxia and the extracellular matrix: Drivers of tumour metastasis. Nat. Rev. Cancer 14, 430–439 (2014).
Fitzmaurice, C. et al. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 32 cancer groups, 1990 to 2015: A Systematic Analysis for the Global Burden of Disease Study Global Burden. JAMA Oncol. 3, 524–548 (2017).
Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, P. D. & Forman D, Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase. No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer. 11, http://globocan.iarc.f (2013).
Tu, K. et al. Fibulin-5 inhibits hepatocellular carcinoma cell migration and invasion by down-regulating matrix metalloproteinase-7 expression. BMC Cancer 14, 938 (2014).
Baglieri, J., Brenner, D. A. & Kisseleva, T. The role of fibrosis and liver-associated fibroblasts in the pathogenesis of hepatocellular carcinoma. Int. J. Mol. Sci. 20, 1723 (2019).
Fattovich, G., Stroffolini, T., Zagni, I. & Donato, F. Hepatocellular carcinoma in cirrhosis: Incidence and risk factors. Gastroenterology 127, 2 (2004).
Lin, D. & Wu, J. Hypoxia inducible factor in hepatocellular carcinoma: A therapeutic target. World J. Gastroenterol. 21, 12171–12178 (2015).
Zhang, Q. et al. Wnt/β-catenin signaling enhances hypoxia-induced epithelial-mesenchymal transition in hepatocellular carcinoma via crosstalk with hif-1α signaling. Carcinogenesis 34, 962–973 (2013).
Wilson, G. K., Tennant, D. A. & McKeating, J. A. Hypoxia inducible factors in liver disease and hepatocellular carcinoma: Current understanding and future directions. J. Hepatol. 61, 1397–1406 (2014).
Anton, D., Burckel, H., Josset, E. & Noel, G. Three-dimensional cell culture: A breakthrough in vivo. Int. J. Mol. Sci. 16, 5517–5527 (2015).
Unger, C. et al. Modeling human carcinomas: Physiologically relevant 3D models to improve anti-cancer drug development. Adv. Drug Deliv. Rev. 79, 50–67 (2014).
Ingram, M. et al. Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor. Vitr. Cell. Dev. Biol. Anim. 33, 459–466 (1997).
Ravi, M., Paramesh, V., Kaviya, S. R., Anuradha, E. & Paul Solomon, F. D. 3D cell culture systems: Advantages and applications. J. Cell. Physiol. 230, 16–26 (2015).
Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. D. & Takayama, S. Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release 164, 192–204 (2012).
Maere, S., Heymans, K. & Kuiper, M. BiNGO: A Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449 (2005).
Shannon, P. et al. Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 10.1101/gr.1239303.metabolite (2003). DOI: 10.1101/gr.1239303.metabolite
Heberle, H., Meirelles, V. G., da Silva, F. R., Telles, G. P. & Minghim, R. InteractiVenn: A web-based tool for the analysis of sets through Venn diagrams. BMC Bioinform. 16, 169 (2015).
Kanehisa, M., Furumichi, M., Sato, Y., Ishiguro-Watanabe, M. & Tanabe, M. KEGG: Integrating viruses and cellular organisms. Nucleic Acids Res. 49, D545–D551 (2021).
Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).
Kanehisa, M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 28, 1947–1951 (2019).
Bindea, G. et al. ClueGO: A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25, 1091–1093 (2009).
Bindea, G., Galon, J. & Mlecnik, B. CluePedia Cytoscape plugin: Pathway insights using integrated experimental and in silico data. Bioinformatics 29, 661–663 (2013).
Goldman, M. J. et al. Visualizing and interpreting cancer genomics data via the Xena platform. Nat. Biotechnol. 38, 675–678 (2020).
Cervantes-Gracia, K. & Husi, H. Integrative analysis of Multiple Sclerosis using a systems biology approach. Sci. Rep. 8, 1–14 (2018).
Luo, Y. D. et al. p53 haploinsufficiency and increased mTOR signalling define a subset of aggressive hepatocellular carcinoma. J. Hepatol. 74, 96–108 (2021).
