Chemoradiation Increases PD-L1 Expression in Certain Melanoma and Glioblastoma Cells

Abstract

Immunotherapy approaches currently make their way into the clinics to improve the outcome of standard radiochemotherapy (RCT). The programed cell death receptor ligand 1 (PD-L1) is one possible target that, upon blockade, allows T cell-dependent antitumor immune responses to be executed. To date, it is unclear which RCT protocol and which fractionation scheme leads to increased PD-L1 expression and thereby renders blockade of this immune suppressive pathway reasonable. We therefore investigated the impact of radiotherapy (RT), chemotherapy (CT), and RCT on PD-L1 surface expression on tumor cells of tumor entities with differing somatic mutation prevalence. Murine melanoma (B16-F10), glioblastoma (GL261-luc2), and colorectal (CT26) tumor cells were treated with dacarbazine, temozolomide, and a combination of irinotecan, oxaliplatin, and fluorouracil, respectively. Additionally, they were irradiated with a single dose [10 Gray (Gy)] or hypo-fractionated (2 × 5 Gy), respectively, norm-fractionated (5 × 2 Gy) radiation protocols were used. PD-L1 surface and intracellular interferon (IFN)-gamma expression was measured by flow cytometry, and IL-6 release was determined by ELISA. Furthermore, tumor cell death was monitored by AnnexinV-FITC/7-AAD staining. For first in vivo analyses, the B16-F10 mouse melanoma model was chosen. In B16-F10 and GL261-luc2 cells, particularly norm-fractionated and hypo-fractionated radiation led to a significant increase of surface PD-L1, which could not be observed in CT26 cells. Furthermore, PD-L1 expression is more pronounced on vital tumor cells and goes along with increased levels of IFN-gamma in the tumor cells. In melanoma cells CT was the main trigger for IL-6 release, while in glioblastoma cells it was norm-fractionated RT. In vivo, fractionated RT only in combination with dacarbazine induced PD-L1 expression on melanoma cells. Our results suggest a tumor cell-mediated upregulation of PD-L1 expression following in particular chemoradiation that is not only dependent on the somatic mutation prevalence of the tumor entity.

Keywords: IFN-gamma; IL-6; PD-L1; checkpoint inhibitor; fractionated radiotherapy; glioblastoma; immunotherapy; melanoma.

Figure 1

Radiation and chemotherapy (CT) treatment scheme for the cell lines B16-F10 (A), GL261-luc2 (B), and CT26 (C). After seeding, cells rested overnight. B16-F10 cells were treated with a single dose of 250µM DTIC on day 1, GL261-luc2 cells with 20µM temozolomide every other day for 5 days, and CT26 cells with single doses of 10 µg/ml irinotecan, 10 µg/ml oxaliplatin, and 400 ng/ml 5-fluorouracil. After CT, cells of all cell lines were irradiated with either a single dose of 10 Gray (Gy) on day 5, 2 × 5 Gy on days 3 and 5 or 5 × 2 Gy. Cells were analyzed 24 or 48 h after the last treatment.

Figure 2

Cell death and programed cell death receptor ligand 1 (PD-L1) surface expression of B16-F10 melanoma cells after radiation and/or chemotherapy. The analyses were performed 24 and 48 h after single and multimodal treatments with the chemotherapeutic agent DTIC, differently fractionated radiotherapy, or radiochemotherapy. Cell death was determined by flow cytometry; vital cells (white) are defined as AxV⁻/7-AAD⁻, apoptotic cells (gray) as AxV⁻/7-AAD⁺, and necrotic ones (dark gray) as 7-AAD⁺ (A). PD-L1 surface expression was determined on vital (B) and apoptotic (C) cells by staining with anti-PD-L1 antibody and consecutive analysis by flow cytometry. DTIC was used at a concentration of 250 µM and recombinant murine interferon-gamma (0.5 ng/ml) served as a positive control (A–C). Joint data of three independent experiments, each performed in triplicates, are presented as mean ± SEM and analyzed by one-tailed Mann–Whitney U test as calculated via Graph Pad Prism. Each treatment was compared to the control (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3

