FLASH radiation reprograms lipid metabolism and macrophage immunity and sensitizes medulloblastoma to CAR-T cell therapy

Abstract

FLASH radiotherapy holds promise for treating solid tumors given the potential lower toxicity in normal tissues but its therapeutic effects on tumor immunity remain largely unknown. Using a genetically engineered mouse model of medulloblastoma, we show that FLASH radiation stimulates proinflammatory polarization in tumor macrophages. Single-cell transcriptome analysis shows that FLASH proton beam radiation skews macrophages toward proinflammatory phenotypes and increases T cell infiltration. Furthermore, FLASH radiation reduces peroxisome proliferator-activated receptor-γ (PPARγ) and arginase 1 expression and inhibits immunosuppressive macrophage polarization under stimulus-inducible conditions. Mechanistically, FLASH radiation abrogates lipid oxidase expression and oxidized low-density lipid generation to reduce PPARγ activity, while standard radiation induces reactive oxygen species-dependent PPARγ activation in macrophages. Notably, FLASH radiotherapy improves infiltration and activation of chimeric antigen receptor (CAR) T cells and sensitizes medulloblastoma to GD2 CAR-T cell therapy. Thus, FLASH radiotherapy reprograms macrophage lipid metabolism to reverse tumor immunosuppression. Combination FLASH-CAR radioimmunotherapy may offer exciting opportunities for solid tumor treatment.

Extended Data Fig. 1 |. Effects of RT on tumor-associated Mfs, microglia, and NK cells.

Medulloblastoma was genetically engineered in SmoM2 mice, followed by irradiation with FLASH or standard proton beam. Tumors were excised and subjected to flow cytometry analysis. a, Gating strategies for analysis of T cells and Mϕs, corresponding to Fig. 1i–o. b-e, Analysis for b, CD11b⁺F4/80⁺ total Mϕs (n = 5 mice), c, CD45^LowCD11b⁺TMEM119⁺ total microglia (n = 12 mice for no RT group, and n = 11 mice for FLASH and standard RT groups), d, CD86⁺ M1-like (n = 5 mice) and CD206⁺ M2-like microglia (n = 6 mice), and e, NK1.1⁺ NK cells (n = 6 mice). Statistical analysis by one-way ANOVA (mean ± SEM).

Extended Data Fig. 2 |. Effects of RT on human Mf polarization in vitro.

Human PBMC-derived Mϕs were irradiated with FLASH or standard proton beam, followed by treatment with LPS or IL-4. a, Experimental procedure. b,c, After treatment with b, LPS or c, IL-4, cells were analyzed by flow cytometry. Left, representative cell sortings. Right, quantified results (n = 3 human participants, mean ± SEM). b, Statistical analysis by two-tailed Student’s t test. c, Statistical analysis by one-way ANOVA.

Extended Data Fig. 3 |. Effects of irradiated Mfs on T cell functions in vitro.

Human PBMC-derived Mϕs cells were irradiated by FLASH or standard proton beam and treated with IL-4 for 2 days. Human PBMC-derived CD3⁺ T cells were stimulated with CD3/CD28 beads for 3 days, and loaded with CFSE. Treated Mϕs and T cells were incubated for 2 days, followed by flow cytometry analysis. a, CFSE was analyzed in CD3⁺ T cells. Left, representative cell sortings. Right, quantified results (n = 3 human participants, mean ± SEM). Statistical analysis by one-way ANOVA. b, CD25 expression was analyzed in CD3⁺ T cells. Left, representative cell sortings. Right, quantified results (n = 3 human participants, mean ± SEM). Statistical analysis by one-way ANOVA.

Extended Data Fig. 4 |. Effects of RT on ROS generation, PPARg activity and oxLDL production in human Mfs.

Human PBMC-derived Mϕs were irradiated with FLASH or standard proton beam. a, Total ROS were analyzed at different time post-irradiation (mean ± SEM, n = 3 human participants). b, PPARγ activity was measured 24 h after irradiation (mean ± SEM, n = 5 human participants). Statistical analysis by one-way ANOVA. c, Human PBMC-derived Mϕs were irradiated with FLASH or standard proton beam, followed by treatment with or without IL-4. Cell lystes were subjected to oxLDL analysis (mean ± SEM, n = 6 human participants). Statistical analysis by one-way ANOVA.