Petrelli, A. et al. MicroRNA/gene profiling unveils early molecular changes and nuclear factor erythroid related factor 2 (NRF2) activation in a rat model recapitulating human hepatocellular carcinoma (HCC). Hepatology 59, 228–241 (2014).
Zhang, C. et al. The identification of key genes and pathways in hepatocellular carcinoma by bioinformatics analysis of high-throughput data. Med. Oncol. 34, 101 (2017).
Ohnishi, S., Murakami, T., Moriyama, T., Mitamura, K. & Imawari, M. Androgen and estrogen receptors in hepatocellular carcinoma and in the surrounding noncancerous liver tissue. Hepatology 6, 440–443 (1986).
Battello, N. et al. The role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells. Cancer Metab. 4, 3 (2016).
Liang, H. et al. Interleukin-6 and oncostatin M are elevated in liver disease in conjunction with candidate hepatocellular carcinoma biomarker GP73. Cancer Biomark. 11, 161–171 (2012).
Martinez-Quetglas, I. et al. IGF2 is up-regulated by epigenetic mechanisms in hepatocellular carcinomas and is an actionable oncogene product in experimental models. Gastroenterology 151, 1192–1205 (2016).
Kutmon, M. et al. PathVisio 3: An extendable pathway analysis toolbox. PLoS Comput. Biol. 11, 2 (2015).
Yang, X. et al. VEGF-B promotes cancer metastasis through a VEGF-A-independent mechanism and serves as a marker of poor prognosis for cancer patients. Proc. Natl. Acad. Sci. U. S. A. 112, E2900–E2909 (2015).
Sulas, P. et al. A large set of miRNAs is dysregulated from the earliest steps of human hepatocellular carcinoma development. Am. J. Pathol. 188, 785–794 (2018).
Guo, W. et al. MiR-199a-5p is negatively associated with malignancies and regulates glycolysis and lactate production by targeting hexokinase 2 in liver cancer. Hepatology 62, 1132–1144 (2015).
Eferl, R. & Wagner, E. F. AP-1: A double-edged sword in tumorigenesis. Nat. Rev. Cancer 3, 859–868 (2003).
Behnke, M., Reimers, M. & Fisher, R. The expression of embryonic liver development genes in hepatitis c induced cirrhosis and hepatocellular carcinoma. Cancers (Basel). 4, 945–968 (2012).
Rudalska, R. et al. In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer. Nat. Med. 20, 1138–1146 (2014).
Kimlin, L. C., Casagrande, G. & Virador, V. M. In vitro three-dimensional (3D) models in cancer research: An update. Mol. Carcinog. 52, 167–182 (2013).
Luo, D., Wang, Z., Wu, J., Jiang, C. & Wu, J. The role of hypoxia inducible factor-1 in hepatocellular carcinoma. BioMed Res. Int. 2014, 2 (2014).
Shen, G. & Li, X. The multifaceted role of hypoxia-inducible factor 1 (HIF1) in lipid metabolism. In Hypoxia and Human Diseases (InTech, 2017). 10.5772/65340. DOI: 10.5772/65340
Mahata, B. et al. Tumors induce de novo steroid biosynthesis in T cells to evade immunity. Nat. Commun. 11, 1–15 (2020).
Sheppard, E. C., Morrish, R. B., Dillon, M. J., Leyland, R. & Chahwan, R. Epigenomic modifications mediating antibody maturation. Front. Immunol. 9, 2 (2018).
Martin, A., Chahwan, R., Parsa, J. Y. & Scharff, M. D. Somatic Hypermutation: The Molecular Mechanisms Underlying the Production of Effective High-Affinity Antibodies. Molecular Biology of B Cells (Elsevier Ltd, 2014). 10.1016/B978-0-12-397933-9.00021-7. DOI: 10.1016/B978-0-12-397933-9.00021-7
Hynes, R. O. Integrins: Bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).
