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MyD88-mediated chimaeric antigen receptor macrophages suppress brain metastasis using target-specific phagocytosis

Nature Biomedical Engineering (2026) Cite this article

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Abstract

Metastatic brain disease occurs in up to 30% of patients with lung, melanoma and breast cancers, and the median survival time remains less than a year. Treating these patients is a challenge because surgical approaches are limited and most chemotherapeutic drugs and immunotherapies are ineffective at crossing the blood–brain barrier (BBB). Given the unique abilities of macrophages to cross the BBB and exert their phagocytic function on tumour cells, we genetically engineer macrophages that express a chimaeric antigen receptor (CAR) targeting mesothelin (MSLN). To specifically target metastatic brain tumours, we fused the cells with the immune signalling molecule MyD88. This chimaeric antigen receptor macrophage (CARMA) penetrates the BBB and decreases brain metastasis growth in a humanized mouse model. MSLN–CARMA shows antigen-specific phagocytosis activity against tumour cells and exhibits a bystander effect by releasing TNF to act on surrounding tumour cells lacking the tumour antigen. These features of CARMA represent advantages over other immune therapies and CARMA may serve as a promising therapeutic tool for the treatment of brain metastasis.

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Fig. 1: Expression of MSLN in brain metastasis.
The alternative text for this image may have been generated using AI.
Fig. 2: CARMA suppressed growth of MSLN+ brain metastasis cells by activating phagocytic ability.
The alternative text for this image may have been generated using AI.
Fig. 3: Effect of different routes of CARMA injection on brain metastasis in vivo.
The alternative text for this image may have been generated using AI.
Fig. 4: Single-cell sequencing analysis of brain metastasis tumour after treatment with CARMA.
The alternative text for this image may have been generated using AI.
Fig. 5: CARMA suppressed the growth of MSLN− tumour cells through bystander effect.
The alternative text for this image may have been generated using AI.
Fig. 6: Efficacy of CARMA in suppressing brain metastasis progression in vivo.
The alternative text for this image may have been generated using AI.
Fig. 7: CARMA constructed from primary monocytes suppressed primary tumour and brain metastasis in vivo.
The alternative text for this image may have been generated using AI.

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Data availability

The single-cell-sequencing dataset in this study has been previously reported92. Our generated single-cell RNA-sequencing data are available in the NCBI Gene expression Omnibus database (GEO) with accession number GSE234832 (ref. 92). The analysis codes for survival analysis are shared in GitHub at https://github.com/QSong-github/BrMet (ref. 93) and in Zenodo. To investigate the influence of MSLN on the progression of lung cancer, we retrieved data on the overall survival and MSLN expression for patients at stages 1–4 from the TCGA lung cancer cohort. Patients lacking overall survival or MSLN expression data in the cohort were excluded. These patients were subsequently grouped into MSLN-High and MSLN-Low cohorts based on the median MSLN expression. The overall survival data for these two groups were analysed using Prism 9.0. We employed Kaplan–Meier survival analysis to determine the relationship between overall survival and MSLN gene expression in lung cancer patients. The references for the Kaplan–Meier survival analysis used in the study can be accessed in public repositories on the following websites: https://www.ncbi.nlm.nih.gov/gds, https://www.tfcheckpoint.org and http://kmplot.com/analysis/index.php?p=service. We obtained gene expression profiles from GEO datasets89,90,91 (GSE123902, GSE137797, GSE200563) and analysed the expression of MSLN in lung cancer and its association with brain metastasis. For the tumour antigen analysis, the references used in the study are available in a public repository provided by the Human Protein Atlas data (https://www.proteinatlas.org). To analyse immune cell populations in brain metastasis and primary CNS tumours, we utilized data from the URECA and TIMER 2.0 datasets. These datasets can be accessed in public repositories on the following websites: http://kmplot.com/analysis/, http://ureca-singlecell.kr/ and http://timer.comp-genomics.org/. We downloaded all immune cell profiles from URECA and examined the proportions of different immune cell populations in lung cancer brain metastasis. Source data are provided with this paper.

