Breast cancer stem cells convert anti-tumor CD4+ T cells to pro-tumor T regulatory cells: Potential role of exosomal FOXP3

Cancer progression and its treatment-response are regulated by the tumor microenvironment (TME). Tumor-initiating cancer stem cells (CSCs) remain in constant communication with the TME, and modulate it through several mechanisms. Here, from in-silico findings and analyzing breast cancer patient tissue-derived data, CSCs and Tregs were found to be positively correlated. Furthermore, our in-silico analyses highlighted a positive relationship between CSC genes and Treg signature marker, FOXP3, even in cancer cell lines that do not contain any T cell or Treg cells, thus raising the possibility of CSCs expressing FOXP3. Validating our hypothesis, higher expression of FOXP3, both at mRNA and protein levels, was observed in breast CSCs than non-stem cancer cells. Since a small population of CSCs initiate tumor in immune cell-dominated TME, we aimed at exploring whether breast CSCs directly transfer FOXP3 to CD4⁺T cells to generate immunosuppressive Treg cells. First, our search revealed CSC-derived exosomes (CDEs) generated CD4⁺CD25⁺FOXP3⁺ Tregs at an early time-point of 24 h, which were immunosuppressive in nature. Next, detecting presence of FOXP3 protein in CDEs showed a strong possibility of FOXP3 transfer through CDEs. This was supported by detecting elevated FOXP3 levels from 12 h in translation inhibitor-treated T cells upon CDE-exposure. Finally, exosomes derived from FOXP3 attenuated-CSCs furnished lower FOXP3 in T cells than control CDEs. This mechanism was validated in in-vivo murine model. Together these results indicate a hitherto unexplored role of CSC-derived FOXP3 in reprogramming T cells into immunosuppressive Treg cells.