Chimeric antigen receptors containing FcγRIIa significantly increase targeted phagocytosis in macrophages

FcγRs are the primary receptors responsible for engulfing particles bound by antibodies, with the immune receptor tyrosine activation motif (ITAM) playing a key role in this process. To increase the phagocytic function of CARs, we developed receptors by fusing the scFv targeting HER2 (P1h3) with the transmembrane and intracellular domains of FcγRI (FcεRIγ), FcγRIIa, and FcγRIIc, which all contain ITAMs. These recombinant CAR sequences were inserted downstream of the SFFV promoter in a third-generation lentiviral vector, while GFP was cloned under the PGK promoter (Fig. 1A).

Fig. 1

The FcγRIIa domain enhances CAR-mediated tumor phagocytosis. A Construction of HER2-targeting CAR macrophages incorporating diverse FcγR domains. B Flow cytometry histograms illustrating the lentivirus transfection efficiency in THP-1 cells. C Fluorescence-activated cell sorting (FACS) analysis of the phagocytic efficiency of the GFP+ macrophages. GFP- and CAR-modified macrophages were cocultured with mCherry+ SKOV3 cells at an effector-to-target ratio (E:T) of 1:1 for 1 h before flow cytometry analysis. D Structural diagram of CAR and CAR-shSIRPα. Flow cytometry histograms depicting CAR and CAR-shSIRPα expression levels, with GFP (E) and His-tagged recombinant HER2 (F) in sorted THP-1 cells. G Immunoblot analysis of SIRPα protein levels in untransduced (UTD), CAR, and CAR-shSIRPα macrophages. H FACS analysis of SIRPα expression levels in unstained (USD), untreated (UTD), CAR, and CAR-shSIRPα macrophages, as well as UTD, CAR, and CAR-shSIRPα macrophages cocultured with SKOV3 cells for 24 h

To generate CAR-modified macrophages, we first optimized the differentiation conditions for THP-1 human leukemic monocytes. Mature macrophages induced with 20 ng/mL phorbol-12-myristate-13-acetate (PMA) expressed the conventional macrophage markers CD11b and CD14 and maintained high viability (Supplementary Fig. 1a, b). Compared with untransduced (UTD) macrophages, lentiviral infection achieved approximately 65% efficiency in these macrophages (Fig. 1B), with no significant differences observed in terms of proliferation, cell cycle, or viability, regardless of PMA induction (Supplementary Fig. 1c–i). To evaluate the phagocytic capacity of CAR-modified macrophages, we cocultured CAR-Ms with HER2-positive SKOV3 cells that consistently expressed mCherry. Flow cytometry analysis revealed minimal phagocytosis by control GFP macrophages, whereas P1h3-FcεRIγ, P1h3-FcγRIIa, and P1h3-FcγRIIc macrophages efficiently engulfed tumor cells. Notably, P1h3-FcγRIIa macrophages presented the highest phagocytic ratio, suggesting that the presence of FcγRIIa in the CAR structure enhances targeted phagocytosis the most (Fig. 1C). Therefore, we utilized P1h3-FcγRIIa as the chimeric receptor in subsequent experiments, referred to as “CAR.” In addition to the use of the lentiviral system for CAR delivery, we also investigated a replication-incompetent adenoviral vector (Ad5f35), guided by Klichinsky’s study [8]. Our findings showed that macrophage infection efficiency was dependent on the adenoviral dose; however, cell viability decreased significantly at higher multiplicities of infection (MOIs) (Supplementary Fig. 2b–d). We ultimately opted for the lentiviral system in subsequent experiments because of its acceptable infection efficiency, cell viability, and cost-effectiveness.

To further enhance the phagocytic activity of CAR-modified macrophages against tumors, we incorporated a shRNA expression element targeting SIRPα downstream of the GFP sequence in the lentiviral vector and designated it CAR-shSIRPα (Fig. 1D). Postsorting, the proportion of GFP-positive CAR and CAR-shSIRPα macrophages exceeded 83% (Fig. 1E). Moreover, fluorescence-activated cell sorting (FACS) analysis revealed that approximately 90% of the sorted CAR and CAR-shSIRPα macrophages expressed CAR molecules, which was correlated with the presence of GFP expression levels in the CAR (Fig. 1F). Western blot analysis revealed a significant reduction in SIRPα expression in CAR-shSIRPα macrophages compared with that in UTD and CAR macrophages (Fig. 1G and Supplementary Fig. 2e). Importantly, CD47 expression was extremely high in SKOV3, SKBR3, DLD-1, and ASPC1 tumor cells (Supplementary Fig. 2a), and coculture with these tumor cells significantly increased SIRPα expression in UTD and CAR macrophages, whereas CAR-shSIRPα macrophages maintained low SIRPα levels (Fig. 1H). These findings indicate effective inhibition of CD47-SIRPα signaling by CAR-shSIRPα macrophages.