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Acknowledgements

We thank J. Massagué for providing the H2030- and PC9-BrM cell lines. This work was supported by the National Institutes of Health grants RO1CA173499, R01CA185650 and R01CA205067, and by Department of Defense grant W81XWH2110075 (to K. Watabe). This study also utilized the Cancer Center Shared Resources, including Cancer Genomics, Tumor Tissue and Pathology, Cell Engineering, Flow Cytometry, and Biostatistics and Bioinformatics, which are supported by the Comprehensive Cancer Center of Wake Forest University NCI, National Institutes of Health Grant (P30CA012197). W.Z. was supported by the Hanes and Willis Family Professorship in Cancer. Additional support for Q.S. and W.Z. were provided by a Fellowship to W.Z. from the National Foundation for Cancer Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute. S.-Y.W. was supported by the T32 training grant (T32CA247819) and the NIH Pathway to Independence Award (K99CA276895).

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Author notes

  1. These authors contributed equally: Shih-Ying Wu, Abhishek Tyagi.

Authors and Affiliations

  1. Department of Cancer Biology, Wake Forest Baptist Medical Center, Winston-Salem, NC, USA

    Shih-Ying Wu, Abhishek Tyagi, Kerui Wu, Eleanor C. Smith, Lance D. Miller, Wei Zhang, Ashok Pullikuth, Fei Xing, Ravindra Pramod Deshpande, Dan Zhao, Yin Liu, Jee Won Kim & Kounosuke Watabe

  2. Department of Radiation Oncology, Wake Forest Baptist Medical Center, Winston-Salem, NC, USA

    Shih-Ying Wu & Michael Chan

  3. Department of Health Outcomes and Biomedical Informatics, University of Florida College of Medicine, Gainesville, FL, USA

    Qianqian Song

  4. Nirogy Therapeutics, Natick, MA, USA

    Sambad Sharma

  5. Duke Department of Pathology, Duke University School of Medicine, Durham, NC, USA

    Bo-Syong Pan & Hui-Kuan Lin

  6. Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan

    Jung-Shun Lee

  7. Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA

    Michael H. Soike

  8. Department of Medicine, Section of Oncology and Hematology, Wake Forest Baptist Medical Center, Winston-Salem, NC, USA

    Jimmy Ruiz

  9. Section of Hematology and Oncology, W. G. (Bill) Hefner VA Medical Center, Salisbury, NC, USA

    Jimmy Ruiz

  10. Department of Biostatistical Sciences, Wake Forest Baptist Medical Center, Winston-Salem, NC, USA

    Jeff Chou

  11. Wake Forest University, Winston-Salem, NC, USA

    Alexandra Parson

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Contributions

S.-Y.W. and K. Watabe designed the study and wrote the manuscript. S.-Y.W., S.S. and K. Wu conducted experiments and acquired, analysed and interpreted the data. S.S., A.T. and K. Wu performed animal experiment. S.-Y.W. and K. Wu performed intracardiac injections. J.-S.L., E.C.S., D.Z., R.P.D., J.W.K. and F.X. performed clinical sample collection and analysis. S.-Y.W., B.-S.P. and H.-K.L. designed and performed the CRISPER KO TNF in monocytes. S.-Y.W., A.P., F.X., M.C., M.H.S., J.R. and Y.L. performed single-cell sequencing. S.-Y.W., Q.S., J.C., W.Z., L.D.M., A.P. and M.C. analysed the results of single-cell sequencing. S.-Y.W., S.S., K. Wu, A.T., D.Z., R.P.D., E.C.S. and K. Watabe reviewed and edited manuscript and interpreted the data. S.-Y.W. and K. Watabe supervised the study.

Corresponding authors

Correspondence to Shih-Ying Wu or Kounosuke Watabe.

Ethics declarations

Competing interests

H.-K.L. is a consultant for Stablix, Inc. and Chang Gung University of Science and Technology.

Peer review

Peer review information

Nature Biomedical Engineering thanks Saar Gill and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Macrophage infiltration and the expression of MSLN in lung cancer.

a, Analysis of epithelial and immune cell population in brain metastasis of lung cancer using the single cell sequencing data of URECA (http://ureca-singlecell.kr/). b, Population of different immune cells in brain tumors was analyzed using the PRECOG dataset30. Population analysis of different immune cells in brain tumors [Astrocytoma (n = 341), glioblastoma (n = 697), meningioma (n = 68), and oligodendrogliomas (n = 276)] was performed using the PRECOG dataset30. c, The expression levels of CD14+ monocyte/macrophage and Reg-TAMs markers (CD163, Arg1, Arg2) were investigated in the tumor adjacent normal brain (Non-tumor) and brain metastatic (BM) lesions of lung cancer patients using the TIMER2.0 dataset. Unpaired two-tailed t-test: *, p = 0.04. (n = 5 / group). d, Analysis of mRNA expression of MSLN in different brain regions and in distant organs using Human Protein Atlas data (https://www.proteinatlas.org/). e, Analysis of the expression of MSLN and other lung cancer tumor antigens in the brain metastatic samples using single-cell RNA sequencing. The data are presented as the mean ± SD.