Park, C. C. et al. β1integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo. Cancer Res. 66, 1526–1535 (2006).
White, D. E. et al. Targeted disruption of β1-integrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell 6, 159–170 (2004).
Schaffner, F., Ray, A. M. & Dontenwill, M. Integrin ??5??1, the fibronectin receptor, as a pertinent therapeutic target in solid tumors. Cancers 5, 27–47 (2013).
Grzesiak, J. J. & Bouvet, M. Determination of the ligand-binding specificities of the alpha2beta1 and alpha1beta1 integrins in a novel 3-dimensional in vitro model of pancreatic cancer. Pancreas 34, 220–228 (2007).
Kugler, M. C., Wei, Y. & Chapman, H. A. Urokinase receptor and integrin interactions. Curr. Pharm. Des. 9, 1565–1574 (2003).
Tyndall, J., Kelso, M., Clingan, P. & Ranson, M. Peptides and small molecules targeting the plasminogen activation system: Towards prophylactic anti-metastasis drugs for breast cancer. Recent Pat. Anticancer. Drug Discov. 3, 1–13 (2008).
Shain, K. H. et al. β1 integrin adhesion enhances IL-6-mediated STAT3 signaling in myeloma cells: Implications for microenvironment influence on tumor survival and proliferation. Cancer Res. 69, 1009–1015 (2009).
Kesanakurti, D., Chetty, C., Dinh, D. H., Gujrati, M. & Rao, J. S. Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma. Oncogene 32, 327–340 (2013).
Lee, K. W., Yeo, S. Y., Sung, C. O. & Kim, S. H. Twist1 is a key regulator of cancer-associated fibroblasts. Cancer Res. 75, 73–85 (2015).
Lin, Z. Y., Chuang, Y. H. & Chuang, W. L. Cancer-associated fibroblasts up-regulate CCL2, CCL26, IL6 and LOXL2 genes related to promotion of cancer progression in hepatocellular carcinoma cells. Biomed. Pharmacother. 66, 525–529 (2012).
Khawar, I. A. et al. Three dimensional mixed-cell spheroids mimic stroma-mediated chemoresistance and invasive migration in hepatocellular carcinoma. Neoplasia 20, 800–812 (2018).
Liu, J. et al. Cancer-associated fibroblasts provide a stromal niche for liver cancer organoids that confers trophic effects and therapy resistance. Cell. Mol. Gastroenterol. Hepatol. 11, 407–431 (2021).
Enguita-Germán, M. & Fortes, P. Targeting the insulin-like growth factor pathway in hepatocellular carcinoma. World J. Hepatol. 6, 716–737 (2014).
Pan, J. H. et al. Role of exosomes and exosomal microRNAs in hepatocellular carcinoma: Potential in diagnosis and antitumour treatments (Review). Int. J. Mol. Med. 41, 1809–1816 (2018).
Kim, H. R., Roe, J. S., Lee, J. E., Cho, E. J. & Youn, H. D. P53 regulates glucose metabolism by miR-34a. Biochem. Biophys. Res. Commun. 437, 225–231 (2013).
Lu, L., Chen, Y. & Zhu, Y. The molecular basis of targeting PFKFB3 as a therapeutic strategy against cancer. Oncotarget 8, 62793–62802 (2017).
Shi, W.-K. et al. PFKFB3 blockade inhibits hepatocellular carcinoma growth by impairing DNA repair through AKT. Cell Death Dis. 9, 428 (2018).
Li, S. et al. By inhibiting PFKFB3, aspirin overcomes sorafenib resistance in hepatocellular carcinoma. Int. J. Cancer 141, 2571–2584 (2017).
Taniguchi, C. M. et al. Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nat. Med. 19, 1325–1330 (2013).
Yano, H. et al. PHD3 regulates glucose metabolism by suppressing stress-induced signalling and optimising gluconeogenesis and insulin signalling in hepatocytes. Sci. Rep. 8, 1–16 (2018).
