IGF2 Promotes Colorectal Cancer Progression And Up-regulates PD-L1 On Tumor Cells Under Hypoxia

Objective To explore the role of IGF2 signaling in colorectal cancer and its role on antitumor immune response,and to provide reference and theoretical basis for IGF2 to become a possible target for clinical treatment of colorectal cancer.Methods 1.Immunohistochemical staining and database analysis The expression of IGF2 in tissue array consisting 101 samples of colorectal tumor tissues and para-tumor tissues was detected by immunohistochemistry and double-blind interpretated by two pathologists to explore the expression level of IGF2 in colorectal cancer patients.Influence of IGF2 on survival of colorectal cancer patients were analyzed(data were downloaded from GSE24551).2.Subcutaneous tumor model and flow cytometry IGF2 overexpressed cell lines,including CT26-IGF2 and MC38-IGF2,and coordinate control cell lines,including CT26-Con and MC38-Con were established via lentivirus.The overexpression efficacy was verified by real-time q PCR and ELISA.Then,tumor cells were injected subcutaneously into immunecompetent BALB/c or C57BL6 mice.The tumor volume was measured with an electronic caliper every 2–3 days in two dimensions(length and width).The infiltration of CD3+,CD4+ and CD8+ T cells in tumor tissues and the expression of functional molecules IFN-γ and TNF-α,as well as the infiltration of inhibitory cells MDSC,Treg and TAMS and the expression of immune checkpoint PDL1 were determined by flow cytometry on day 13/15 post tumor challenge.3.Recombinant mouse IGF2 protein was used to treat tumor cells Wild type cell line MC38 were cultured in vitro and stimulated with 500ng/ml recombinant mouse IGF2 protein.The expression level of PD-L1 on tumor cells was detected by quantitative Real-time PCR and Western blot.4.Treatment of tumor cells with recombinant mouse IGF2 protein under hypoxia PD-L1 and HIF-1α expression level was detected under hypoxia after stimulation of wild-type cell lines by IGF2 recombinant protein.Results 1.IGF2 was highly expressed in human colorectal cancer and was associated with prognosis Microarray data showed that IGF2 expression was higher in human colorectal cancer tissues compared with para-carcinoma tissues.Higher IGF2 expression correlated with poorer prognosis of patients with colorectal cancer.2.IGF2 promoted the progression of colorectal cancer by inhibiting antitumor immune response IGF2 overexpression of cell lines could promote the progression of colorectal cancer.The infiltration CD3+T cells,CD8+T cells and expression of functional cytokine of CD8+T cells in the tumor tissue of CT26-IGF2 group were decreased.PD-L1 expression on tumor cells was increased in CT26-IGF2 group.Suggesting that IGF2 could hamper anti-tumor immunity and promote the progression of colorectal cancer through the PD-1/PD-L1 pathway.3.IGF2 synergized with hypoxia up-regulated the expression level of PD-L1 on tumor cells The expression level of PD-L1 on IGF2 overexpressed cells and wild-type tumor cells treating with mouse recombinant IGF2 protein did not change significantly.Under hypoxia,flow cytometry and PCR analysis showed that the expression level of PD-L1 was up-regulated on wild-type tumor cells treating with mouse recombinant IGF2 protein and IGF2-overexpressed cell lines.4.HIF-1α pathway was potentially involved in IGF2-driven PD-L1 expression MC38 wild-type tumor cells treated with recombinant mouse IGF2 protein and cultured in hypoxia condition,It was found that the expression level of PD-L1 was upregulated simultaneously,and the expression level of hypoxia inducible factor-Ⅰ(HIF-1α)was also upregulated statistically.Conclusions 1.The expression of IGF2 was high in colon cancer samples,and its expression level was closely related to the prognosis of colorectal cancer.Patients with high IGF2 expression had a worse prognosis.2.IGF2 promotes the progression of colorectal cancer by inhibiting the infiltration and function of CD8+T cells and inhibiting the anti-tumor immune response.3.IGF2 up-regulated PD-L1 expression under hypoxia context.