Extended Data Fig. 2 Cytotoxic effect of CARMA on tumor cell growth.

a, Protein expressions of CD3 and CD28 in THP-1 human monocyte cell line and PBMC-derived primary human monocytes (Mono). b, The procedure of CARMA generation. The details of the procedure were provided in the Methods section. Image was created with BioRender.com. c, The expression of CD74+ Inflam-TAMs and CD206+ Reg-TAMs before or after CD14 microbeads isolation. (n = 5 / group). d, mRNA and protein expression of INF-γ, TNF-α and IL-1β on monocyte before or after CD14 microbeads isolation. (n = 5 / group). e, Efficiency of virus infection of Flag-labeled anti-MSLN-CAR constructs to the human primary (Pri) macrophage (MΦ) and N-CARMA and anti-MSLN-CARMA were examined by FACS. Significance was calculated using One-way ANOVA with Tukey’s multiple comparisons test: (e) ***, p = 0.0001 (n = 4 / group). f, The Flag protein expressions on the primary T, primary macrophage, Anti-MSLN-CAR-T, and anti-MSLN-CARMA (MyD-CARMA) cells were examined by western blot. g, MSLN binding ability in control N-CARMA and anti-MSLN-MyD-CARMA. h, Morphological changes of monocyte/macrophage that were treated with PBS (Veh), 1 nM CSF1, GM-CSF, or PMA. i, Cell viability of primary macrophage (Mϕ) and different CARMA cells after the treatment with PBS, CSF1 (1 nM), GM-CSF (1 nM) or PMA (1 nM) for 24 h. i, We calculated cell viability by dividing the absorbance of the wells containing cells treated with various cytokines by the average absorbance of the control wells. (n = 5 / group). j, Expression of Inflam-TAMs/Reg-TAMs phenotype on CARMA with PBS, CSF1, GM-CSF, or PMA treatment for 24 h. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 4 / group). k, The expression of MSLN in the tumor and CARMA. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). l, The MyD-CAR construct in lentivirus was introduced into the PBMC-derived primary human monocyte by viral infection, and respective CAR-monocyte cells were generated. MyD-monocytes were treated with PBS, CSF1, PMA, or GM-CSF. After 24 h, the cells were washed with PBS and they were co-cultured with luciferase-labeled H2030BrM cells (E/T ratio: 30:1) followed by assaying cell viability by IVIS. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 3 / group). m, Real-Time Cytotoxicity Assay (RTCA) was used to evaluate cytotoxic function of CARMA. Target cells (H2030BrM) were first added to the culture wells, and cell index was recorded. After 25 h, effector cells (N-, CD3-, and MyD-CARMA) were added at a 30:1 (Effector: Target cells) ratio. The percentage of specific cell lysis was shown in the right panel. One-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.001. (n = 4 / group). n, Primary N-, CD3-, or MyD-CARMA were cultured with luciferase-labeled H2030BrM cells (E/T ratio: 10:1) followed by assaying long-tern effect of cell viability by IVIS. Two-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.0063 and ***, p = 0.0001. (n = 4 / group). The data are presented as the mean ± SD.

Source data

Extended Data Fig. 3 Effect of CARMA on brain metastasis.

