The high-plasticity states of tumor cells lead to drastic phenotypic changes, such as epithelial-mesenchymal transition (EMT), enabling high invasive and metastatic potential. Tumor cells may occupy a spectrum along an epithelial-mesenchymal axis, where the two ends are mixed by a hybrid partial EMT (pEMT) state. Colorectal cancer (CRC) is the second-third leading cause of cancer-related death. Gene expression-based CRC clustering identified a group (CMS4) with EMT, fibroblast (CAF) accumulation, invasion, and decreased survival, representing an aggressive subtype with high plasticity and no effective treatment. In this work, we aimed at characterizing pEMT by using the organoid technology and co-cultures. Patient-derived organoids (PDO) maintain the cellular heterogeneity of the original tissue, representing a popular tool for cancer research.

PDOs were isolated from patients with CRC and cultured in the absence/presence of fibroblasts in different extracellular matrices (ECM), such as the laminin-rich Matrigel or collagen-I. Cells were characterized by imaging and statistical analysis, flow cytometry, whole-mount immunostaining and by RT-qPCR at the RNA level.

IL1R1 was shown to be a marker of iCAFs in CRC, a subpopulation of fibroblasts with inflammatory phenotype. Whereas immune cell-derived cytokines, such as IL-17A, TNF-alpha, IL-22 or IFN-gamma had no effect on fibroblast polarization, co-culturing fibroblasts with CRC organoids resulted in the decrease in the percentage of IL1R1+ cells. Thus, tumor cells actively shift CAFs towards the myCAF phenotype that are a major source of collagens. Interestingly, co-culturing with fibroblasts resulted in an increase in EMT marker levels without the loss of epithelial identity, suggesting a pEMT phenotype, but with no invasion. pEMT with cellular migration was only induced when changing extracellular matrix to collagen-I. This resulted in a loss of organoid circularity and CRC organoids acquired an invasive phenotype. Furthermore, modifying the collagen-based ECM by including other proteins had only a limited effect on the PDO phenotype.

Tumor cells with the most metastatic capacity reside in a pEMT state that can be characterized by the co-presence of mesenchymal and epithelial markers. Thus, this malignant cell population should be a primary target of therapies. Fibroblasts induce pEMT in CRC cells, however, this is coupled with an invasive phenotype only in a permissive ECM niche, such as the presence of collagen-I. These results highlight the importance of targeting ECM components in CRC. Funding: OTKA137554 (National Research, Development and Innovation Office, Hungary), TKP2021-EGA-24 (Ministry of Innovation and Technology of Hungary). Ethical permission: TUKEB 2015, 51323-4/2015/EKU.