Figure 2.

Senescent tumor cells exclude CD8⁺ T cells. A) Ex vivo culture. p16^INK4A positive or negative CRC tissues were cocultured with the GFP lentivirus infected isolated primary CD8⁺ T cells for 24 h and then stained with p16^INK4A, CD8, and GFP. The number of GFP positive cells was counted and presented as a bar graph. B) Senescent tumor cells inhibited CD8⁺ T cell migration. SW480 cells were treated with H₂O₂ (200 × 10⁻⁶ m) for 3 days and analyzed for SA‐β‐Gal expression (upper panel) and SASP expression (middle panel). T cell migration assay. Isolated primary CD8⁺ T cells were cocultured with SW480 (control or H₂O₂ treated) for 3 h, and the number of migrated CD8⁺ T cells was counted. C) Microdissection analysis. CRC tissues were serially dissected and stained with p16^INK4A and toluidine blue. p16^INK4A positive and negative regions were microdissected and then analyzed for mRNA expression (N = 5, upper panel). The expression in RNA sequencing indicates the relative values of the p16^INK4A positive region compared with those of the p16^INK4A negative region. D) CXCL12 expression in p16^INK4A expressing senescent tumor cells. p16^INK4A positive and negative CRC tissues were serially dissected and stained with p16^INK4A and CXCL12 antibody, respectively. “1” and “2” indicate the high magnification views of the original figure. Results are presented as mean ± SD. The p value was calculated by the Mann–Whitney U test A) or Kruskall–Wallis test B) or χ ² test D). N and n indicated the number of cases and independent experiments, respectively.