The Inhibition of Platelets Restore Anti-Tumor Immune Response to Ovarian Cancer and Its Therapeutic Implication

The interactions between platelets and cancer cells activate platelets and enhance tumor growth. The ability of a cancer cell line to activate platelets in vitro, tumor cell-induced platelet aggregation (TCIPA), predicts the in vivo aggressiveness of that particular cancer cell line. We have found that platelets extravasate into tumor microenvironment. We also have shown that ovarian cancer cells secret ADP that activate platelets by binding to P2Y12 (an ADP receptor on platelets). In turn, activated platelets enhance proliferation of ovarian cancer cells and tumor growth. Activated platelets release TGFβ to the tumor microenvironment (TME) that enhances cancer cell proliferation in a TGFβ dose-dependent manner. Platelet TGFβ also functions as an immune modulator by enhancing differentiation of T cells toward T-reg. Platelets not only increase cancer cell proliferation but also alter the immune response to cancer by suppressing the function of tumor-infiltrating T cells. The presence of tumor-infiltrating lymphocytes (TIL) is associated with a significant improvement in the progression-free survival (PFS) and the overall survival (OS) of patients with ovarian cancer. In the current study, we investigated the effect of platelet inhibition on promoting the immune response to tumors and the therapeutic benefit of combining anti-platelet reagents with checkpoint inhibitors (CPIs) in murine models of ovarian cancer.

We have shown before, P2Y12 deficient (P2Y12^-/-) and platelet specific TGFβ knockout (TGFβfl/fl;PF4-cre) mice developed smaller tumors compared to control tumor-bearing mice in a murine ovarian cancer model. To determine the effect of anti-platelet reagent in platelet extravasation into the TME, we counted the number of platelets in the TME in four groups of tumor-bearing mice: control, aspirin-treated (ASA), platelet infused (Plt) and aspirin-treated + platelet-infused (Asp Plt) mice. Platelet extravasation increased after platelet infusion and reduced after aspirin treatment. The groups with a higher number of extravasated platelets developed larger tumors compared to those with less extravasated platelets.

We investigated the role of platelet in regulation of immune response to tumor using mice with platelet specific defects in murine models of ovarian cancer. We immune profiled tumors induced in mice with platelet-specific TGFβ deficiency, P2Y12 deficient mice, and aspirin or Ticagrelor-treated mice. Overall platelet functional defects was associated with an increase in the number of T cells, DC, MØ and NK and a decrease in the number of MDSCs in tumors..

We investigated the effect of immune check point inhibitors (CPI) on growth of murine ovarian cancer. We administrated 200µg of combination of CPIs (anti-CTLA4/ anti-PD-L1) into tumor-bearing mice. Combined CPI therapy had a minimal effect on the tumor burden (control=0.53± 0.082g vs. CPIs=0.33± 0.04g, p=0.15). Although CPI-treatment increased the number of CD8 cells inside tumors, it also increased the expression of VISTA (a negative) with an overall non-significant effect on the tumor growth. To investigate the effect of platelet inhibition on expression of checkpoint regulatory proteins in the TME, the expression of VISTA was examined in the tumors resected from TGFβfl/fl; PF4-cre tumor-bearing mice by quantitative RT-PCR (qRT-PCR), and was compared to that in tumors resected from WT tumor bearing mice. Tumors from TGFβfl/fl; PF4-cre mice expressed less VISTA mRNA (control= 1 vs. TGFβfl/fl; PF4-cre= 0.04 ± 0.01, p < 0.0001). Interestingly, P2Y12 deficiency and antiplatelet reagents (ticagrelor and aspirin) also reduced expression of VISTA in tumors. This is the first evidence for the effect of platelet inhibition on the expression of a checkpoint regulator in tumors.

We hypothesize that platelet inhibition enhances anti-tumor immune response and can be used as an adjuvant to checkpoint inhibitors in immunotherapies for ovarian cancer.