Fig. 4

PIWIL1 overexpression-induced MDSCs in the tumor microenvironment of HCC. The tumor and surrounding hepatic tissues (within 5 mm from the tumor) of orthotopic HCC mice were collected. The population of analysis of MDSCs was analyzed by flow cytometry; PIWIL1 overexpression significantly accumulated the PMN-MDSCs in the surrounding hepatic tissues (a), while the M-MDSCs population was suppressed (b); We then used anti-Ly6G to deplete the PMN-MDSCs population. Intraperitoneal injection of anti-Ly6G antibody (200 mg/kg, every 4 days) successfully suppressed the PMN-MDSCs population (c) and attenuated the PIWIL1-induced HCC growth (d) and tumor size (e); f EdU was intraperitoneally injected into the mice (10 mg/kg). Five hours after injection, mice were sacrificed and the EdU-incorporated PMN-MDSCs were analyzed by flow cytometry. Increased EdU incorporation was observed in PMN-MDSCs of PIWIL1-overexpressing orthotopic tumor; g BMDMs were isolated and stained by PKH26PCL. The PKH26PCL-positive BMDMs were then intraperitoneally injected (1 × 10⁷ cells/mice) 48 h before sacrifice. The infiltration of PKH26PCL-positive cells was significantly increased in the hepatic tissues surrounding PIWIL1-overexpressing HCC; the PMN-MDSCs in the hepatic tissues surrounding wild type or PIWIL1-overexpressing HCC were sorted and co-cultured with simulated CD8 + cytotoxic T cells. PMN-MDSCs from PIWIL1-overexpressing HCC had a more potent ability in inhibiting the expression h Ki67, a marker of cell proliferation, and i Granzyme B (GranB), a marker of activation of co-cultured stimulated CD8 + cytotoxic T cells. All experiments were performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001