a, Expressions of Inflam-TAMs/Reg-TAM markers were examined in isolated N-, CD3- or MyD-CARMA cells after co-culturing with PC9BrM (E/T ratio: 30:1). The value of qRT-PCR in each figure was normalized using actin as a control. The y-axis indicates arbitrary unit. Significance was calculated using One-way ANOVA with Tukey’s multiple comparisons test: CD74: *, p = 0.012; ***, p = 0.0001; CD80: *, p = 0.023; ***, p = 0.0001; CD86: ***, p = 0.0001; ***, p = 0.0001; HLA-B: *, p = 0.041; ***, p = 0.0001; HLA-C: ***, p = 0.0001; ***, p = 0.0001; CD163: ***, p = 0.0001; ***, p = 0.0001; CD204: ***, p = 0.0001; ***, p = 0.0001; CD206: ***, p = 0.0001; ***, p = 0.0001; RHEB: **, p = 0.007; APOE: **, p = 0.0013. (n = 5 / group). b, c, After 24 tumor co-culture of CARMA and PC9BrM cells, CARMA were isolated from the co-culture using CD14 microBeads. The macrophage polarization of CARMA cells was examined by FACS using antibody to CD74+ (Inflam-TAMs) and CD206+ (Reg-TAMs). One-way ANOVA with Tukey’s multiple comparisons test: (b) *, p = 0.03; ***, p = 0.0001; (c) *, p = 0.04; ***, p = 0.0001. (n = 5 / group). d, CARMA cells were co-cultured with PC9BrM for 24 h followed by examining the mRNA expression of Inflam-TAMs-related cytokines (INF-γ, TNF-α, and IL-1β). One-way ANOVA with Tukey’s multiple comparisons test: INF-γ: ***, p = 0.0001; TNF-α: *, p = 0.01; **, p = 0.001; ***, p = 0.0001; IL-1β: ***, p = 0.0001. (n = 5 / group). e, N-, CD3-, and MyD-CARMA cells were incubated with PC9BrM for 24 h and the protein level of TNF-α in conditioned medium was measured by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.03; ***, p = 0.0001. (n = 5 / group). f, CARMA cells were incubated with PKH26-labeled PC9BrM cells for 24 h, followed by measurement of the monocyte and macrophage phagocytic activity by FACS. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.014; ***, p = 0.0001; *, p = 0.02; ***, p = 0.0001 (From left to right). (n = 5 / group). g-j, Scheme of the in vivo experiment (g). H2030BrM cells were transplanted intracranially into the nude mice. After 7 days of tumor transplantation, the mice received 1 ×105 human primary N(MyD)-CAR (N-CAR) monocyte (h), primary MyD-CAR monocyte (i), or primary CAR-T (j) that were all luciferase-labeled, by intracardiac injection, and the mice were scarified and checked for the luciferase intensity at the endpoint (Day 35). k, ex vivo BLI signals were examined for various organs at the endpoint after injection of luciferase-labeled CAR-T, N(MyD)-CARMA (N-CARMA), or MyD-CARMA. One-way ANOVA with Tukey’s multiple comparisons test: Brain: ***, p = 0.0001; ***, p = 0.0001; Liver: **, p = 0.002; **, p = 0.003; Spleen: ***, p = 0.0001; ***, p = 0.0001 (From left to right). (n = 3 / group). l, Population of microglia (Tmem119+/CD45−) and circulating macrophage (CD14+/CD45+) in brain metastatic region. t-test: ***, p = 0.001. (n = 4 / group). m, After one week of H2030BrM transplantation, mice received N-CAR-monocyte (N-CARMA), CD3-CAR-monocyte (CD3-CARMA), or MyD-CAR-monocyte (MyD-CARMA) by intracardiac injection. At the endpoint (Day 35), the brains were removed from the mice injected with or without CARMA. The cells were dissociated, and they were isolated using human CD14+ microbeads. They were then stained with human CD14 and Flag antibodies followed by analysis of FACS. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.019; **, p = 0.0028; ***, p = 0.0001. (n = 5 / group). n, Human primary monocytes were transduced with the Flag-conjugated CAR construct. After 24 h, the CAR-transduced primary monocyte cells were washed with PBS and cultured in fresh regular medium for 3 days. The primary Flag+ CAR-monocytes (CARMA) were isolated by FACS. To test the efficacy of CARMA, Luciferase labeled-tumor cells (H2030BrM cells) were transplanted into nude mice by intracranial injection. After one week of tumor transplantation, the mice were treated with different concentrations of N-CARMA, CD3-CARMA or MyD-CARMA cells every two weeks by intracardiac injection. Quantitative data of in vivo brain tumor. Two-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 9 / group). o, Ex vivo BLI signals in the brain at the endpoint. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 8 / group). p, Percentage of brain metastases of the mice that received different doses of primary N-CARMA, CD3-CARMA, or MyD-CARMA. Chi Square test: ***, p = 0.0001. (n = 8 / group). q, Metastatic lesions in the brain were removed from the mice used in (n) and the protein expression of TNF-α in the brain of each group was measured by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001 (n = 8 / group). The data are presented as the mean ± SD.