Inhibition of SIRPα enhances the antitumoral M1-like polarizing phenotype of CAR-modified macrophages

Macrophages exhibit significant phenotypic and functional diversity. While M1-like macrophages possess antitumor properties, most tumor-associated macrophages (TAMs) undergo polarization toward a tumor-promoting M2-like phenotype in response to signals from the tumor microenvironment (TME), contributing to rapid tumor progression. Therefore, maintaining an antitumoral phenotype in CAR-modified macrophages following incubation with tumor cells is essential to ensure sustained antitumor activity. To this end, we performed phenotypic characterization of CAR macrophages and CAR-shSIRPα macrophages with and without antigen stimulation. Our qPCR and flow cytometry results indicated that CAR macrophages and CAR-shSIRPα macrophages presented significantly higher expression levels of CD80, CD86, and TNF-α than control macrophages (UTD and GFP) in the resting state. The macrophages in which SIRPα was knocked down presented the highest levels of CD80, CD86 and TNF-α expression (Fig. 2A–C). Coculture with SKOV3 cells led to a notable increase in M1-like polarization markers in CAR-modified macrophages. Consistent with the resting-state phenotype, CAR-shSIRPα macrophages presented higher expression levels of CD80, CD86, and TNF-α than CAR-Ms did (Fig. 2D–F). The upregulation of CD80, CD86, and HLA-DR in CAR-modified macrophages was further confirmed by FACS analysis (Fig. 2G, H). Although coculture with SKOV3 cells increased the expression of the M2-like phenotype markers CD163 and CD206, CAR-M and CAR-shSIRPα-M presented significantly lower expression levels of CD163 and CD206 than did UTD macrophages. The lowest expression levels were observed in CAR-shSIRPα-M under both resting and antigen-stimulated conditions (Fig. 2I, J). Furthermore, CAR-M and CAR-shSIRPα-M exhibited increased secretion of IL-1β, IFN-γ, and TNF-α, regardless of tumor antigen stimulation. Notably, CAR-shSIRPα-M presented the highest capacity for proinflammatory cytokine secretion (Fig. 2K–M). These results illustrate that CAR-M and CAR-shSIRPα-M display an antitumor M1-like polarization phenotype and that SIRPα inhibition further enhances the ability of CAR-modified macrophages to present antigens and secrete proinflammatory cytokines.

Fig. 2

CAR-shSIRPα-treated macrophages exhibit an enhanced M1-like polarization phenotype. qRT‒PCR analysis of CD80 (A), CD86 (B), and TNF-α (C) expression levels in UTD, GFP, CAR, and CAR-shSIRPα macrophages. qRT‒PCR analysis of CD80 (D), CD86 (E), and TNF-α (F) expression levels in UTD-stimulated, and SKOV3-cocultured CAR- and CAR-shSIRPα-stimulated macrophages. G–J FACS analysis of M1- and M2-like phenotypic markers in resting macrophages and macrophages cocultured with SKOV3 cells. Expression levels of CD80, CD86, and HLA-DR in resting (G) and SKOV3-cocultured (H) UTD, CAR, and CAR-shSIRPα macrophages. Expression of CD163 and CD206 in resting (I) and SKOV3-cocultured (J) UTD, CAR, and CAR-shSIRPα macrophages. ELISA analysis of IL-1β (K), INF-γ (L), and TNF-α (M) production in UTD, CAR, and CAR-shSIRPα macrophages with or without coculture with SKOV3 cells. The data are presented as the means ± s.e.m. of three technical replicates. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001

Silencing of SIRPα in CAR-modified macrophages results in significant antigen-specific phagocytosis and cytotoxicity against HER2-positive tumor cells in vitro

To explore the specific phagocytic effect of CAR-modified macrophages on HER2-positive tumor cells, we assessed HER2 antigen expression in various tumor cell lines. High HER2-positive rates were observed in the human SKOV3, SKBR3, and ASPC1 cancer cell lines, with SKOV3 cells demonstrating the highest HER2 antigen expression level. Conversely, human DLD-1 cancer cells presented low HER2 abundance (Fig. 3A). Subsequently, DLD-1 cells and mouse B16, MC-38, and ID8 cancer cells were transduced with lentiviruses carrying truncated human HER2 lacking intracellular domains (Fig. 3A). The externally introduced truncated HER2 antigens enabled CAR-modified macrophages to selectively target tumor cells while avoiding interference from HER2 signaling. Furthermore, lentiviral transduction enabled the stable expression of mCherry and luciferase in these cell lines, facilitating both in vitro and in vivo experiments.

