Chimeric antigen receptor macrophage therapy for breast tumours mediated by targeting the tumour extracellular matrix

Abstract

Background: The extracellular matrix (ECM) is essential for malignant tumour progression, as it is a physical barrier to various kinds of anticancer therapies. Matrix metalloproteinase (MMPs) can degrade almost all ECM components, and macrophages are an important source of MMPs. Studies using macrophages to treat tumours have shown that macrophages can enter tumour tissue to play a regulatory role.

Methods: We modified macrophages with a designed chimeric antigen receptor (CAR), which could be activated after recognition of the tumour antigen HER2 to trigger the internal signalling of CD147 and increase the expression of MMPs.

Results: Although CAR-147 macrophage treatment did not affect tumour cell growth in vitro compared with control treatment. However, we found that the infusion of CAR-147 macrophages significantly inhibited HER2-4T1 tumour growth in BALB/c mice. Further investigation showed that CAR-147 macrophages could reduce tumour collagen deposition and promote T-cell infiltration into tumours, which were consistent with expectations. Interestingly, the levels of the inflammatory cytokines TNF-α and IL-6, which are key factors in cytokine release syndrome, were significantly decreased in the peripheral blood in CAR-147 macrophage-transfused mice.

Conclusion: Our data suggest that targeting the ECM by engineered macrophages would be an effective treatment strategy for solid tumours.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Construction of the chimeric antigen receptor CAR-147 targeting HER2. a The design of a chimeric antigen receptor (CAR-147) for macrophages is shown. b Human HER2 surface expression on 4T1 cells was measured. c CAR-147 surface expression on Raw264.7 cells was measured. d Control Raw264.7 (CON) cells and CAR-147 Raw264.7 (147) cells were cocultured with HER2-4T1 (HER2) cells at a ratio of 1:1 for 24 h, and the expression of MMP genes was assessed by RT-qPCR (n = 6). e Control Raw264.7 (CON) cells and CAR-147 Raw264.7 (147) cells were cocultured with HER2-4T1 (HER2) cells at a ratio of 1:2 for 24 h, and the expression of MMP genes was assessed by RT-qPCR (n = 6). f Control Raw264.7 (CON) cells and CAR-147 Raw264.7 (147) cells were cocultured with HER2-4T1 (HER2) cells at a ratio of 1:2 for 24 or 48 h, and the expression of MMP genes was assessed by RT-qPCR (n = 6). g Control Raw264.7 (CON) cells or CAR-147 Raw264.7 (147) cells were cocultured with wild-type 4T1 (CON) cells or HER2-4T1 (HER2) cells at a ratio of 1:1 for 24 h, and the expression of MMP genes was assessed by RT-qPCR (n = 6). All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 2

The effect of CAR-147 macrophages on tumour growth in vivo. a In vivo imaging of luciferase-expressing HER2-4T1 tumour-bearing mice after intravenous injection of DiR-labelled Raw264.7 cells. b Quantitative analysis of the DiR fluorescence signal in the liver (n = 5). c Quantitative analysis of the DiR fluorescence signal at the tumour site (n = 5). d The experimental schedule for tumour implantation, macrophage infusion and bioluminescence imaging (BLI) monitoring. e BLI was performed on day 7 to assess tumour burden. Control Raw264.7 (CON) cells (1 × 106), CAR-147 Raw264.7 (147) cells (1 × 106), or PBS were injected i.v. on day 8 and day 15, and mice were followed with serial BLI. f The weight of tumours from control Raw264.7 cell- or CAR-147 Raw264.7 cell-treated mice was analysed. The data presented are pooled from two independent experiments. Each symbol indicates one mouse (n = 10). g The spleen weight of tumour-bearing mice was analysed. h The body weight of tumour-bearing mice was measured every 3 days. i Blood serum samples and tumour homogenates were collected from control Raw264.7 cell- or CAR-147 Raw264.7 cell-treated mice and analysed for proinflammatory cytokine release (IL-6, IFNγ, TNFα, IL-1β, IL-12) by ELISA (n = 5). All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 3

CAR-147 macrophages can promote T-cell infiltration into tumours. a Flow cytometric analysis and quantification of CD45+ tumour-infiltrating leucocytes (TILs) (n = 10). b Flow cytometric analysis and quantification of CD3+ T cells in the TIL population (n = 10). c Immunofluorescence analysis of CD3+ T-cell infiltration in tumour tissues from control Raw264.7 cell- and CAR-147 Raw264.7 cell-treated animals. d Flow cytometric analysis and quantification of MDSCs (Gr-1+CD11b+) in the TIL population (n = 10). e In total, 2 × 105 HER2-4T1 cells were transplanted into BALB/c nude mice. BLI was performed to assess tumour burden. f Tumour growth from control Raw264.7 cell- or CAR-147 Raw264.7 cell-treated BALB/c nude mice was analysed by measuring volumes every 2 days. g The weight of tumours from control Raw264.7 cell- or CAR-147 Raw264.7 cell-treated BALB/c nude mice was analysed. Each symbol indicates one mouse (n = 8). All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 4

CAR-147 macrophages reduce extracellular matrix deposition. a Representative images of tumour sections stained with Masson’s trichrome to detect extracellular matrix deposition (blue). Scale bar, 100 μm. b Quantification of the extracellular matrix (n = 15). c Analysis of MMP gene expression in tumour tissues from control Raw264.7 cell- and CAR-147 Raw264.7 cell-treated animals by RT-qPCR (n = 5). All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5

CAR-147 macrophages facilitate T-cell infiltration into 3D human tumour spheroids. a CAR-h147 surface expression on THP-1 cells. b Detection of HER2 expression on MDA-MB-453 cells by flow cytometry. c PMA-treated CAR-h147 THP-1 cells were cocultured with MDA-MB-453 tumour cells at a ratio of 1:1 for 48 h, and the expression of MMP genes was assessed by RT-qPCR (n = 6). d Confocal microscopy images of MDA-MB-453/THP-1 spheroids treated with CFSE-labelled Jurkat T cells for 20 h. Scale bars, 100 μm. All values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001