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

In colorectal cancer (CRC), increased numbers of tumor-infiltrating CD4 ⁺ regulatory T (Treg) cells correlate with tumor development and immunotherapy failure, leading to poor prognosis. However, the molecular and cellular mechanisms governing Treg recruitment, expansion, or differentiation remain unclear. Here, we developed an in vitro co-culture system to assess the capacity of CRC tumors to directly modulate Treg cell differentiation. CD4 ⁺ T cells from Foxp3eGFP mice were co-cultured with murine tumor-derived CRC organoids, resulting in a significant increase in Treg cell numbers. This induction of Treg cells was not due to increased proliferation, but rather through differentiation of CD4 ⁺ T cells in a TGFβ-dependent manner. Human CRC tumor organoids similarly induced Treg cells that exhibited enhanced suppressive capacity compared to TGFβ-induced Treg cells. 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 interval and overall survival of CRC patients, highlighting their prognostic potential. Taken together, CRC tumor organoids drive CD4 ⁺ differentiation to Treg cells with a phenotype resembling tumor-infiltrating Treg cells. This model can be applied to both understand the molecular mechanisms by which tumors can directly modulate CD4 ⁺ T cell differentiation and identify approaches to disrupt Treg cell function and stimulate anti-tumor immunity.