Combination of OX40 Co-Stimulation, Radiotherapy, and PD-1 Inhibition in a Syngeneic Murine Triple-Negative Breast Cancer Model

Abstract

Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (α-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and α-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of α-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, α-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-γ, respectively. Together, our results suggest that the combination of α-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.

Keywords: OX40; PD-1; breast cancer; immuno-oncology; immunotherapy; radiotherapy; stereotactic body radiotherapy.

Figure 1

Combination of RT, α-OX40, and PD-1B increases primary and abscopal tumor control and prolongs survival. Primary tumor growth curves of subcutaneous (a) luciferase-tagged and (b) luciferase-untagged 4T1 implants in mice for each group: control, RT, α-OX40, PD-1B, α-OX40 + RT, PD-1B + RT, α-OX40 + PD-1B, and triple combination therapy (each group, n = 5). (c) Representative bioluminescence images of 4T1-luc tumor-bearing mice acquired before and after each treatment. Paired bar graphs comparing primary tumor volumes before and after treatment show total luminosity flux (emitted photons per second) for each group. (d) Images of extracted tumors from luciferase-untagged 4T1 tumor-bearing mice. (e) Secondary (abscopal) tumor growth curves in luciferase-untagged 4T1 tumor-bearing mice for each group (each group, n = 5). (f) Quantitation and representative bioluminescence images of 4T1-luc secondary tumor-bearing mice acquired before and after each treatment. (g) Metastatic lung nodules observed on post-implantation day 31. (h) Survival curves of 4T1-luc tumor-bearing mice for each treatment group (each group, n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations: 4T1-luc, luciferase-tagged 4T1; CON, control; RT, radiotherapy.

Figure 2

Immune cell profiles in the spleen. Representative flow cytometry plots for (a) CD4+ or CD8+ T cells, (b) CD4 + FOXP3+ Tregs, and (c) CD45 + CD103+ dendritic cells. (d–h) Cell population percentages calculated from flow cytometry analyses. ELISpot quantification of (i) IFN-γ, (j) IFN-β, and (k) TNF-α plasma levels. (l) Cytometric bead array results for IL-6 plasma levels. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations: CON, control; RT, radiotherapy; Treg, regulatory T cell; DC, dendritic cell; ns, not significant.

Figure 3

Immune cell profiles in the tumor microenvironment. Representative flow cytometry plots and immunohistochemistry images of (a) CD45+ tumor-infiltrating leukocytes, (b) CD4+ or CD8+ T cells, and (c) CD4 + FOXP3+ Tregs. (d–i) Cell population percentages calculated from flow cytometry analyses and immunohistochemistry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations: CON, control; RT, radiotherapy; Treg, regulatory T cell; ns, not significant.

Figure 4

Expression of exhaustion markers on CD8+ T cells in the tumor microenvironment. The percentage of CD8+ T cells expressing exhaustion markers (PD-1, CTLA-4, TIM-3, and LAG-3) was calculated by flow cytometry. Representative flow cytometry plots of (a) PD-1+ or PD-1- and (b) CTLA-4+ or CTLA-4- on CD8+ T cells. Results are presented as (c) percentage of T cells expressing PD-1 and/or CTLA-4, (d) MFI of TIM-3 in CD8+ T cells, and (e) MFI of LAG-3 in CD8+ T cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations: MFI, mean fluorescent intensity; CON, control; RT, radiotherapy; ns, not significant.

Figure 5

Proliferative T cell compartments in the tumor microenvironment. Representative flow cytometry plots of (a) CD8 + Ki67+ and (b) CD4 + Ki67+ T cells. The percentage of T cells expressing the proliferative marker Ki-67 in (c) CD8+ and (d) CD4+ T cell populations in the tumor microenvironment was calculated by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Abbreviations: CON, control; RT, radiotherapy; ns, not significant.