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

Nature Cancer volume 6, pages 460–473 (2025)Cite this article

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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.

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Fig. 1: FLASH RT shows antitumor effects and skews Mϕs toward a proinflammatory phenotypes in mouse MB.
Fig. 2: FLASH radiation stimulates the capacity for stimulus-dependent proinflammatory polarization in Mϕs.
Fig. 3: FLASH radiation induces less PPARγ expression and immunosuppressive phenotypes in Mϕs.
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.
Fig. 5: GD2 CAR-T cells show robust activity in vitro but minimal therapeutic efficacy in vivo.
Fig. 6: FLASH RT overcomes MB resistance to GD2 CAR-T cell immunotherapy.

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Data availability

Single-cell and bulk RNAseq data were deposited to the National Center for Biotechnology Information’s Gene Expression Omnibus under accession numbers GSE246970 and GSE246969, respectively. All remaining data are available within the article and the Supplementary Information or available from the authors upon request. Source data are provided with this paper.

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Acknowledgements

We are grateful to J. Billings for help with single-cell RNAseq analysis. This work was supported in part by the University of Pennsylvania Abramson Cancer Center Radiation Oncology Translational Center for Excellence (to Y.F.), National Institutes of Health grants R01NS094533 (to Y.F.), R01NS106108 (to Y.F.), R01CA241501 (to J.F.D. and Y.F.), R01HL106108 (to Y.F. and Y.G.), R35CA197616 (to D.G.K), P01CA257904 (to A.J.M., C.K. and D.G.K.) and K08CA256045 (to Z.J.R.) and developmental funds from P30CA014236 (to Z.J.R.) and P50CA190991 Duke SPORE in Brain Cancer (to Z.J.R. and D.G.K.), a Yosemite Award from the American Cancer Society (to Y.F.), a Mark Foundation grant (to A.J.M.), a Pediatric Brain Tumor Foundation grant (to Z.J.R.), a St. Baldrick’s Foundation grant (to Z.J.R.), a Chadtough Defeat DIPG grant (to Z.J.R.) and an Alex’s Lemonade Stand Foundation grant (to Z.J.R.).

Author information

Author notes

  1. These authors contributed equally: Haiwei Ni, Zachary J. Reitman.

Authors and Affiliations

  1. Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA, USA

    Haiwei Ni, Wei Zou, Md Naushad Akhtar, Ritama Paul, Menggui Huang, Duo Zhang, Hao Zheng, Ruitao Zhang, Ruiying Ma, Gina Ngo, Eric S. Diffenderfer, S. Azar Oliaei Motlagh, Michele M. Kim, Andy J. Minn, Jay F. Dorsey, James Metz, Constantinos Koumenis & Yi Fan

  2. Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA

    Zachary J. Reitman & David G. Kirsch

  3. Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA, USA

    Lin Zhang

  4. Mark Foundation Center for Immunotherapy, Immune Signaling, and Radiation, University of Pennsylvania, Philadelphia, PA, USA

    Andy J. Minn, Constantinos Koumenis & Yi Fan

  5. Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Jessica B. Foster

  6. Radiation Medicine Program, Princess Margaret Cancer Centre, Toronto, Ontario, Canada

    David G. Kirsch

  7. Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada

    David G. Kirsch

  8. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    David G. Kirsch

  9. Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Yanqing Gong

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Contributions

H.N. performed the experiments, analyzed the results and produced the figures. Z.J.R. designed the experiments. W.Z. and S.A.O.M. conducted the radiation dosimetry and delivery. M.N.A. and R.M. helped with the T cell assays. R.P. and M.H. contributed to the initial in vivo RT experiments. D.Z. and L.Z. contributed to the bioinformatic analysis. H.Z., R.Z. and G.N. helped with the experimental assays. J.B.F. contributed to the GD2 CAR-T treatment. E.S.D., M.M.K., A.M., J.F.D., J.M. and C.K. contributed to the FLASH physics and RT. D.G.K., Y.G. and Y.F. contributed to the experimental design and data interpretation. D.G.K., Y.G. and Y.F. supervised the project. Y.F. conceptualized the ideas and wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to David G. Kirsch, Yanqing Gong or Yi Fan.

Ethics declarations

Competing interests

D.G.K. is a cofounder of and stockholder in XRAD Therapeutics, which is developing radiosensitizers. D.G.K. is a member of the scientific advisory board and owns stock in Lumicell, a company commercializing intraoperative imaging technology. None of these affiliations represent a conflict of interest with respect to the work of this manuscript. D.G.K. is a coinventor on a patent for a handheld imaging device and is a coinventor on a patent for radiosensitizers. None of these patents are relevant to this manuscript. XRAD Therapeutics, Merck, Bristol Myers Squibb and Varian Medical Systems have provided research support to D.G.K. but this did not support the research described in this manuscript. Z.J.R. is listed as an inventor for intellectual property related to genetic testing for brain tumors that is managed by Duke Office of Licensing and Ventures, which is not relevant to this manuscript. The other authors declase no competing interests.

Peer review

Peer review information

Nature Cancer thanks Niels Bovenschen, Aaron Diaz, Ping-Chih Ho and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

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, CD45LowCD11b+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).

Source data

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.

Source data

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.

Source data

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.

Source data

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.

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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.

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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.

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Ni, H., Reitman, Z.J., Zou, W. et al. FLASH radiation reprograms lipid metabolism and macrophage immunity and sensitizes medulloblastoma to CAR-T cell therapy. Nat Cancer 6, 460–473 (2025). https://doi.org/10.1038/s43018-025-00905-6

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