Fig. 3

CAR-shSIRPα macrophages exhibit enhanced tumor phagocytosis and cytotoxicity. A FACS analysis of HER2 expression levels in human and mouse tumor cell lines. B Confocal microscopy analysis of macrophage phagocytosis. mCherry+ SKOV3 cells were cocultured with GFP, CAR, or CAR-shSIRPα macrophages at an effector-to-target (E:T) ratio of 5:1 for 1 h before visualization. Scale bars represent 20 μm. C–F FACS analysis of targeted tumor phagocytosis by GFP+ macrophages. HER2-negative B16 cells (C) and HER2-positive B16-HER2 cells (E) were cocultured with GFP, CAR, or CAR-shSIRPα macrophages at an E:T ratio of 1:1 for 1 h prior to analysis. Statistical analysis of the phagocytosis efficiency of HER2-negative B16 cells (D) and HER2-positive B16-HER2 cells (F). G Cytotoxic effects of CAR-modified macrophages against HER2-positive tumor cells. mCherry-positive SKOV3 cells were cocultured with GFP, CAR, or CAR-shSIRPα macrophages at an E:T ratio of 5:1 for 24 h. Continuous images were obtained via a Lumascope 720 fluorescence microscope. Scale bars represent 100 μm. H Luciferase-based cytotoxicity analysis of CAR-modified macrophages at E:T ratios of 1:1, 3:1, 5:1, and 10:1 against SKOV3 cells after 24 h of coculture. I Apoptosis analysis of CAR-modified macrophages cocultured with SKOV3 cells. J Statistical analysis of the results presented in (I). The data are presented as the means ± s.e.m. of three technical replicates. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001

Using confocal microscopy, we observed clear antigen-specific phagocytosis of mCherry-positive SKOV3 tumor cells by CAR-modified macrophages (Fig. 3B). Furthermore, B16 and B16-HER2 cells were cocultured with CAR-modified macrophages, and subsequent phagocytosis was quantitatively assessed via flow cytometry to evaluate the specific engulfment of tumor cells by CAR-modified macrophages. The results revealed that GFP control macrophages, as well as CAR-M and CAR-shSIRPα-M, exhibited low levels of phagocytosis of HER2-negative B16 cells (Fig. 3C, D). In contrast, while control macrophages maintained low phagocytosis rates, CAR-M and CAR-shSIRPα-M demonstrated phagocytosis rates of 22.7% and 29.4%, respectively, for HER2-positive B16-HER2 cells (Fig. 3E, F), suggesting that the phagocytic activity of CAR-modified macrophages toward tumors is HER2 specific. Additionally, we conducted a comparative analysis of targeted phagocytosis by CAR-modified macrophages in HER2-positive SKOV3, SKBR3, ID8-HER2, MC38-HER2, and DLD1-HER2 cell lines. Our results illustrate that CAR molecules equip macrophages with effective targeted phagocytic capabilities and that further suppression of SIRPα can enhance this phagocytic effect (Supplementary Fig. 3).

To determine whether knocking down SIRPα universally enhances the phagocytic ability of CAR-modified macrophages, we replaced P1h3 with FMC63, a scFv against human CD19, and constructed CD19-targeting CAR-Ms and CAR-shSIRPα-Ms (Supplementary Fig. 4a). Through coculture phagocytosis experiments, we observed that CAR-shSIRPα-Ms exhibited significantly greater phagocytic activity against CD19-positive human Burkitt lymphoma Raji cells and human diffuse large B-cell lymphoma SU-DHL-4 cells than CAR-Ms did (Supplementary Fig. 4b). These findings suggest that SIRPα knockdown enhances the phagocytic ability of CAR-modified macrophages that target various antigens.

