Optimized fractionated radiotherapy with anti-PD-L1 and anti-TIGIT: a promising new combination

Abstract

Purpose/objective: Radiotherapy (RT) induces an immunogenic antitumor response, but also some immunosuppressive barriers. It remains unclear how different fractionation protocols can modulate the immune microenvironment. Clinical studies are ongoing to evaluate immune checkpoint inhibitors (ICI) in association with RT. However, only few trials aim to optimize the RT fractionation to improve efficacy of these associations. Here we sought to characterize the effect of different fractionation protocols on immune response with a view to associating them with ICI.

Materials/methods: Mice bearing subcutaneous CT26 colon tumors were irradiated using a SARRP device according to different radiation schemes with a same biologically effective dose. Mice were monitored for tumor growth. The radiation immune response (lymphoid, myeloid cells, lymphoid cytokines and immune checkpoint targets) was monitored by flow cytometry at different timepoints after treatment and by RNA sequencing analysis (RNAseq). The same radiation protocols were performed with and without inhibitors of immune checkpoints modulated by RT.

Results: In the absence of ICI, we showed that 18x2Gy and 3x8Gy induced the longest tumor growth delay compared to 1×16.4Gy. While 3x8Gy and 1×16.4Gy induced a lymphoid response (CD8⁺ T-cells, Regulators T-cells), 18x2Gy induced a myeloid response (myeloid-derived suppressor cells, tumor-associated macrophages 2). The secretion of granzyme B by CD8⁺ T cells was increased to a greater extent with 3x8Gy. The expression of PD-L1 by tumor cells was moderately increased by RT, but most durably with 18x2Gy. T cell immunoreceptor with Ig and ITIM domains (TIGIT) expression by CD8⁺ T-cells was increased with 3x8Gy, but decreased with 18x2Gy. These results were also observed with RNAseq. RT was dramatically more effective with 3x8Gy compared to all the other treatments schemes when associated with anti-TIGIT and anti-PD-L1 (9/10 mice in complete response). The association of anti-PD-L1 and RT was also effective in the 18x2Gy group (8/12 mice in complete response).

Conclusion: Each fractionation scheme induced different lymphoid and myeloid responses as well as various modulations of PD-L1 and TIGIT expression. Furthermore, 3x8Gy was the most effective protocol when associated with anti-PD-L1 and anti-TIGIT. This is the first study combining RT and anti-TIGIT with promising results; further studies are warranted.

Keywords: Colorectal cancer; Immune response; Immunotherapy; Radiotherapy fractionation.

Fig. 1

Effect of fractionation of RT on CT26 tumors grafted onto immunodepressed (a, b) or immunocompetent (c, d) mice. Growth of irradiated tumors in immunodeficient BALB/C nude mice (a) (n = 6 mice per group) or immunocompetent BALB/C mice (c) (n = 10–12 mice per group) treated with: 0Gy (black), 1×16.4Gy (red), 3x8Gy (blue), 18x2Gy (purple). The averages are expressed ± SEM The average time for the tumor volume to reach 1500 mm³ in each group is shown for immunodepressed mice (b) or immunocompetent mice (d). Not significant (NS); *p < 0.05; **p < 0.01. Non-parametric Mann-Whitney test was used

Fig. 2

Immunomonitoring of lymphoid cells and myeloid cells after radiotherapy. Ten days after the injection of CT26 colon murine cancer, mice were assigned in 4 groups: control (at day 7), 1×16.4Gy (red), 3x8Gy (blue), 18x2Gy (purple) (a). Seven, 14 and 30 days after the beginning of RT, flow cytometry monitoring (FCM) was performed on dissociated tumors. Lymphoid panel analysis (b) including: T-cells, CD8⁺ T cells, CD4⁺ T cells, Treg T cells, CD8⁺ T cells/CD4⁺ T cells ratio, CD8⁺ granzyme⁺ (grz). Myeloid panel analysis (c) including: myeloid cells, myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM) 2, TAM 1, TAM1/TAM2 ratio. All data are shown with box and whiskers with min to max values obtained from 8 independent samples per point (duplicate, n = 8 per condition). *p < 0.05. Non-parametric Mann-Whitney test was used

Fig. 3

Heatmaps showing differentially expressed genes at day 7 after the end of treatment tumors (CT26 model) between at least one condition and control group. Illustration of gene expression with s-value < 0.005 and absolute shrink lock-fold change threshold of one (Z-score): control (black), 1×16.4Gy (red), 3x8Gy (blue), 18x2Gy (purple). Experimental groups contained 4 mice per condition

Fig. 4

Efficacy evaluation of immunotherapy (anti-PD-L1 and/or anti-TIGIT) and different fractionation schemes of radiotherapy (RT) in CT26 model. Induction of the expression of PD-L1 (cd274 gene) (a) or TIGIT (b) using RNA sequencing analysis (left) (7 days after the beginning of RT and 7 days after the end of RT for the 18x2Gy scheme) and flow cytometry monitoring (FCM) (right) (7, 14 days after the beginning of RT and 7 days after the end of RT (day 30) for the 18x2Gy scheme): control (black), 1×16.4Gy (red), 3x8Gy (blue), 18x2Gy (purple). Growth of irradiated tumors in mice treated with 0Gy, 1×16.4Gy, 3x8Gy, 18x2Gy with IgG or anti-PD-L1 and/or anti-TIGIT (c). Complete response (CR) ratio indicates the number of mice free from the irradiated tumor. Mean ± SEM for 18x2Gy (purple) and 3x8Gy (blue) are shown at the bottom of the Fig. X axes express the number of days since the beginning of RT. Y axes express the tumor volume (mm³). Experimental groups contained at least 8 mice per group. Not significant (NS); *p < 0.05; **p < 0.01, ***p < 0.001. Non-parametric Mann-Whitney test was used

Fig. 5

Survival curves after immunotherapy (anti-PD-L1 and/or anti-TIGIT) and fractionated radiotherapy (RT) in CT26 model. Survival curves of mice treated with 3x8Gy (a), 18x2Gy (b) with IgG or anti-PD-L1 and/or anti-TIGIT. X axes express the number of days since the beginning of RT. Y axes express the percentage survival of mice in each group. Experimental groups contained at least 10 mice per group. Log-rank test was used