Combination PD-1 blockade and irradiation of brain metastasis induces an effective abscopal effect in melanoma

Abstract

Nearly half of melanoma patients develop brain metastases during the course of their disease. Despite advances in both localized radiation and systemic immunotherapy, brain metastases remain difficult to treat, with most patients surviving less than 5 months from the time of diagnosis. While both treatment regimens have individually shown considerable promise in treating metastatic melanoma, there is interest in combining these strategies to take advantage of potential synergy. In order to study the ability of local radiation and anti-PD-1 immunotherapy to induce beneficial anti-tumor immune responses against distant, unirradiated tumors, we used two mouse models of metastatic melanoma in the brain, representing BRAF mutant and non-mutant tumors. Combination treatments produced a stronger systemic anti-tumor immune response than either treatment alone. This resulted in reduced tumor growth and larger numbers of activated, cytotoxic CD8⁺ T cells, even in the unirradiated tumor, indicative of an abscopal effect. The immune-mediated effects were present regardless of BRAF status. These data suggest that irradiation of brain metastases and anti-PD-1 immunotherapy together can induce abscopal anti-tumor responses that control both local and distant disease.

Keywords: Melanoma; antibody therapy; immunomodulation; immunotherapy; radiotherapy.

Figure 1.

Combination radiation and anti-PD-1 therapy induces an abscopal effect in contralateral flank tumors. A. 1.5 × 10⁵ B16-F10 tumor cells were injected in to each flank of wildtype C57BL/6 mice. Nine days later, mice received 150 µg anti-PD-1 antibody or IgG via intraperitoneal injection. After five days, indicated mice received 8 Gy in 4 fractions to one flank tumor. Growth of the irradiated (left) and non-irradiated contralateral tumor (right) was followed for 20 days. B. At the conclusion of the experiment, tumor tissues were harvested and digested to a single cell suspension with collagenase/hyaluronidase. T cell phenotype and frequency was determined using staining for readout by flow cytometry. Indicated study is representative of the independent experiments. *P < 0.05 for the indicated comparisons at day 20 in A. and between anti-PD-1 and anti-PD-1 + head irradiation groups in B.

Figure 2.

Irradiation and PD-1 blockade therapy induce an additive effect in a model of metastatic melanoma of the brain. A. Graphical representation of the experimental setup and treatment schedule for head irradiation experiments. B. Mice receiving concomitant D4M tumors implanted intracranial and in the flank were treated with IgG or anti-PD-1 antibody followed by four doses of 2 Gy irradiation to the head according to the experimental plan in A. Mice were then followed for 25 days post irradiation, and survival is indicated by the Kaplan-Meier plot. C. Growth in flank tumor size as measured by volume for the indicated groups is represented. On day 20, IVIS bioluminescence imaging of the animals was performed and graphs represent the intensity of luminescence of flank tumors (D.) and brain tumors (E.). Representative bioluminescence images at this time point. (F.) *p < 0.05 and **p < 0.01 for comparisons between the indicated groups.

Figure 3.

Abscopal effects induced by radiation and anti-PD1 combination therapy are associated with increased anti-tumor immunity. A. At the conclusion of the previous experiment, brain tissue was sectioned and T cell markers stained for immunofluorescence microscopy. B. Quantification of the number of cells in at least four independent fields for each treatment group. C. Flank tumor tissue was harvested and dissociated by collagenase/hyaluronidase digestion and immune cell markers were stained for readout and quantification by flow cytometry. Ki-67 expression was assessed on the indicated T cell subsets by permeabilization and staining followed by readout by flow cytometry. D-I. T cell subset percentages in non-irradiated flank tumor tissue were compared to total cell recovery to determine total number of T cells and ratio of CD8T cells to regulatory CD4T cells (Treg). CD4⁺/CD8⁺ percentages in D. and G. are of total CD45⁺ cells. J. CD8⁺ T cells from non-irradiated flank tumors were restimluated ex vivo with plate-bound anti-CD3 and anti-CD28 antibodies followed by intracellular cytokine staining for interferon-gamma (IFNγ) staining. K. Expression of the indicated markers of cytotoxic T cell function on CD8⁺ T cells from non-irradiated flank tumor tissue were stained following permeabilization and read-out by flow cytometry. *p ≤ 0.5 and **p ≤ 0.01 for indicated comparisons between anti-PD-1 and anti-PD-1 plus head irradiation treatment groups according to a student’s t test.

Figure 4.

Effect of combination BM irradiation and PD-1 treatment on regulatory immune cell populations in distant flank tumors. A. Tissue from flank D4M tumors were harvested at the conclusion of the experiment described in Figure 3, dissociated by collagenase/hyaluronidase digestion, and stained for regulatory T cell markers before being quantified by flow cytometry. Regulatory T cells were defined as CD25^hi Foxp3⁺ cells among CD3⁺ CD4⁺ T cells. B. Based on the percentages of the flow stain, the numbers of Treg per tumor and the ratio of these cells to the number of CD8⁺ T cells was determined (B, C). D. The percentage of myeloid derived suppressor cells (MDSCs) was determined in the tumors by anti-body staining and flow cytometry. Cells were gated on CD45⁺ Gr1⁺ CD11b⁺ populations. *p ≤ 0.5 for the indicated comparisons to the anti-PD-1 plus irradiation group.

Figure 5.

Anti-PD-1 treatment and irradiation induces an abscopal effect in B16 tumors. B16 tumors were established intracranially and subcutaneously in the flank similar to those using D4M cells. Brain (A.) and flank (B.) tumor growth was followed by bioluminescence imaging and graphs represent percent in growth. C. D. At the conclusion of the experiment, tumors were harvested and dissociated by collagenase/hyaluronidase digestion followed by staining for CD8⁺ T cell markers. Cells were then read-out and quantified by flow cytometry. *p < 0.5 and **p < 0.01 for the indicated comparisons between treatment groups. For the tumor growth graph. *p < 0.05 for the combination group vs. others at the final time point using ANOVA analysis.