Next, we investigated the cytotoxicity of CAR-modified macrophages to HER2-positive tumor cells. Our results showed that within 24 h of coculture, CAR-modified macrophages effectively eliminated SKOV3 cells (Fig. 3G). Furthermore, cytotoxicity experiments at various effector-to-target (E:T) ratios demonstrated that both CAR and CAR-shSIRPα macrophages had significant dose-dependent killing effects on HER2-positive tumor cells (Fig. 3H and Supplementary Fig. 5a–d). Additionally, coculture with CAR-modified macrophages resulted in notable induction of apoptosis in tumor cells, with CAR-shSIRPα demonstrating an even more pronounced proapoptotic effect (Fig. 3I, J and Supplementary Fig. 5e–j). Overall, these findings suggest that CAR-modified macrophages exhibit promising antitumor effects against HER2-positive tumor cells in vitro.

CAR-modified macrophages exhibit potent antitumor activity in patient-derived organoids

Compared with traditional tumor cell lines, PDOs provide a more accurate representation of tumor phenotypes and characteristics. To validate the targeted killing capability of CAR-modified macrophages, primary tumors from gallbladder and pancreatic cancer patients were isolated, and stable, expandable organoids were subsequently generated. Bright-field observations and hematoxylin and eosin (HE) staining revealed the characteristic features of the spherical organoids. Immunohistochemistry confirmed the concurrent expression of HER2 and CA199 tumor antigens in the constructed PDOs (Fig. 4A, B, and Supplementary Fig. 6a). Coculturing macrophages with PDOs revealed that CAR-modified macrophages adhered to the PDOs and efficiently engulfed the tumor cells (Fig. 4C, D, Supplementary Fig. 6b–e). After 48 h of coculture, Apopxin™ Green was added to detect apoptosis, revealing that PDO tumor cells died when cocultured with CAR-shSIRPα macrophages (Fig. 4E). Bright-field observations and HE staining revealed considerable disruption of the PDO structure within 72 h, particularly in CAR- and CAR-shSIRPα-treated macrophages. Notably, CAR-shSIRPα macrophages had a more significant damaging effect on HER2-positive PDOs (Fig. 4F, G). ELISAs revealed elevated levels of IL-1β and TNF-α in the coculture medium of PDOs and CAR-modified macrophages, with the highest concentrations recorded in the CAR-shSIRPα macrophages (Fig. 4H–K).

Fig. 4

CAR-modified macrophages effectively eradicate tumor patient-derived organoids (PDOs). Cultivation of gallbladder cancer PDOs (A) and pancreatic cancer PDOs (B). The morphology of the PDOs was examined via bright-field microscopy. Additionally, hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining were performed to assess the expression of HER2 and CA199 in the PDOs. Scale bars represent 20 μm for bright-field and HE-stained images and 50 μm for IHC-stained images. C Confocal imaging of the accumulation of CAR-modified macrophages around PDOs. PDOs were cocultured with CAR- and CAR-shSIRPα-treated macrophages (green) for 12 h. Nuclei were stained prior to confocal imaging. Scale bars represent 20 μm. D Phagocytosis of PDOs by CAR-shSIRPα macrophages. PDOs were stained with CellTracker (red) and cocultured with CAR-shSIRPα macrophages (green) for 48 h before confocal imaging. Scale bars represent 50 μm. E Cytotoxic effects of CAR-shSIRPα macrophages on PDO cells. PDOs labeled with CellTracker (red) were cocultured with CAR-shSIRPα macrophages that did not express GFP for 48 h, after which Apopxin™ Green was added to identify apoptotic cells before confocal imaging. Scale bars represent 50 μm. Destruction of the PDO structure by CAR-modified macrophages. Gallbladder cancer PDOs (F, scale bars represent 100 μm) or pancreatic cancer PDOs (G, scale bars represent 20 μm) were cocultured with GFP, CAR, and CAR-shSIRPα macrophages. Bright-field microscopy and HE were performed at the specified time points. H–K Identification of proinflammatory cytokines in the supernatants of the cocultures. Gallbladder cancer PDOs (H, J) and pancreatic cancer PDOs (I, K) were cocultured with GFP, CAR, and CAR-shSIRPα macrophages for 72 h. The supernatant was analyzed via ELISA to measure the levels of IL-1β (H, I) and TNF-α (J, K). For all panels, *P < 0.05, **P < 0.01, ***P < 0.001

Inhibition of SIRPα enhances inflammatory signaling, triggers the cGAS-STING pathway, and augments the production of ROS and NO

