TY - JOUR
T1 - Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer
AU - Strating, Esther
AU - Verhagen, Mathijs P
AU - Wensink, Emerens
AU - Dünnebach, Ester
AU - Wijler, Liza
AU - Aranguren, Itziar
AU - De la Cruz, Alberto Sanchez
AU - Peters, Niek A
AU - Hageman, Joris H
AU - van der Net, Mirjam M C
AU - van Schelven, Susanne
AU - Laoukili, Jamila
AU - Fodde, Riccardo
AU - Roodhart, Jeanine
AU - Nierkens, Stefan
AU - Snippert, Hugo
AU - Gloerich, Martijn
AU - Rinkes, Inne Borel
AU - Elias, Sjoerd G
AU - Kranenburg, Onno
N1 - Copyright © 2023 Strating, Verhagen, Wensink, Dünnebach, Wijler, Aranguren, De la Cruz, Peters, Hageman, van der Net, van Schelven, Laoukili, Fodde, Roodhart, Nierkens, Snippert, Gloerich, Rinkes, Elias and Kranenburg.
PY - 2023/5/16
Y1 - 2023/5/16
N2 - BACKGROUND: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation.METHODS: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation.RESULTS: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFβ1, VEGFA and lactate, and potently inhibited T cell proliferation.CONCLUSION: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.
AB - BACKGROUND: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation.METHODS: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation.RESULTS: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFβ1, VEGFA and lactate, and potently inhibited T cell proliferation.CONCLUSION: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.
KW - Humans
KW - Cancer-Associated Fibroblasts/metabolism
KW - Coculture Techniques
KW - Reproducibility of Results
KW - Colonic Neoplasms/pathology
KW - Tumor Microenvironment
U2 - 10.3389/fimmu.2023.1053920
DO - 10.3389/fimmu.2023.1053920
M3 - Article
C2 - 37261365
SN - 1664-3224
VL - 14
SP - 1053920
JO - Frontiers in immunology
JF - Frontiers in immunology
ER -