Source data

Extended Data Fig. 4 Infiltration of PBMC-derived CARMA into the brain metastasis region.

a, Experimental procedure to test anti-tumor effect of PBMC-derived CARMA in the humanized mouse model. Mice were engrafted with human PBMC cells. After human myeloid engraftment was confirmed, H2030BrM cells were transplanted into the brain by intracranial injection. After seven days, 1 × 105 PBMC-derived N-CARMA or MyD-CARMA were injected intracardially every 2 weeks until the endpoint. Brain tumors were harvested at the endpoint, and they were dissociated, and single cell RNA sequencing was performed. Scale bar: 100 µm. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 3 / group). b, Population of human CD45+ cells in humanized mice one month after receiving PBMC engraftment. c, The positive signal intensity of different circulating human immune cells in the blood of humanized mouse. d, The human CD45+/Flag+ cell were detected by FACS. Unpaired two-tailed t-test: ***, p = 0.0004. (n = 5 / group). e, Mice were engrafted with human PBMC cells. After the confirmation of human myeloid engraftment, H2030BrM cells were transplanted into the brain by intracranial injection. After seven days, 1 × 105 cells of PBMC-derived primary monocyte (Pri. MΦ), PBMC-derived N-CAR monocyte (N-CARMA), or PBMC-derived MyD-CAR monocyte (MyD-CARMA) were injected intracardially every 2 weeks until the endpoint. Upper panels: BLI images of representative mice from each experimental group at the endpoint. Lower panels: ex vivo image of brain metastasis. f, g, Quantitative data of in vivo (f) and ex vivo (g) brain tumor. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 6 / group). h, Percentage of brain metastasis in each group was measured. Chi Square test: ***, p = 0.00001. (n = 9 / group). i, j, Percentage of Reg-TAMs (i) and Inflam-TAMs (j) in the tumor was measured. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 9 / group). k, The expression of TNF-α in brain metastasis in the mice that were treated with primary monocyte, N-, or MyD-monocyte was measured. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 9 / group). l, Expression of Flag in the tumor brain of humanized mouse implanted with CARMA. The data are presented as the mean ± SD.

Source data

Extended Data Fig. 5 Effect of CAR on cell viability and polarization of CARMA and T cell trafficking.

a, b, Proliferation rate of Ki67+/cFMS+ cells (a) and cell viability (b) of the primary macrophage and different CARMA were examined using FACS and MTS. (n = 4 / group). c, Infiltration index of primary macrophage or different CARMA was measured using the boyden chamber. The CARMA were seeded on the upper chamber and H2030BrM cells were seeded on the lower chamber (E/T ratio: 30:1). After 72 h of incubation, the macrophages in the lower chamber were examined by FACS. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). d, Expression of lysosome capacity in primary macrophage and CARMA. e, Polarization phenotype in N- and MyD-CARMA before or after CAR insertion. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 4 / group). f, Expressions of TNF-α, IL-1β, and INF-γ in primary macrophage, N-CARMA and MyD-CARMA cells that were co-cultured with H2030BrM were quantified by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.002; *, p = 0.018; *, p = 0.013. (n = 6 / group). g-i, Expressions of TNF-α, IL-1β, and INF-γ in MyD-CARMA cells before or after CAR insertion and CARMA-H2030BrM co-culture were quantified by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.03; ***, p = 0.001 (n = 5 / group). j, k, H2030BrM cells were co-cultured with PBMC-derived CARMA (E/T ratio: 30:1) for 72 h. The primary CARMA cells were then isolated from the co-culture and the expressions of CXCL9 (j) and CXCL10 (k) of the isolated CARMA cells were examined by qRT-PCR. One-way ANOVA with Tukey’s multiple comparisons test: (j) *, p = 0.03; **, p = 0.0012; ***, p = 0.0001; (k) ***, p = 0.0001 (n = 5 / group). l, Primary T cells were co-cultured with H2030BrM-pretreated primary CARMA (E/T ratio: 30:1) for 24 h. The T cells were then isolated from the co-culture, and the expression of CXCR3 of the T cells was examined by qRT-PCR. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.03; ***, p = 0.0001. (n = 5 / group). m, H2030BrM cells were co-cultured with primary CARMA (E/T ratio: 30:1) for 72 h. CARMA cells were then separated from the co-culture and incubated with primary T cells that were labeled with cell proliferation dye. The T cell proliferation rate was then examined by FACS. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). n, H2030BrM cells were co-cultured with primary CARMA (E/T ratio: 30:1) for 24 h. CARMA cells were then separated from the co-culture and seeded in the lower chamber, and the primary T cells were seeded in the upper chamber. After 3 days of co-culture, the primary T cells were isolated from the lower chamber and examined for the chemotactic index. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). o, The luciferins-labeled H2030BrM cells were co-cultured with ‘IL-4-activated primary T cell only (Tumor + pri T)’ or ‘CARMA plus IL-4-activated primary T cells (Tumor + CARMA + pri. T)’ for 0, 2, 5 days, and the tumor cell growth was examined by IVIS. One-way ANOVA with Tukey’s multiple comparisons test: Day 2: **, p = 0.015; **, p = 0.014; Day 5: *, p = 0.02; **, p = 0.009; ***, p = 0.0001. (n = 5 / group). The data are presented as the mean ± SD.

