Oncolytic Reovirus and Immune Checkpoint Inhibition as a Novel Immunotherapeutic Strategy for Breast Cancer

Abstract

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8⁺ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials.

Keywords: PD-1; breast cancer; immune checkpoint inhibition; immunotherapy; oncolytic viruses; reovirus.

Figure 1

Reovirus has both direct oncolytic effects and induces an inflammatory immune response in breast cancer cells. (A) ED₅₀ of established human and murine breast cancer cell lines infected with serial dilutions of reovirus (RV) multiplicity of infection (MOI) and incubated for 48 h. Cytotoxicity was detected by measuring mitochondrial NADPH dehydrogenase using a (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST) assay. N = 3 per group. (B) EMT6 cells infected with ED₅₀ (7.37 MOI) of UV-irradiated dead reovirus (DV) or live reovirus (LV) for 48 h, taken with a Zeiss Axiovert 200M microscope at 10× zoom. Scale bar = 50 μm. (C) EMT6 cells +/− ED 50 (7.37 MOI) of DV or LV and incubated for 24 h. Chemokine and cytokine levels in supernatants from EMT6 cells were determined by luminex analysis. N = 3 per group. (D) Dendritic cell or (E) Lymphocyte migration in response to cytokine secretion from EMT6 infected or not by RV using of a Transwell^® migration assay. N = 4 per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = standard error of the mean (SEM) of three independent experiments.

Figure 2

Reovirus modulates PD-L1 expression on breast cancer cell lines. Human (A) MDA-MB-468 (B) Hs 578T and murine (C) 4T1 (D) EMT6 breast cancer cell lines were either treated with ED₅₀ RV +/− IFN-γ or DV +/− IFN-γ. Expression of surface PD-L1 was analyzed via surface flow cytometry. N = 3 per group.(E) EMT6 or (F) 4T1 cells were incubated with UV-inactivated supernatant from 4T1 or EMT6 cells, respectively, previously treated with RV or DV for 24 h. PDL-1 expression was analyzed by surface flow cytometry. N = 3 per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = SEM of three independent experiments.

Figure 3

Reovirus combined with PD-1 inhibition results in decreased tumor burden and improved overall survival in the EMT6 murine model. Balb/C mice were implanted with EMT6 (2 × 10⁵ cells) into the right mammary fat pad and treated with phosphate buffered saline (PBS), anti-PD-1 antibody (200 ug i.p.), RV (5 × 10⁸ PFU i.t.) or a combination of these agents. RV was administered four times (days 6, 9, 12 and 14) following tumor implantation and anti-PD-1 antibody was given six times (days 14. 17, 20, 23, 26 and 29). (A) Tumor size was followed with caliper measurements every three days starting from day 9. PBS N = 15, RV N = 13, PD-1 N = 13, RV + PD-1 N = 14. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by two-way. Error bars = SEM of replicates within each group. (B) Kaplan–Meier survival plot of mice in each treatment group. PBS N = 10, RV N = 8, PD-1 N = 8, RV + PD-1 N = 9. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by log rank test. Error bars = SEM of replicates within each group.

Figure 4

Reovirus combined with PD-1 inhibition enhances splenic immune stimulatory cells while preventing accumulation of tumor immune suppressor cells. Pooled splenocytes (A–D) and tumor single-cell suspensions (E) from EMT6 tumor–bearing mice treated as per Figure 2A were immunophenotyped by flow cytometry. (A) CD4⁺ T cells, (B) CD8⁺ T cells, (C) Effector CD4⁺ memory T cells, (D) Effector CD8⁺ memory T cells, (E) T-regulatory cells. N = 5 mice per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = SEM of experimental replicates. Source of cells indicated in parentheses. CD8⁺ cells were separated from pooled spleens of EMT6 tumor-bearing mice treated as per Figure 2A and stimulated with EMT6 cells. (F) Percentage of EMT6-specific IFN⁺ cells determined by ELISPOT assay and (G) representative quantification. N = 3 mice per group. *** p ≤ 0.001 by one-way ANOVA. Error bars = SEM of experimental replicates.

Figure 5

Reovirus combined with PD-1 inhibition significantly enhances IFN-γ, TNF-α and IL-2 production by CD4 and CD8 T cells. Splenocytes were stimulated with Ionomycin for 12 h with Brefeldin A added in the last two hours. Surface and intracellular flow cytometric analysis were performed and stained for markers specific for CD4 T cells (A,C,E) and CD8 T cells (B,D,F). Cytokine secretion for each population was then analyzed [IFN-γ (A/B), TNF-α (C/D) and IL-2 (E/F)]. N = 5 per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = SEM of experimental replicates.

Figure 6

Survival advantage of combination therapy relies on presence of CD8⁺ T cells. (A,B) Kaplan–Meier plot demonstrating overall survival (OS )for mice pretreated with depleting CD8a (A) or CD4a (B) antibodies (i.p.) followed by treatment as in Figure 3A,B. ** p ≤ 0.01 and * p ≤ 0.05 by log-rank test. N = 5 mice. Results from Figure 3B included in panel A,B as reference. (C,D) Cohorts of pretreated mice demonstrating cure (Combination: N = 6, RV: N = 3) and a cohort of treatment-naïve mice (N = 5) were challenged with EMT6 (1 × 10⁵ cells) into the opposite (left) mammary fat pad as initial tumor inoculation. (C) Tumor size was followed with caliper measurements every three days starting from day 9. (D) Kaplan–Meier survival plot of mice in each treatment group. ***p ≤ 0.001. Error Bars = SEM of experimental replicates.