Gemcitabine-facilitated modulation of the tumor microenvironment and PD-1/PD-L1 blockade generate a synergistic antitumor effect in a murine hepatocellular carcinoma model

Gemcitabine can alter the immunogenic microenvironments, and the effect of gemcitabine plus programmed death-1 (PD-1)/ programmed death-ligand 1 (PD-L1) blockade in hepatocellular carcinoma (HCC) is investigated.Subcutaneous H22-green fluorescent protein (GFP) cells inoculation model was constructed and treated with gemcitabine, anti-PD-1 antibody (αPD-1), or the combination every four days when the tumor volumes reached about 50 mm3. Four days after the final treatment, primary tumor tissues were resected and dissociated, which were further subcutaneously injected on the contralateral side to construct the HCC relapse model. The infiltrated proportion of immune cells and PD-1 expression were quantified by flow cytometry. The relative content of transforming growth factor (TGF)-β, interleukin (IL)-12p70, and interferon (IFN)-γ were detected by the enzyme-linked immunosorbent assay (ELISA). Tumor volume and the number of tumor-free mice were evaluated.Gemcitabine treatment can effectively increase the total proportion of infiltrating immune cells, reduce the proportion of myeloid-derived suppressor cells (MDSCs) and macrophages, and increase T cells proportion without significant growth inhibition. While after gemcitabine treatment, PD-L1 expression on tumor cells and PD-1 on T cells were significantly up-regulated. Subcutaneous tumors volume were reduced considerably after gemcitabine plus αPD-1 treatment compared with gemcitabine (P<0.01) or αPD-1 monotherapy (P<0.001) with the increased proportion of IL-2+CD8+T, CD8+T central memory cells (TCM), CD4 TCM, up-regulated IL12p70 and IFN-γ secretion, and down-regulated TGF-β. Gemcitabine plus αPD-1 blockade could inhibit the relapse tumor model as indicated with down-regulated tumor volume and increased number of tumor-free mice.Gemcitabine up-regulates the proportion of intratumor CD8+T and the relative expression of PD-1/PD-L1, and the combination of PD-1/PD-L1 blockade can further inhibit the growth and the relapse of HCC.