To elucidate the potential mechanisms underlying the targeted engulfment and killing of tumor cells by CAR-modified macrophages, we analyzed the expression profiles of control, CAR, and CAR-shSIRPα macrophages. Transcriptomic sequencing data were subjected to Gene Ontology (GO) analysis, which revealed that, compared with those in the UTD control group, CAR macrophages presented enrichment of genes associated with biological processes such as the regulation of NF-κB signaling, the response to IL-1, the inflammatory response, and phagocytosis (Fig. 5A). Conversely, CAR-shSIRPα-M presented significant enrichment of genes related to biological processes such as cytokine-mediated signaling pathways, the inflammatory response, endocytosis, ROS metabolic processes, and superoxide metabolic processes (Fig. 5B). Furthermore, compared with CAR macrophages, CAR-shSIRPα macrophages not only presented further enrichment of genes linked to inflammatory responses, phagocytosis, and antigen presentation but also presented additional enrichment of genes associated with processes such as the regulation of T-cell proliferation, the regulation of ROS metabolism, and the regulation of glycolysis (Fig. 5C). Enrichment analysis via the Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) revealed that, compared with control macrophages, CAR-modified macrophages were predominantly enriched in inflammatory signaling pathways, including the TNF-α, NF-κB, MAPK, PI3K-Akt, and TLR signaling pathways (Supplementary Fig. 7a–d). In contrast, compared with CAR-shSIRPα macrophages, CAR-shSIRPα macrophages presented enriched signaling pathways related to cytokines, chemokines, phagocytosis, and TLR signaling (Supplementary Fig. 7e, f).

Fig. 5

Enhanced inflammatory signaling, cGAS-STING pathway activation, and ROS and NO production in CAR-shSIRPα macrophages. SKOV3 cells cocultured with GFP, CAR, or CAR-shSIRPα macrophages were sorted via flow cytometry and subsequently subjected to RNA sequencing. Gene Ontology (GO) term enrichment analysis of differentially expressed genes (DEGs) was conducted for CAR vs. GFP (A), CAR-shSIRPα vs. GFP (B), and CAR-shSIRPα vs. CAR (C). D Heatmap analysis of DEGs related to polarization and glycolysis in GFP, CAR, and CAR-shSIRPα macrophages. E Lactate levels were detected in the supernatants of GFP, CAR, and CAR-shSIRPα macrophages cocultured with SKOV3 cells for 24 h. Immunoblot analysis of PFKFB3 and LDHA (F) and cGAS-STING signaling (G) protein levels in GFP, CAR, and CAR-shSIRPα-sorted macrophages stimulated with SKOV3 cells at an E:T ratio of 10:1 for 24 h. H Luciferase-based cytotoxicity analysis of CAR-modified macrophages at E:T ratios of 10:1 against SKOV3 cells after 24 h of coculture, with or without the STING inhibitor H-151. (I) ELISA analysis of INF-β production in CAR-modified macrophages at E:T ratios of 10:1 against SKOV3 cells after 24 h of coculture with or without the STING inhibitor H-151. J Heatmap analysis of DEGs related to NADPH oxidase complex-related genes in GFP, CAR, and CAR-shSIRPα macrophages. K Immunoblot analysis of iNOS protein levels in GFP-, CAR-, and CAR-shSIRPα-sorted macrophages stimulated with SKOV3 cells. L NO production by GFP, CAR, and CAR-shSIRPα macrophages with or without recombinant HER2 stimulation. *P < 0.05, **P < 0.01

In particular, both CAR- and CAR-shSIRPα-treated macrophages presented increased expression of M1 proinflammatory genes and significantly elevated expression of genes associated with glycolysis, which was particularly pronounced in CAR-shSIRPα-treated macrophages (Fig. 5D). Given the crucial role of glycolysis in M1 macrophage polarization, lactate secretion was quantified in each group. The results indicated that CAR-modified macrophages presented higher levels of lactate expression than control macrophages did, while CAR-shSIRPα macrophages presented the highest lactate expression levels (Fig. 5E). Moreover, Western blot analysis revealed that coincubation with recombinant HER2 protein resulted in a noticeable increase in the expression of key glycolytic enzymes, namely, PFKBR3 and LDHA, in both CAR-M and CAR-shSIRPα-M (Fig. 5F). This observation implies that CAR-modified macrophages may enhance the M1 phenotype through enhanced glycolysis.