Cell death and programed cell death receptor ligand 1 (PD-L1) surface expression of GL261-luc2 glioblastoma cells after radiation and/or chemotherapy. The analyses were performed 24 and 48 h after single and multimodal treatments with the chemotherapeutic agent temozolomide (TMZ), differently fractionated radiotherapy, or radiochemotherapy. Cell death was determined by flow cytometry; vital cells (white) are defined as AxV⁻/7-AAD⁻, apoptotic cells (gray) as AxV⁻/7-AAD⁺, and necrotic ones (dark gray) as 7-AAD⁺ (A). PD-L1 surface expression was determined on vital (B) and apoptotic (C) cells by staining with anti-PD-L1 antibody and consecutive analysis by flow cytometry. TMZ was used at a concentration of 20µM and recombinant murine interferon-gamma (0.5 ng/ml) served as a positive control (A–C). Joint data of three independent experiments, each performed in triplicates, are presented as mean ± SEM and analyzed by one-tailed Mann–Whitney U test as calculated via Graph Pad Prism. Each treatment was compared to the control (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4

Cell death and PD-L1 surface expression of CT26 colorectal cancer cells after radiation and/or chemotherapy (CT). The analyses were performed 24 and 48 h after single and multimodal treatments with CT consisting of 10 µg/ml irinotecan, 10 µg/ml oxaliplatin, and 400 ng/ml 5-fluorouracil, differently fractionated radiotherapy, or radiochemotherapy. Cell death was determined by flow cytometry; vital cells (white) are defined as AxV⁻/7-AAD⁻, apoptotic cells (gray) as AxV⁻/7-AAD⁺, and necrotic ones (dark gray) as 7-AAD⁺ (A). PD-L1 surface expression was determined on vital (B) and apoptotic (C) cells by staining with anti-PD-L1 antibody and consecutive analysis by flow cytometry. Recombinant murine interferon-gamma (0.5 ng/ml) served as a positive control (A–C). Joint data of three independent experiments, each performed in triplicates, are presented as mean ± SEM and analyzed by one-tailed Mann–Whitney U test as calculated via Graph Pad Prism. Each treatment was compared to the control (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5

Intracellular interferon (IFN)-gamma levels after norm-fractionated radiation and/or chemotherapy (CT). B16-F10 melanoma (A) and GL261-luc2 glioblastoma cells (B) were analyzed for intracellular IFN-gamma expression after treatment with either CT with DTIC or temozolomide, 5 × 2 Gy norm-fractionated radiotherapy, or chemoradiation. Data of two independent experiments, each performed in triplicates, are presented as mean ± SEM and analyzed by one-tailed Mann–Whitney U test as calculated in Graph Pad Prism. Each treatment was compared to the control (*p < 0.05; **p < 0.01).

Figure 6

IL-6 release after norm-fractionated radiation and/or chemotherapy (CT). Supernatants of B16-F10 melanoma (A) and GL261-luc2 glioblastoma cells (B) were analyzed for the concentration of IL-6 by ELISA after treatment with either CT with DTIC or temozolomide, 5 × 2 Gy norm-fractionated radiotherapy, or chemoradiation. Two independent experiments each conducted in technical duplicates were performed. Data are presented as mean ± SEM and analyzed by one-tailed Mann–Whitney U test as calculated in Graph Pad Prism. Each treatment was compared to the control (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 7

In vivo growth and PD-L1 surface expression of B16-F10 tumors after fractionated irradiation and in combination with DTIC treatment. Growth (A) and PD-L1 surface expression (B) of B16-F10 tumors in wild-type C57BL/6 mice are displayed. The tumors were initiated on day 0, left untreated or were locally irradiated on day 8, 9, and 10 with the clinically relevant dose of 2 Gray using a linear accelerator. An additional group of mice received DTIC (2 mg/mouse) 2 h after the irradiation at day 8 and 10. For determination of tumor growth (A) an electronic caliper was used (n ≥ 8 mice/group; data are presented as mean ± SEM). PD-L1 surface expression on the tumor cells (each dot represents the values obtained from an individual tumor of a single mouse; the mean value is displayed as line) (B) was analyzed by flow cytometry at day 13. Statistics was analyzed by one-tailed Mann–Whitney U test as calculated via Graph Pad Prism.