New Mechanism in Anti Tumor Activity of Poly (I:C): Modulation of Tumor-Associated Myeloid Derived Suppressor Cells in Murine Model of Breast Cancer

Abstract

Background: Polyinosinic- polycytidilic acid {Poly (I:C)} a ligand for TLR-3, has potents effects on activating CD8+ T cells and NK cells and has been used as an immunotherapy adjuvant. Although it is thought TLR-3 signaling is crucial for enhancing cell-mediated immune responses, the effect of Poly (I:C) on immunosuppressive Myeloid derived suppressor cells (MDSC) in tumor-bearing animal is not known. MDSCs are one of the main immunosuppressive factors in cancer, and abnormal accumulation of MDSCs an important mechanism of tumor immune evasion. Here, we describe a novel mechanism for the immuno-stimulatory activity of Poly (I:C): a dramatic reduction of absolute numbers of tumor-infiltrating MDSCs and reduced immunosuppressive function of MDSCs.Methods: 4T1-luciferase tumor cells were implanted into mammary glands of female BALB/c mice (1´106 cells per mouse, n = 5/group). Poly (I:C) was administrated on day 7th (200 mg/mouse) and day 10th (100 mg/mouse) after tumor inoculation (D0), continued with one boost dose. Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Mice were euthanized when tumors had reached to 100-200 mm3. Flow cytometry measured CD11b+ Gr-1+ MDSC, and T-cells in the tumors of tumor-bearing mice.Results: Treatment with Poly I:C increased overall survival accompanied by a reduction in tumor burden as measured by total flux/s (p =0.0217) and numbers of MDSCs in ressected tumor samples (p=0.0286). 4T1 tumors grew more slowly in Poly (I:C) treated mice vs PBS-treated group with the lower numbers of CD11b+Gr-1+ cells in Poly (I:C)- treated animals (Figure1). Both granulocytic and monocytic subsets of tumor infiltrating MDSCs (CD11b+ Gr-1+, and Ly6G+/hi or Ly6C+/hi) and CD11b+ Gr-1+ F4/80+ macrophages were significantly reduced of multiple time points after Poly (I:C) treatment compared with control mice. The decrease in MDSC subsets was paralleled with a significant increase in tumor –infiltrating T cells (TIL)s and decreased numbers of CD4+ CD25+ Foxp3+ T regulatory in the tumor. Moreover, Poly (I:C) treatment significantly increased the percentage of CD4+ and CD8+ TNF-a+ T cells in spleen of tumor-bearing mice after reactivation with anti CD3/CD28 antibody. Intracellular staining of pro inflammatory cytokines in splenocyte- derived MDSCs showed higher amount of TNF-a and IL-1b, 5 days after Poly (I:C) treatment indicating MDSCs differentiation into M1 macrophage. Purified splenocyte-derived MDSCs co-cultured with Poly (I:C) (20 mg/ml) showed significant increase in mean fluorescence index (MFI) for MHC class II and co-stimulatory molecules CD80 and CD86 supporting increase d effectiveness as APC.Discussion: These data demonstrate a new mechanism of Poly (I:C) that can lead to alterations in numbers and differentiation of MDSCs resulting in increasing local response of the immune system to tumor with concomitant decrease in tumor volume in a murine model of breast cancer. Also our data support the hypothesis that Poly (I:C) has global effects on the immunosuppressive network in the tumor micro-environment. Considering the role of MDSCs in cancer promotion and backed by data showing alteration in MDSCs quantity and function in 4T1-tumor-bearing hosts, Poly (I:C) treatment can effectively augment the activity of cancer immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.