Tumor-derived DNA is capable of escaping from phagolysosomes into the cytosol, where it is recognized by cGAS, thereby activating the cGAS-STING signaling pathway. Consistent with these findings, our study revealed significant upregulation of the expression of cGAS, STING, phosphorylated STING, IRF3, and phosphorylated IRF3—key transcription factors within this cascade—in CAR-modified macrophages following coincubation with tumor cells, particularly in SIRPα-inhibited CAR-Ms (Fig. 5G). Furthermore, research indicated that the specific STING inhibitor H-151 substantially reduced the cytotoxicity of CAR-modified macrophages against tumor cells (Fig. 5H) and diminished the secretion of IFN-β by both CAR-Ms and CAR-shSIRPα-Ms upon coincubation with tumor cells (Fig. 5I). These findings highlight the significant role of the cGAS-STING signaling pathway in the antitumor response mediated by CAR-modified macrophages.

Analysis of transcription in CAR-modified macrophages revealed significant enrichment of the ROS and superoxide metabolism signaling pathways. To investigate the contributions of ROS and superoxide to the antitumor effect of CAR-modified macrophages, we examined the expression levels of genes related to the NADPH oxidase complex. Our results indicated notable upregulation of genes such as NOX1, NOX2, NOX3, NCF1, NCF2, RAC1, and RAC2 in CAR-modified macrophages, particularly in CAR-shSIRPα-Ms (Fig. 5J). Furthermore, both the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) were significantly increased in CAR-modified macrophages after coincubation with tumors (Fig. 5K, L). This increase in ROS and NO production offers a plausible explanation for the phagocytosis-independent cytotoxic activity observed in CAR-modified macrophages (Fig. 3I, J).

CAR-shSIRPα macrophages demonstrate significant in vivo antitumor activity

To evaluate the efficacy of CAR-modified macrophages in vivo, various tumor models have been established in nude mice. Initially, an intraperitoneal tumor model was developed using SKOV3 cells. One week after implantation, the mice were randomly divided into four treatment groups: the PBS group, the UTD control group, the Ad-CAR group (generated through adenovirus infection), and the Lenti-CAR group (generated through lentivirus infection). Live imaging examinations were conducted on tumor-bearing mice at 1, 3, 6, 9, and 12 weeks posttumor initiation (Supplementary Fig. 8a, b). Tumors exhibited rapid growth in the PBS and UTD treatment groups. In the PBS group, more than half of the mice died within five weeks of tumor growth, whereas 80% of the tumor-bearing mice in the UTD group did not survive beyond 10 weeks. In contrast, substantial growth inhibition of intraperitoneal tumors was observed in the Ad-CAR and Lenti-CAR treatment groups, with only 20% and 10% of the mice, respectively, surviving within ten weeks of tumor development (Fig. 6A). At 100 days posttumor inoculation, 20% and 40% of the mice in the respective treatment groups survived. Statistical analysis revealed a slightly greater therapeutic effect of Lenti-CAR macrophages than of Ad-CAR macrophages (Fig. 6B). Consequently, lentivirus-infected macrophages were used in subsequent in vivo experiments.

Fig. 6

Potent antitumor activity of CAR-shSIRPα macrophages in vivo. A, B Lenti-CAR and Ad-CAR macrophages were used to treat nude mice bearing peritoneal SKOV3 tumors. A The tumor burden was assessed via bioluminescence imaging (BLI), and representative images at different time points were presented. B Kaplan‒Meier curve showing the survival of the mice (n = 5 mice per group). C The tumor burden in the B16-HER2 cell subcutaneous injection model was evaluated via BLI, and representative images at different time points were shown (n = 5 mice per group). D IHC staining and TUNEL immunofluorescence staining were performed on tumor tissue sections from the B16-HER2 cell subcutaneous injection mouse model to examine the expression of Ki67, CD31, and active caspase-3, as well as apoptosis, in the tumor tissues. Scale bars represent 100 μm. E Polarization markers for M1 (CD80 and CD86) and M2 (CD163 and CD206) infiltrating macrophages were analyzed via FACS. F, G Tumor-bearing mice were euthanized four weeks after receiving caudal vein transfusions of CAR-modified macrophages in the B16-HER2 lung metastasis model. F Representative macroscopic images of lungs excised from the specified treatment groups at the end of the experiment. G Lung metastatic burden was assessed via HE staining. Scale bars represent 1 mm (n = 7 mice per group). For all panels, *P < 0.05, **P < 0.01, ***P < 0.001