Extended Data Fig. 5 |. Effects of RT on trancriptional factor activity in vitro.

Mouse BM-derived Mϕs (pooled from 3 mouse samples for each group) were irradiated with FLASH or standard proton beam, and subjected to analysis with a transcriptional factor profiling assay. The activity of 96 transcriptional factors was expressed as the fold of no RT group. a, Heatmap. b, Ranked activity.

Extended Data Fig. 6 |. Combination of RT with CAR T cell therapy in a syngeneic mouse glioma model.

Glioma was induced in mice by orthotopic transplantation with GL261 mouse glioma cells, followed by FLASH or standard RT and GD2 CAR-T cell therapy. a, Experimental procedures. b, Animal survival was monitored for 60 days (n = 10 mice). Statistical analysis by a two-tailed Log-rank Mantel-Cox test. c, Tumor volume was measured by bioluminescence imaging (n = 10 mice, mean ± SEM). Note: after standard RT plus CAR T cell treatment, one mouse developed neurological symptoms at late day 26 and was imaged at day 27. Statistical analysis by two-way ANOVA.

Extended Data Fig. 7 |. Effects of RT on CAR T infiltration and activity in vivo.

a-c, 5 days after irradiation, SmoM2 mice were treated with GD2 CAR-T cells. a, Experimental procedures. b,c, Tumors were excised 7 days after CAR-T cell therapy, followed by flow cytometry analysis. (b, Analysis of GFP⁺ CAR-T cells (n = 6 mice, mean ± SEM). Statistical analysis by one-way ANOVA. c, Analysis of IFN-g⁺, Ki-67⁺, Lag-3⁺, PD-1⁺ and Tim-3⁺ GFP⁺ CAR T cells (n = 4 mice, mean ± SEM). Statistical analysis by two-way ANOVA. d-e, 3 days after irradiation, SmoM2 mice were treated with GD2 CAR-T cells. d, Experimental procedures. e,f, Tumors were excised 3 days after CAR-T cell therapy, followed by flow cytometry analysis. e, Analysis of GFP⁺ CAR T cells (n = 3 mice, mean ± SEM). Statistical analysis by one-way ANOVA. f, Analysis of IFN-g⁺, Ki-67⁺, Lag-3⁺, PD-1⁺ and Tim-3⁺ GFP⁺ CAR T cells (n = 3 mice, mean ± SEM). Statistical analysis by two-way ANOVA.

Fig. 1 |. FLASH RT shows antitumor effects and skews Mϕs toward a proinflammatory phenotypes in mouse MB.

MB was genetically engineered in SmoM2 mice, followed by irradiation with FLASH or standard proton beam. a, Experimental procedure. b, After irradiation with a 10-Gy proton beam, animal survival was monitored (n = 11–14 mice; specific n value of each group listed in the figure). Statistical analysis by log-rank test. c–h, Single-cell RNAseq analysis (pooled from n = 3 mice for each group). c, UMAP analysis of transcriptome gene signature in all tumor-derived cells. Left, integration of RNAseq data from samples irradiated with FLASH or standard proton beam. Right, distribution of cell clusters. d, UMAP analysis of transcriptome gene signature in microglia and leukocytes. e, Top regulated genes in microglia and leukocytes. FC, fold change. f, CD86, CD80, CD163 and CD206 (Mrc1) expression in CD11b⁺ myeloid cells. g, Top altered genes in CD11b⁺ myeloid cells. h, Arginase 1 (Arg1) and MCP1 (Ccl2) expression in CD11b⁺ myeloid cells. i–o, Flow cytometry analysis for CD45⁺ hematopoietic cells (i), CD86⁺ M1-like Mϕs (j) and CD206⁺ M2-like Mϕs (k) in CD11b⁺F4/80⁺ Mϕs, CD45⁺CD3⁺ T cells in total cells (l) and CD4⁺ (m) and CD8⁺ (n) T cells in CD45⁺CD3⁺ T cells and ratio of CD8⁺/CD4⁺ T cells (o) (n = 5 mice, mean ± s.e.m.). Statistical analysis by one-way ANOVA. i, Left, representative cell sortings. Right, quantified results.

