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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (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.)

The liver has a complex and hierarchical segmental organization of arteries, portal veins, hepatic veins and lymphatic vessels. In-depth imaging of liver vasculature and malignancies could improve knowledge on tumor micro-environment, local tumor growth, invasion, as well as metastasis. Non-invasive imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI) and positron-emission transmission (PET) are routine for clinical imaging, but show inadequate resolution at cellular and subcellular level. In recent years, tissue clearing - a technique rendering tissues optically transparent allowing enhanced microscopy imaging - has made great advances. While mainly used in the neurobiology field, recently more studies have used clearing techniques for imaging other organ systems as well as tumor tissues. In this study, our aim was to develop a reproducible tissue clearing and immunostaining model for visualizing intrahepatic blood microvasculature and tumor cells in murine colorectal liver metastases. CLARITY and 3DISCO/iDISCO+ are two established clearing methods that have been shown to be compatible with immunolabelling, most often in neurobiology research. In this study, CLARITY unfortunately resulted in damaged tissue integrity of the murine liver lobes and no specific immunostaining. Using the 3DISCO/iDISCO+ method, liver samples were successfully rendered optically transparent. After which, successful immunostaining of the intrahepatic microvasculature using panendothelial cell antigen MECA-32 and colorectal cancer cells using epithelial cell adhesion molecule (EpCAM) was established. This approach for tumor micro-environment tissue clearing would be especially valuable for allowing visualization of spatial heterogeneity and complex interactions of tumor cells and their environment in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Frenkel, Poghosyan, van Wijnbergen, van den Bent, Wijler, Verheem, Borel Rinkes, Kranenburg and Hagendoorn.)