Subcutaneous tumor models were created in nude mice via B16-HER2 or ID8-HER2 tumor cells. One week after tumor establishment, the mice were administered PBS, UTD, CAR-M, or CAR-shSIRPα-M via intravenous injection (Supplementary Fig. 8c, d). In vivo imaging over four consecutive weeks in the B16-HER2 tumor model revealed notable inhibition of tumor growth attributed to both CAR-M and CAR-shSIRPα-M. Notably, CAR-shSIRPα-M exhibited the most promising therapeutic effects among the treatment groups (Fig. 6C and Supplementary Fig. 8e). Similar results were observed in the ID8-HER2 tumor model (Supplementary Fig. 8f–h). Immunohistochemical analysis of tumor tissues revealed high levels of Ki67 and CD31 in the PBS and UTD treatment groups, indicating increased tumor proliferation and angiogenesis. Conversely, reduced expression levels of Ki67 and CD31 were observed in the groups treated with CAR and CAR-shSIRPα macrophages (Fig. 6D). Furthermore, few apoptotic cells were detected in the tumor tissues of the PBS and UTD treatment groups. In contrast, significant cleaved caspase-3 staining and TUNEL-positive apoptotic cells were observed in the groups treated with CAR-modified macrophages, particularly in those treated with CAR-shSIRPα macrophages (Fig. 6D). Additionally, mice in the PBS and UTD groups presented significant weight increases due to tumor growth, whereas the weight changes in the CAR and CAR-shSIRPα macrophage-treated groups were not statistically significant (Supplementary Fig. 9a). By the end of the treatment, no apparent abnormalities were noted in the vital organs of the nude mice across all groups (Supplementary Fig. 9b), indicating that the in vivo use of CAR macrophages is safe.

To evaluate the phenotype of the macrophages infiltrating the tumor tissue, we isolated the tumor tissue five days after the intravenous injection of the macrophages and analyzed the polarization markers of the GFP-positive macrophages. Our analysis revealed that, compared with those in the UTD group, infiltrating CAR- and CAR-shSIRPα-treated macrophages presented increased levels of the M1 polarization markers CD80 and CD86, whereas the expression of the M2 polarization markers CD163 and CD206 was significantly reduced (Fig. 6E). This M1 phenotype was particularly pronounced in CAR-shSIRPα-treated macrophages, suggesting that the knockdown of SIRPα may help sustain the M1-polarized state of CAR-Ms in vivo.

To establish a nude mouse model of lung metastasis, B16-HER2 cells were injected intravenously via the tail vein for seven days. Subsequently, the mice were injected with PBS, UTD, CAR macrophages, or CAR-shSIRPα macrophages. After three weeks, the mice were euthanized, and their lung tissues were dissected for observation (Supplementary Fig. 10a, b). Widespread melanized tumor cells were observed on the lung surfaces of the mice in the PBS and UTD treatment groups, whereas only limited lung tumor metastases were present in the CAR-modified macrophage treatment group, with virtually no metastases in the CAR-shSIRPα macrophage treatment group (Fig. 6F). Consistent results were obtained upon examination of lung tissue sections via HE staining (Fig. 6G). Additionally, while there were no significant differences in body weight among the groups during the treatment period (Supplementary Fig. 10c), the ratio of lung weight to body weight was significantly lower in the CAR and CAR-shSIRPα macrophage treatment groups than in the control groups (Supplementary Fig. 10d). Overall, these findings demonstrated a favorable in vivo therapeutic effect of CAR macrophages, which can be further enhanced through the suppression of SIRPα expression.

CAR-modified macrophages promote functional T-cell tumor infiltration

Humanized mouse models of cancer, which involve immunodeficient mice coengrafted with human tumors and immune cells, serve as vital tools in immuno-oncology research with potential clinical applications. We established a human immune system (HIS) mouse model by intravenously injecting human peripheral blood mononuclear cells (hu-PBMCs) into NCG mice (Supplementary Fig. 11a). Flow cytometric analysis performed two weeks after hu-PBMC injection revealed an average of 23.98% CD45+ cells and 19.05% CD3+ cells, indicating that T cells were the predominant component involved in the immune system reconstitution of HIS mice (Supplementary Fig. 11b, c). Following hu-PBMC engraftment for one week, HER2-positive APSC1 cells were subcutaneously transplanted into NCG mice. The adoptive transfer of CAR-modified macrophages was performed two weeks after tumor inoculation, and hCD45+ and hCD3+ T cells in the peripheral blood of tumor-bearing HIS mice were analyzed two weeks after CAR-modified macrophage treatment (Supplementary Fig. 11d–f). In vivo imaging revealed comparable tumor volumes among all groups before CAR macrophage treatment; however, notable tumor growth inhibition was evident after two weeks of treatment in the mice that received CAR macrophages and those that received CAR-shSIRPα macrophages (Fig. 7A). Four weeks after tumor formation, HIS mice were euthanized, and flow cytometric analysis of tumor tissues revealed minimal infiltration of CD45+ cells in the PBS- and UTD-treated groups, in contrast to the substantial increase in CD45+ cell infiltration in the groups treated with CAR-modified macrophages, particularly in the CAR-shSIRPα-treated group (Fig. 7B, C). Furthermore, the CAR-shSIRPα-treated group presented the highest proportion of IFN-γ+ cytotoxic T cells in the tumor tissues (Fig. 7D, E). Immunofluorescence staining and flow cytometry of tumor tissues revealed significantly greater numbers of CD3+ and CD8+ cells in both the CAR- and CAR-shSIRPα macrophage-treated groups than in the PBS- or UTD-treated groups, along with decreased Ki67 expression and increased apoptotic cell counts within the tumor tissues (Supplementary Fig. 12).