Source data

Extended Data Fig. 6 Bystander effect of CARMA.

a, N-CARMA or MyD-CARMA cells were cultured with H2030BrM (E/T ratio: 30:1) for 24 h. CM was collected and analyzed by using the cytokine/growth factor array (Raybio). b, Expressions of Inflam-TAMs-related cytokines in CM were examined by ELISA after the co-culture of primary CARMA and H2030BrM cells. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0002; ***, p = 0.00023; **, p = 0.0021; *, p = 0.03; **, p = 0.0001. (n = 3). c and d, The expression of TNF-α, IL-1β, or INF-γ receptor on tumor population in human brain metastatic sample (c) and humanized brain metastatic mouse sample (d). e, mRNA expression of TNF-α, IL-1β or INF-γ receptor on H2030BrM cell. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.001. (n = 5). f, Cell viability of H2030BrM after one week of recombinant TNF-α, IL-1β or INF-γ treatment. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.001. (n = 5). g, The CM derived from the co-culture of MyD-CARMA and H2030BrM cells was treated with anti-TNF-α, anti-IL-1β, or anti-INF-γ antibody to deplete each cytokine in CM. H2030BrM cells were then treated with the cytokine-depleted CM followed by assaying the colony-forming ability. One-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.001; ***, p = 0.0001. (n = 4). h, Scheme of experimental design. NCI-H522MSLN+/GFP+ and NCI-H522MSLN-/RFP+ cells were mixed and intracranially transplanted into mice. After 7 days, the mice received 1 ×105 PBMC-derived primary N-, CD3-, or MyD-CAR monocyte by intracardiac injection every 2 weeks for 35 days. i, Quantitative data of in vivo brain metastasis of lung cancer. Two-way ANOVA with Tukey’s multiple comparisons test: ##, p = 0.01; ###, p = 0.001; **, p = 0.01. (n = 6-8 / group). j, Ex vivo BLI signals in the brain at the endpoint. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.05; ***, p = 0.0001. (n = 9 / group). k, Kaplan–Meier analysis of brain tumor free survival was performed. Two-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.05; ***, p = 0.001. (n = 7-9 / group). l, The metastatic lesions in the mouse brain in Extended Data Fig. 6h were sectioned and subjected to immunohistochemical analysis for GFP (MSLN+) and RFP (MSLN−). Scale bar: 100 μm. m-o, Metastatic brain tumors in Extended Data Fig. 6h were isolated from the brain and examined by FACS for the population of CD45+ macrophage (m), Reg-TAMs (n), and Inflam-TAMs (o). One-way ANOVA with Tukey’s multiple comparisons test: (m) *, p = 0.04; ***, p = 0.0001; (n) **, p = 0.01; ***, p = 0.0001; (o) ***, p = 0.0001. (n = 6 / group). p, Metastatic tumors in the brain were removed from the mice in Extended Data Fig. 6h. The expression of TNF-α in the dissociated mouse brain from the primary N-, CD3-, or MyD-CARMA treated groups were measured by ELISA. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.012; ***, p = 0.0001. (n = 6 / group). The data are presented as the mean ± SD.

