Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer

Abstract

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

Keywords: Cancer; Dendritic cell-based immunotherapy; HIF-1α; Hypoxia.

Fig. 1

Effect of HIF-1α inhibition on the expression of HIF-1 downstream genes. BALB/c mice bearing 4T1 tumors were left untreated or administered PX-478 (i.p at a dose of 40 mg/kg, every other day for 2 weeks). Tumors and lymph nodes were excised on day 12 after initiating treatment. a HIF-1α, VEGF, and Foxp3 transcript levels in tumors and lymph nodes were measured by real-time RT-PCR, calculated relative to housekeeping gene β-actin. Data are expressed as the mean ± SEM of four mice per group. *p < 0.05 by Mann–Whitney U test. b HIF-1α and Foxp3 protein synthesis of tumors was determined by western blotting as described in Materials and Methods. β-actin was used as a loading control. c Immunohistochemical analyses of representative sections obtained from untreated and PX-478 treated mice stained with an antibody directed against mouse HIF-1α (inset, negative staining control)

Fig. 2

Delay of tumor growth and enhanced survival in mice treated with DC and PX-478. a schematic of the experimental design followed to evaluate the effects of HIF-1α inhibition combined with vaccination in the 4T1 breast cancer model. BALB/c mice bearing 4T1 tumors were left untreated or administered PX-478 (as described), DC vaccine (10⁶ cells, subcutaneously around the tumor site, day 7 of tumor inoculation), or PX-478 plus vaccine, with b tumor size (mean ± SEM) reported in mm³ followed for up to 25 days (left) and tumor growth rate reported in mm³ per 48 h (right). *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA). c Survival of the animals in each group was monitored and the respective Kaplan–Maier curves are given; (n = 6 mice for control and combination therapy group, n = 5 for single therapies)

Fig. 3

DC vaccination accompanied by HIF-1α blockade enhances T cell effector functions. The ability of CD8⁺ T cells to recognize 4T1 tumor cells evaluated by a GrB production (ELISA) and b CD107 translocation assay (described in Materials and Methods). c 2 × 10⁶ CFSE-labeled splenocytes isolated from tumor-bearing mice were treated in vitro with tumor lysate in CM (80 μg/ml, 5 days) and the proliferation ability of CD3 stained cells (reported as division index) was evaluated by flow cytometry. d VEGF production measured in the supernatant of antigen stimulated splenocytes (four mice per group) by ELISA. e Splenic T cells isolated from tumor-bearing mice were cocultured with CFSE-labeled splenic T cells from a tumor antigen immunized mouse and tumor antigen pulsed or un-pulsed DCs. Reduction in proliferative activity indicated by CFSE dilution, was considered as suppressor activity of splenic T cells. A representative out of three independent experiments is depicted;*p < 0.05; **p < 0.01 and ***p < 0.001; n = 4

Fig. 4

Impact of HIF-1α inhibition in combination with DC vaccine on T helper cell subsets. IFN-γ, IL10 and IL-17 production measured in the supernatant of tumor lysate antigen stimulated splenocytes (80 µg/ml) by ELISA. A representative out of three independent experiments is depicted; *p < 0.05; **p < 0.01 and ***p < 0.001; n = 4