Fig. 2 |. FLASH radiation stimulates the capacity for stimulus-dependent proinflammatory polarization in Mϕs.

Mouse BM-derived Mϕs were irradiated with FLASH or standard proton beam, followed by treatment with LPS or IL-4. a, Experimental procedure. b–e, After treatment with LPS, cells were analyzed by flow cytometry (b), RT–PCR (c) or ELISA (d,e). b, Left, representative cell sortings. Right, quantified results (n = 3 mice, mean ± s.e.m.). c, Quantified results of RT–PCR (n = 3 mice, mean ± s.e.m.). d, ELISA results for IL-1β expression (n = 9 mice, mean ± s.e.m.). e, ELISA results for TNF expression (n = 9 mice, mean ± s.e.m.). b–e, Statistical analysis by one-way ANOVA. f,g, After treatment with IL-4, cells were analyzed by flow cytometry (f) and RT–PCR (g). f, Left, representative cell sortings. Right, quantified results (n = 3 mice, mean ± s.e.m.). Statistical analysis by one-way ANOVA. g, Quantified results of RT–PCR (n = 3 mice, mean ± s.e.m.). Statistical analysis by two-tailed Student’s t-test.

Fig. 3 |. FLASH radiation induces less PPARγ expression and immunosuppressive phenotypes in Mϕs.

Mouse BM-derived Mϕs were irradiated with 5-Gy FLASH or standard proton beam, followed by treated with LPS (M1) or IL-4 (M2). a–e, RNA was extracted and analyzed by RNAseq (n = 3 samples per group, pooled from three mice). a, Principal component analysis of all of the mapped genes. b, Expression of M1 and M2 marker genes in unirradiated Mϕs treated with or without LPS or IL-4 (mean ± s.e.m.). Statistical analysis by two-way ANOVA. c, Expression of immunosuppression-associated and proinflammation-associated genes in irradiated M1 or M2 Mϕs. Left, heat map of gene expression. Right, quantified changes over unirradiated cells (average percentage change). d, Expression of M2 polarization-associated TFs in unirradiated Mϕs treated with or without LPS or IL-4 (mean ± s.e.m.). Statistical analysis by one-way ANOVA. e, Expression of PPAR TFs in irradiated control, M1 Mϕs or M2 Mϕs. Left, heat map of gene expression. Right, quantified changes over unirradiated cells (average percentage change). f, Treated cells were analyzed by immunoblot. This experiment was repeated independently twice with similar results.

Fig. 4 |. FLASH radiation inhibits oxidase expression and oxLDL generation to reduce PPARγ activity and arginase 1 expression, while standard radiation induces redox-dependent PPARγ activation and arginase 1 expression in Mϕs.

a,b, Mouse BM-derived Mϕs were irradiated with 5-Gy FLASH or standard proton beam. a, Total ROS were analyzed at different times after irradiation (mean ± s.e.m., n = 4 mice). b, PPARγ activity was measured 24 h after radiation (mean ± s.e.m., n = 4 mice). Statistical analysis by one-way ANOVA. c,d, Mouse Mϕs were treated with TEMPO or DMTU, followed by FLASH or standard irradiation. c, PPARγ activity was measured 24 h after radiation (mean ± s.e.m., n = 4 mice). Statistical analysis by two-way ANOVA. d, Cell lysate was immunoblotted. This experiment was repeated independently twice with similar results. e–g, Mouse Mϕs were irradiated with 5-Gy FLASH or standard radiation. e, Cell lysates were subject to oxLDL analysis (mean ± s.e.m., n = 7 mice, pooled from two experiments). Statistical analysis by one-way ANOVA. f, RNA was extracted and analyzed by RNAseq (n = 3 samples, pooled from three mice; total of 27 mice). Left, heat map of oxidase gene expression. Right, quantified results. Statistical analysis by two-way ANOVA. g, RNA was extracted and analyzed by RT–PCR (mean ± s.e.m., n = 3 mice). Statistical analysis by one-way ANOVA. h, Mouse BM-derived Mϕs were treated with TEMPO or DMTU, followed by FLASH or standard radiation. Cell lysates were subjected to oxLDL analysis (mean ± s.e.m., n = 3 mice). Statistical analysis by one-way ANOVA.

