Second-degree labeling via surface interactions reveals short range contacts between CSC, their tEVs^CSC and MHC-II–Mac, PD-1+ T cells in the CSC niche.

(Ai) Illustrative schemes of SrtA enzymatic activity. SrtA present on the outer membranes of CSC and tEVsCSC catalyzes the transfer of the reporter protein mSca on CSC niche cells. SrtA recognizes the sequence LPETGG. (Aii) Scheme summarizing experimental design to uncover the immune component in the CSC niche. Since only CSC and tEVsCSC express SrtA, this strategy allows to label host cells with mSca reporter based on their proximity to CSC and their EVs. (B) Overlaid plot representation of different cell populations present in tumors from mice bearing genetically modified mEER cells analyzed by flow cytometry. Two plots from n = 4 are presented for each group. (C) Representative graphs showing the % of CD63-eGFP+ immune cells (that is, immune cells binding to tEVs) in analyzed tumors for each of the analyzed subsets with respect to all CD63-eGFP+ CD45+ immune cells. (D) Representative graphs showing the % of mSca+ immune cells (that is, immune cells in the proximity of CSC and their tEVsCSC) for each of the analyzed subsets with respect to all mSca+ CD45+ immune cells. (E) Summary graph presenting the % of immune cell subsets presenting CD63-eGFP+ and mSca+ fluorescence in tumors carrying modified mEER ALDH1A1:SrtA/mSca-LPETGG:BDP:CD63-eGFP tumor cells test. Percentage of CD63-eGFP+ cells for each immune subset represents the % of cells that had interacted with tEVs. mSca+ cells for each immune subset represent the % of cells within the CSC niche. (F) CSC niche-specificity index indicating the % from tEVstotal-immune cell subsets interactions that correspond to specific CSC niche-immune cell subsets interactions. Two-way ANOVA Holm-Sidak´s multiple comparison test and multiple t tests were used for statistical analysis. BDP: Bi-directional promoter.