CD8(+) T cells cell-in-cell mediates colorectal cancer progression via P38 activation

Cell-in-cell(CIC) refers to the active internalization of one or more living cells into another. However, the biological significance and clinical implications of CIC between immune cells and cancer remain unclear. Here, we showed CIC by immunohistochemistry, real-time imaging and multiplex tissue immunofluorescence staining. CD8(+) T cell CIC counts were correlated with lymph node metastasis,T stage, and differentiation in stage I-III CRC. We found CIC counts >4.25 per high-power (magnification 400×) field positively affected metastasis-free survival in stage I-III CRC. Functional experiments demonstrated CD8(+) T CICs increased the migration and invasion in vitro and in vivo. Notably, CD8(+) T CICs were accompanied by greater activation of p38. In addition, blocking p38 using SB203580 significantly suppressed the migration and invasion of CD8(+) T CICs in vitro and in vivo. Moreover, phosphorylated-p38 overexpressing CD8(+) T CICs showed increased expression of cytokeratin 13 (KRT13); however, CD8(+) T CICs pretreated with SB203580 exhibited a significant suppression of KRT13. In summary, Our findings demonstrate that CD8(+) T CIC could be one of the mechanisms of CRC progression. P38 contributes to CD8(+) T “in-cell progression,” and KRT13 may participate in P38- induced “in-cell progression.” HCT8 cell lines stained with 2μM Green (Invitrogen) were incubated with a 2:1 ratio of live PBMCs stained with 1μM Cell-Tracker Red (Invitrogen) at 37˚C and 5% (v/v) CO2 for 8 hours. Subsequently, double-positive cells, representing HCT8 cells that had engulfed PBMCs, were sorted using flow cytometry and designated as CIC cells. The CIC cells were then treated with 10 μM SB203580 for 24 hours, resulting in the SB_CIC cells.