Lu, C. et al. Integrated analysis reveals critical glycolytic regulators in hepatocellular carcinoma. Cell Commun. Signal. 18, 2 (2020).
Zhang, Q. et al. A novel hypoxia gene signature indicates prognosis and immune microenvironments characters in patients with hepatocellular carcinoma. J. Cell. Mol. Med. 25, 3772–3784 (2021).
Dittmer, J. The Biology of the Ets1 Proto-Oncogene. Mol. Cancer 2, 2 (2003).
Ozaki, I. et al. Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res. 60, 6519–6525 (2000).
Ma, N. et al. MicroRNA-129-5p inhibits hepatocellular carcinoma cell metastasis and invasion via targeting ETS1. Biochem. Biophys. Res. Commun. 461, 618–623 (2015).
Placencio, V. R. & DeClerck, Y. A. Plasminogen activator inhibitor-1 in cancer: Rationale and insight for future therapeutic testing. Cancer Res. 75, 2969–2974 (2015).
Mitxelena, J. et al. An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability. Nucleic Acids Res. 10.1093/nar/gky218 (2018). DOI: 10.1093/nar/gky218
Yim, K. H. W., Hrout, A. A., Borgoni, S. & Chahwan, R. Extracellular vesicles orchestrate immune and tumor interaction networks. Cancers 12, 1–23 (2020).
Cui, L., Hu, Y., Bai, B. & Zhang, S. Serum miR-335 level is associated with the treatment response to trans-arterial chemoembolization and prognosis in patients with hepatocellular carcinoma. Cell. Physiol. Biochem. 37, 276–283 (2015).
Wang, F., Li, L., Piontek, K., Sakaguchi, M. & Selaru, F. M. Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma. Hepatology 67, 940–954 (2018).
Wu, J. & Zhu, A. X. Targeting insulin-like growth factor axis in hepatocellular carcinoma. J. Hematol. Oncol. 4, 2 (2011).
Akazawa, Y. et al. M-CSF receptor antagonists inhibit the initiation and progression of hepatocellular carcinoma in mice. Anticancer Res. 39, 4787–4794 (2019).
Landskron, G., De La Fuente, M., Thuwajit, P., Thuwajit, C. & Hermoso, M. A. Chronic inflammation and cytokines in the tumor microenvironment. J. Immunol. Res. 2014, 2 (2014).
Zhang, Z., Lei, B., Chai, W., Liu, R. & Li, T. Increased expression of insulin-like growth factor-1 receptor predicts poor prognosis in patients with hepatocellular carcinoma. Medicine 98, e17680 (2019).
Jiang, L. et al. Association of PHD3 and HIF2α gene expression with clinicopathological characteristics in human hepatocellular carcinoma. Oncol. Lett. 15, 545–551 (2018).
Nyga, A., Cheema, U. & Loizidou, M. 3D tumour models: Novel in vitro approaches to cancer studies. J. Cell Commun. Signal. 5, 239–248 (2011).
Vidic, S. et al. PREDECT protocols for complex 2D/3D cultures. Methods Mol. Biol. 1888, 1–20 (2019).
Santo, V. E. et al. Drug screening in 3D in vitro tumor models: Overcoming current pitfalls of efficacy read-outs. Biotechnol. J. 12, 1600505 (2017).
Salerno, A., Cesarelli, G., Pedram, P. & Netti, P. A. Modular strategies to build cell-free and cell-laden scaffolds towards bioengineered tissues and organs. J. Clin. Med. 8, 1816 (2019).
Paterson, K., Zanivan, S., Glasspool, R., Coffelt, S. B. & Zagnoni, M. Microfluidic technologies for immunotherapy studies on solid tumours. Lab Chip 21, 2306–2329 (2021).
Redondo, P. A., Pavlou, M., Loizidou, M. & Cheema, U. Elements of the niche for adult stem cell expansion. J. Tissue Eng. 8, 2041731417725464 (2017).
Edmondson, R., Broglie, J. J., Adcock, A. F. & Yang, L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev. Technol. 12, 207–218 (2014).
