Tumor-derived colorectal cancer organoids induce a unique Treg cell population through direct modulation of CD4+ T cell differentiation.

To investigate the capacity for CRC tumor-organoids to influence Treg cell fate specification in vitro, we established a transwell co-culture model utilizing CD4+ T cells together with CRC tumor-organoids in which we study murine tumor-organoid induced Treg (mTO-iTreg) cell differentiation. As control, murine CD4+ T cells were cultured in transwell inserts and treated with TGFβ to induce Treg (TGFβ-iTreg) cells. To define the transcriptional identity of in vitro mTO-iTreg cells we performed bulk RNA-sequencing. RNA-sequencing analysis identified distinct transcriptional profiles between CRC organoid-induced Treg cells and TGFβ-induced Treg cells, with upregulation of key functional signature genes linked to CRC Treg cells in vivo. High expression of genes upregulated in CRC organoid-induced Treg cells correlates with shorter progression free intervaland overall survival of CRC patients, highlighting their prognostic potential. We then performed RNA sequencing of 3 different cells at different time points. Firstly, Foxp3 eGFP- CD4+ T cells (n=3) were isolated on day 0, prior to co-culture establishment; secondly, CD4+ Foxp3 eGFP+ TGFβ-iTreg cells (n=3) were isolated on day 5 of culture; and thirdly, CD4+ Foxp3 eGFP+ mTO-iTreg cells (n=3) were isolated from the co-culture on day 5. Diferential gene expression analysis of RNAseq data for Foxp3 eGFP- CD4+ T cells, CD4+ Foxp3 eGFP+ TGFβ-iTreg cells and CD4+ Foxp3 eGFP+ mTO-iTreg cells was performed.