Fig. 2. CAFs in the murine TME are CD73^hi cells with a superior capacity for ADO generation than TILs.

a EG7 tumors established s.c. in C57BL/6 WT mice for 15 days were used for assessment of cellular constituents of the EG7 TME via FACS as CD11b⁺ myeloid cells, CD45⁺CD11b⁻ EG7-tumors (large) and lymphocytes (small cells), and CD45⁻stroma. CD45⁻ cells were further defined as GP38⁺CD31⁻ CAFs and GP38⁻CD31⁺ BECs. CD73 expression in each cellular subset in the TME was examined (red histogram) as compared with the isotype control for each population (gray). b Relative percentage of each cellular subset within the TME determined via FACS was summarized. n = 6. c IF staining was employed to examine CD73⁺ cell (red) bio-distribution in correlation with ER-TR7⁺ (green) fibroblastic stroma and DAPI nuclear counterstain (blue). Co-localization of CD73^high and ER-TR7⁺ cells was illustrated via merging the images. d The relative area occupied by CD3⁺ T, CD11b⁺ myeloid cells, and ER-TR7⁺ CAFs (red) in the EG7 TME was determined via a computer-assisted software following masking it to white/gray signals and calculated as the percentage of total area. e CD11b⁺, CD3⁺ cells, and CAFs were purified from the EG7 TME via FACSort. Their capacity for ADO generation within 2 h in the presence of 200 μM AMP was compared. f Purified CD8 T cells were activated by α-CD3/α-CD28 beads in CAF-conditioned medium (CAF-CM), pre-incubated with or without 200 μM AMP. Changes in the cell surface expression of CD69 and CD25 were examined and compared with the 100 μM ADO control via FACS 15 h post-activation. The viable CD69⁺CD25⁺CD8⁺ T cells were calculated and summarized to the right. Error bars depict mean ± SEM. p values were determined via two-tail unpaired Student’s t-test. All experiments were repeated at least two times. Source data are provided in the Source Data file.