Fig. 7

CAR-shSIRPα macrophages promote T-cell infiltration and antitumor immunity. A The tumor burden in humanized immune system (HIS) mice treated with CAR-modified macrophages was assessed via BLI, with representative images presented at different time points following tumor implantation (n = 5 mice per group). B, C The percentage of hCD45+ cells in the tumor tissues of tumor-bearing HIS mice was measured 2 weeks after treatment, with each point representing one mouse (n = 3 per group). D, E The percentage of IFN-γ+ cells among hCD8+ T cells in the tumor tissue of tumor-bearing HIS mice was assessed 2 weeks posttreatment, with each point representing one mouse (n = 3 per group). F The tumor burden in immunocompetent C57BL/6 mice treated with CAR-modified macrophages was evaluated via BLI, with representative images shown at different time points (n = 5 mice per group). G CD3+ T, CD4+ T, and CD8+ T cells were detected via FACS in tumor tissues from tumor-bearing C57BL/6 mice. H IHC staining of CD3, CD8, and active caspase-3 was conducted on tumor tissue sections from tumor-bearing C57BL/6 mice. Scale bars represent 100 μm. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001

To analyze the distribution and function of CAR-modified macrophages in vivo more comprehensively, we constructed mouse CAR-Ms and CAR-shSIRPα-Ms via the murine iBMDM cell line. Given that mice do not express FcγRIIa, we utilized FcεRIγ as the intracellular domain to construct a murine CAR. For the experiments assessing the distribution of CAR-modified macrophages in vivo, we created CAR-Luc2 and CAR-shSIRPα-Luc2 constructs, which express luciferase 2 fused with the CAR (Supplementary Fig. 13a). In B16-HER2 subcutaneous tumor-bearing mice, we monitored the distribution of CAR-Luc2 and CAR-shSIRPα-Luc2 macrophages in major organs and tumors after tail vein injection. Our findings revealed that CAR-Luc2 and CAR-shSIRPα-Luc2 macrophages were enriched primarily in the lungs during the first three days, with minimal distribution in the liver. By day five postinjection, these cells had migrated to the liver, and some were present in the tumor tissue. Over time, an increasing number of CAR-Luc2 and CAR-shSIRPα-Luc2 macrophages were found in the tumor tissue. At 15 days after tail vein injection, CAR macrophages were still detectable in the tumor tissue and liver but were consistently undetectable in the spleen or kidneys (Supplementary Fig. 13b).

To further substantiate the promoting effect of CAR-modified macrophages on T-cell tumor infiltration, we established a B16-HER2 subcutaneous tumor model in immunocompetent C57BL/6 mice, and CAR-modified iBMDMs were administered intravenously one week after tumor inoculation. Consistent with the results in the nude mouse models, both CAR- and CAR-shSIRPα iBMDMs significantly inhibited tumor cell growth compared with the PBS and UTD controls, with CAR-shSIRPα iBMDMs demonstrating a more pronounced therapeutic effect (Fig. 7F). Importantly, in the tumor tissues of the mice treated with CAR-shSIRPα iBMDMs, we detected increased numbers of CD3-positive and CD8-positive T cells, along with increased expression of activated caspase-3 (Fig. 7G, H). These findings align with the experimental results from the HIS mouse tumor model, indicating that CAR-shSIRPα macrophages can enhance tumor-killing ability by promoting T-cell infiltration. Overall, these results provide compelling evidence that targeting SIRPα in CAR-modified macrophages not only augments their antitumor efficacy but also fosters a more robust immune response through T-cell recruitment in the tumor microenvironment.