Fig. 5 |. GD2 CAR-T cells show robust activity in vitro but minimal therapeutic efficacy in vivo.

a,b, Normal brain and tumor tissues were excised from WT and SmoM2 mice. a, Tissue sections were immunostained with anti-GD2 antibody (n = 4 mice). Representative images are shown. Scale bars, 50 μm. b, Tissue-derived single-cell suspensions were immunostained and analyzed by flow cytometry. Left, representative sortings. Right, quantified results (n = 3 mice, mean ± s.e.m.). Statistical analysis by two-tailed Student’s t-test. c–g, GD2 CAR-T therapy in mice. c, Preparation of CAR-T cells. Mouse T cells were retrovirally transduced to express control or GD2 CAR. Representative sortings are shown, d, Mouse tumor cells were isolated from MB tumors and incubated with control or GD2 mouse CAR-T cells, followed by cell lysis assay (n = 6 assays, mean ± s.e.m.). Statistical analysis by two-way ANOVA. e–g, SmoM2 mice were treated with or without control or GD2 CAR-T cells. e, Experimental procedures. f, Animal survival was monitored. Statistical analysis by log-rank test. g, Mice were imaged by bioluminescence. Left, representative images. Dashed circles indicate the brain area. Right, quantified luminescence signals in the brain area (n = 4 mice, mean ± s.e.m.). Statistical analysis by one-way ANOVA.

Fig. 6 |. FLASH RT overcomes MB resistance to GD2 CAR-T cell immunotherapy.

SmoM2 mice were irradiated and treated with control or GD2 CAR-T cells. a, Experimental procedures. b, Animal survival was monitored (n = 10 mice). Statistical analysis by log-rank test. c, CAR-T cell infiltration was imaged by bioluminescence. Left, representative images. Right, quantified results on days 1 and 3 after CAR-T cell injection (n = 5–15 mice; specific n value of each group listed in the figure; mean ± s.e.m.). Statistical analysis by two-way ANOVA. d–g, Tumors were excised 3 days after CAR-T cell therapy, followed by flow cytometry analysis. d, CD45⁺ hematopoietic cells. Left, representative cell sortings. Right, quantified results (n = 10 mice, mean ± s.e.m.). Statistical analysis by one-way ANOVA. e, GFP⁺ CAR-T cells. Left, representative cell sortings. Right, quantified results (n = 3 mice, mean ± s.e.m.). Statistical analysis by one-way ANOVA. f, IFNγ⁺, GranzB⁺ and Ki67⁺ GFP⁺ CAR-T cells. Quantified results (n = 4 mice, mean ± s.e.m.). Statistical analysis by two-way ANOVA. g, PD1⁺ and Tim3⁺ GFP⁺ CAR-T cells. Quantified results (n = 4 mice, mean ± s.e.m.). Statistical analysis by two-way ANOVA. h, A schematic model. FLASH radiation abrogates expression of oxidases including Mpo and Alox12 and slightly stimulates ROS generation in Mϕs, culminating in decreases in oxLDL production and PPARγ activation, which in turn increases T cell activity through reduced arginase 1 (Arg1) expression and enhances Mϕ M1-like proinflammatory polarization, eventually overcoming tumor resistance to T cell-based cancer immunotherapy. Standard RT also abolishes oxidase expression but robustly enhances ROS production in Mϕs, resulting in increases in oxLDL production and PPARγ activation, which drives Mϕ M2-like anti-inflammatory polarization and induces tumor resistance to immunotherapy.