Source data

Extended Data Fig. 7 Effect of knockdown of TNF-α on the growth of NCI-H522MSLN+/GFP+ and NCI-H522MSLN-/RFP+ cells.

a, Efficiency of the knockout (KO) of TNF-α in CARMA cells. CRISPR/Cas9 KO plasmids consist of scrambled or 3 sets of TNF-α-specific guide RNA sequences and GFP reporter. The plasmid was transduced to CAR-THP-1 human monocyte cells. The scrambled GFP+ and KO TNF-α GFP+ cell were isolated by FACS. b, The transfection efficiency of isolated CAR-THP-1 in Extended Data Fig. 7a was examined by measuring GFP-positive signal (Right panel). Scale bar: 100 µm. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 4 / group). c, The expression of TNF-α was measured by qRT-PCR. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 5 / group). d, Scheme of testing the effect of KO of TNF-α on bystander killing effect. MSLN+ cells were co-cultured with scramble or KO TNF-α CARMA (E/T ratio: 30:1) for 72 h, and CARMA CM was collected. MSLN-negative cells were then treated with the CM. e-g, The MSLN-negative cells (NCI-H522) were treated with the CM that were collected from Scrambled or KO TNF-α CARMA cells that were co-cultured with MSLN-positive tumor cells (NCI-H522MSLN+), followed by assaying the cell viability (e), colony formation ability (f), and cancer stem cell population (g) of the MSLN-negative cells. One-way ANOVA with Tukey’s multiple comparisons test: (e) ***, p = 0.0001; (f) *, p = 0.02; ***, p = 0.0001; (g) *, p = 0.03; ***, p = 0.0001. (n = 5 / group). h, NCI-H522MSLN+ cells were labeled with GFP while NCI-H522MSLN− cells were labeled with RFP. Tumor cells were then co-cultured with primary MyD-CARMA with or without knockdown of TNF-α at a 30:1 effector/target cells ratio for 72 h. Growth of the NCI-H522MSLN+/GFP+ and NCI-H522MSLN-/RFP+ cells was observed by microscopy, and representative images were shown. Scale bar: 100 µm. i-k, Percentage of NCI-H522MSLN+/GFP+ and NCI-H522MSLN-/RFP+ cells were examined by flow cytometry. One-way ANOVA with Tukey’s multiple comparisons test: (i) ***, p = 0.0001. (n = 5 / group); (j) ***, p = 0.0001. (n = 6 / group). The data are presented as the mean ± SD.

Source data

Extended Data Fig. 8 Expression of TNF-α and bystander effect of CARMA and CAR-T.

a, Kaplan–Meier analysis was performed for overall survival of lung cancer patients in relation to CD47 (left panel) and SIRPα (right panel) expression using the TCGA dataset. b, Expression of CD47 on primary and metastatic brain tumor from lung adenocarcinoma patients was examined using the GSE123902 dataset89. Unpaired two-tailed t-test: ***, p = 0.0001. [Primary (n = 40); BM (n = 19)]. c, Expression of SIRPα on primary N-, CD3-, and MyD-CARMA was examined by qRT-PCR. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0005; ***, p = 0.0001. (n = 3 / group). d, Expression of SIRPα on CD45+ macrophages was examined in the regions of brain metastasis and adjacent normal areas of lung cancer patients. Scale bar: 200 μm. e, Luciferase-labeled H2030BrMMSLN+ cells [Target cells (T)] were co-cultured with the different ratios of CAR-T or MyD-CARMA [Effector cells (E)] for one or three days and the cell viability was measured by IVIS bioluminescence imaging. One-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 3 / group). f, g, Expressions of TNF-α gene and TNF-α protein were examined by qRT-PCR (f) and ELISA (g) after the co-culture of primary T cells, primary macrophage (Mϕ), CAR-T, and MyD-CARMA with or without H2030BrM cells (E/T ratio: 30:1) for 24 h. One-way ANOVA with Tukey’s multiple comparisons test: **, p = 0.01; ***, p = 0.001. (n = 5 / group). h-j, Scheme of testing the effect of CAR-T and CARMA on bystander killing effect (h). NCI-H522MSLN− cells were labeled with RFP (NCI-H522MSLN-/RFP+). NCI-H522MSLN− cells that have ectopic overexpression of MSLN and were labeled with GFP and designated as NCI-H522MSLN+/GFP+. The NCI-H522MSLN+/GFP+ and NCI-H522MSLN-/RFP+ cells were then co-cultured (1:1 ratio). The primary T cells, primary macrophage, CAR-T, and MyD-CARMA cells were incubated with the mixed-culture of NCI-H522MSLN+/GFP+ and NCI-H522MSLN-/RFP+ for 72 h, and the percentage of surviving NCI-H522MSLN+/GFP+ (i) and NCI-H522MSLN-/RFP+ (j) was analyzed by FACS. One-way ANOVA with Tukey’s multiple comparisons test: *, p = 0.04; **, p = 0.01; ***, p = 0.0001. (n = 4 / group). The data are presented as the mean ± SD.

