Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment
Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada
Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada
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Research Article Immunology Oncology

Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment

Abstract

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap–pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap–pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap–pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap–pretreated CAR-T cells in human trials.

Authors

Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada

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Figure 2

A combination of metformin and rapamycin (Met+Rap) promotes the persistent function of CAR-T cells in hypoxic condition.

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A combination of metformin and rapamycin (Met+Rap) promotes the persiste...
(A) The design of the coculture experiment. (B) The number of viable tumor cells after coculture under normoxic condition (left) or hypoxic condition (right). Data are presented as mean ± SD. (C) The design for evaluating metabolic regulators. (D) The number of viable tumor cells on day 2 (left), day 4 (middle), and day 6 (right). Data are presented as mean ± SD. (E) Representative immunoblot of phosphorylation of AMPKα, S6 ribosomal protein, and Akt, and the expression of total AMPKα, total S6 ribosomal protein, total Akt, and PGC-1α in pretreated CD8+ CAR-T cells. The bar charts represent quantitative comparisons between the groups (n = 3/group). For Akt, blots were run on parallel gels using the same samples. Data are presented as mean ± SD. (F) Left: OCR of CAR-T cells was measured by Seahorse XFe96 analyzer on day 0. Data presented as mean ± SEM. Right: Spare respiratory capacity levels were calculated. Data are presented as mean ± SD. (G) Flow cytometric plots for CD44 and CD62L in CD8+ and CD4+ CAR-T cells on day 0. (H) The bar graphs illustrate the comparative frequencies of the CD44+CD62L+ cell population across the groups (n = 3/group). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001 by 1-way ANOVA followed by Tukey’s multiple-comparison test (B, D, E, and H) or unpaired, 2-tailed t test (F).