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Extended Data Fig. 9 Brain MRI demonstrates minimum neurotoxicity of CARMA.

a, T2-weighted coronal images of a mouse brain at multiple planes. Diagram showing the planes of imaging in a mouse brain, starting with the striatum and continuing through the substantia nigra. T2 analysis for measuring volume and microstructure of brain. ROIs indicate the various brain regions for quantitative analysis. Yellow circle: tumor location. Red areas: hemorrhage region. White arrow: abnormal space. The table shows brain volume in different regions with or without tumor transplantation or CARMA supplement. (n = 5 / group). b, Color images were generated from post-processed imaging data from (a). They provide a visual representation of the quantitative. c, Diffusion Tensor Imaging (DTI) for analysis of axonal organization in the brain after CARMA injection. Red: transvers fibers. Green: anteroposterior fibers. Blue: Craniocaudal fibers. (n = 5 / group). **, p = 0.01: compared to non-tumor transplantation group. d-g, CAR-T and CARMA cells were generated using the second-generation CAR construct. Each H23030BrM transplanted animal in primary CAR-T, primary N-CARMA, and primary MyD-CARMA group was subjected to the object recognition test, and the percentages of investigation time of moved (d) and novel (e) object were measured. f,g, Body weight (f) and liver function (g) of the mice were measured at the end points in each group. Unpaired two-tailed t-test: (d) **, p = 0.006; **, p = 0.003; **, p = 0.002. (n = 6 / group); (g) *, p = 0.03; *, p = 0.03 (From left to right). (n = 4 / group). h, Expression of MSLN in brain metastatic tumor region and tumor-adjacent bronchi tissues. (n = 10 / group). Scale bar: 200 µm. i, Morphology of lung tissue in N-, CD3- or MyD-CARMA injected mice. Scale bar: 200 µm. The data are presented as the mean ± SD.

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Extended Data Fig. 10 Effect of mouse CARMA on brain metastasis in male and female wild-type mouse models.

a, Mouse primary monocytes were transduced with the Flag-conjugated CAR construct. After 24 h, the CAR-transduced primary monocyte cells were washed with PBS and cultured in fresh regular medium for 3 days. To test the efficacy of CARMA, Luciferase labeled-mouse lung cancer tumor cells (LL2-BrM cells) were transplanted into male C57BL/6 mice by intracranial injection. After one week of tumor transplantation, the mice were treated with N-CARMA or MyD-CARMA cells every two weeks by intracardiac injection. Image was created with BioRbender.com. b, Quantitative data of in vivo male brain tumor. Two-way ANOVA with Tukey’s multiple comparisons test: ***, p = 0.0001. (n = 6 / group). c-f Each LL2-BrM transplanted animal in N-CARMA and MyD-CARMA group was subjected to the object recognition test, and the percentages of investigation time of moved (c) and novel (d) object were measured. (e) Liver function and (f) body weight of the mice were measured at the end points in each group. ***, p = 0.0001. (n = 6 / group). g, Luciferase labeled-mouse lung cancer tumor cells (LL2-BrM cells) were transplanted into female C57BL/6 mice by intracranial injection. After one week of tumor transplantation, the mice were treated with N-CARMA or MyD-CARMA cells every two weeks by intracardiac injection. ***, p = 0.0001. (n = 6 / group). h-k Each LL2-BrM transplanted female mice in N-CARMA and MyD-CARMA groups were subjected to the object recognition test, and the percentages of investigation time of moved (h) and novel (i) object were measured. Body weight (j) and liver function (k) of the mice were measured at the end points in each group. ***, p = 0.0001. (n = 6 / group). The data are presented as the mean ± SD. l, Morphology of lung tissue in mouse N- and MyD-CARMA injected wild-type mice. Scale bar: 100 µm. m, Genetically engineered macrophages (CARMA) that express tumor-specific antibody fused with MyD88 drastically decreased brain metastasis growth by showing antigen-specific phagocytosis activity against tumor cells. CARMA also showed bystander killing effect on antigen-negative-tumor cells by secreting cytokine TNF-α. Image was created with BioRender.com.

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Wu, SY., Tyagi, A., Wu, K. et al. MyD88-mediated chimaeric antigen receptor macrophages suppress brain metastasis using target-specific phagocytosis. Nat. Biomed. Eng (2026). https://doi.org/10.1038/s41551-026-01613-x

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