Efficacy evaluation of chimeric antigen receptor-modified human peritoneal macrophages in the treatment of gastric cancer

Abstract

Background: Gastric cancer is one of the most common cancers. Peritoneal carcinomatosis (PC) appears to be the most common pattern of recurrence, and more than half of the GC patients eventually die from PC. Novel strategies for the management of patients with PC are urgently needed. Recently, rapid progress has been made in adoptive transfer therapy by using macrophages as the effector cells due to their capabilities of phagocytosis, antigen presentation, and high penetration. Here, we generated a novel macrophage-based therapy and investigated anti-tumoral effects on GC and potential toxicity.

Methods: We developed a novel Chimeric Antigen Receptor-Macrophage (CAR-M) based on genetically modifying human peritoneal macrophages (PMs), expressing a HER2-FcεR1γ-CAR (HF-CAR). We tested HF-CAR macrophages in a variety of GC models in vitro and in vivo.

Results: HF-CAR-PMs specifically targeted HER2-expressed GC, and harboured the FcεR1γ moieties to trigger engulfment. Intraperitoneal administration of HF-CAR-PMs significantly facilitated the HER2-positive tumour regression in PC mouse model and prolonged the overall survival rate. In addition, the combined use of oxaliplatin and HF-CAR-PMs exhibited significantly augment anti-tumour activity and survival benefit.

Conclusions: HF-CAR-PMs could represent an exciting therapeutic option for patients with HER2-positive GC cancer, which should be tested in carefully designed clinical trials.

Conflict of interest statement

The authors declare no competing interests.

© 2023. The Author(s).

Figures

Fig. 1. Construction of HER2-specific CAR in primary human peritoneal macrophages.

a Schematic diagram of experimental design. b Flow cytometry analysis of human primary peritoneal macrophages marker CD11b and CD68. c Construction of chimeric antigen receptor peritoneal macrophages targeting HER2. d Detection of GFP expression in peritoneal macrophages transfected with CAR plasmid by fluorescence microscopy and flow cytometry. e Flow cytometry analysis of the binding of PE-conjugated HER2 protein with HER2-specific scFv on the surface of CAR-PMs. f Phosphorylation levels of downstream transcription factors Syk, Lyn, AKT of CAR-modified PMs and control cells after HER2 protein stimulation for 48 h.

Fig. 2. Phenotypic and functional characteristics of CAR-modified PMs under antigen stimulation.

a Phenotypic markers were detected by treatment with LPS (100 ng/mL), IL-4 (20 ng/mL), HER2 antigen for 48 h. MHC-II to characterise M1polarization and CD206 for M2 polarisation, respectively. b Assessment of ROS release from various groups of macrophages using the fluorescent probe DCFH-DA. c Expression of genes related to M1 and M2 phenotype was detected by q-PCR after LPS (100 ng/mL) treatment or co-culture with HER2+ gastric cancer cells MKN45 for 48 h. d The effect of LPS (100 ng/mL), IL-4 (20 ng/mL), and HER2 antigen treatment on phagocytosis of peritoneal macrophages after 48 h. e Flow cytometry analysis of T-cell proliferation. HF-CAR-PMs and HG-CAR-PMs were co-cultured with MKN45 for 48 h. Then the cells were co-cultured with CFSE-labelled T cells for 3 days after density gradient centrifugation.

Fig. 3. CAR-modified PMs exhibited antigen-specific cytotoxicity against GC cells in vitro.

a Fluorescence of MKN45 cells at different effector-target ratio (macrophage:tumour = 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10) was detected by spectrophotometer. Assessment of tumour cell killing using MKN45 cells alone as a baseline. b Phagocytosis of GFP-positive macrophages co-cultured with DiR-labelled MKN45 cells. c Apoptosis-related protein expression in MKN45 cells co-cultured with HF-CAR-PMs and HG-CAR-PMs. d Expression of inflammatory factors after co-culture of MKN45 gastric cancer cells with HF-CAR-PMs and HG-CAR-PMs. e Annexin V/PI staining for assessment of MKN45 cell apoptosis in HF-CAR-PMs and HG-CAR-PMs treatment. f Microscopic image of MKN45 tumour spheres. g Microscopic image of GFP labelled HF-CAR-PMs on MKN45 tumour spheres. h Infiltration of GFP labelled HF-CAR-PMs in tumour spheres at different effector/target ratio (macrophage:tumour = 1:3, 1:1, 3:1). The fluorescence intensity was quantified by Image J. i Tumor cell killing activity of HF-CAR-PMs and HG-CAR-PMs derived from other two donors. (Statistical analysis was performed by Student’s t test when only two value sets were compared. One-way ANOVA followed by Dunnett’s test was used when the data involved three or more groups).

Fig. 4. The effect of CAR-modified PMs on tumour growth in vitro.

a The schematic representation of the experimental design. Male BALB/c nude mice (6–8 weeks old) were injected intraperitoneally with 1 × 106 MKN45-Luc cells/each, and seven days later groups were treated with PBS, 1 × 107 cells/100ul per mouse HG-CAR-PMs cells; low-dose 1 × 106, medium-dose 1 × 107 and high-dose 1 × 108 HF-CAR-PMs cells/100 μL per mouse for the HF-CAR-PMs treatment groups. b, c Representative image and quantitative analysis of tumour weight in the vehicle, HG-CAR-PMs, and HF-CAR-PMs treatment groups. d Quantitative analysis of spleen weight for the vehicle, HG-CAR-PMs, and HF-CAR-PMs treatment groups. e Quantitative analysis of fluorescence intensity captured every 7 days for each group. Tumour-bearing mice were injected intraperitoneally with 2 mg of luciferase substrate solution each (Fluorescence intensity range 8 × 107–2 × 109). f Representative bioluminescence imaging and survival curve of each treatment group. Statistical analysis by one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 5. Oxaliplatin combined with HER2-specific CAR macrophages inhibits gastric cancer growth in tumour-bearing mice.

a Schematic diagram of the intraperitoneal dissemination model and experimental design. BALB/c nude mice were injected with 1 × 106 of MKN45-Luc cells. Seven days later, mice were treated with PBS, HG-CAR-PMs, HF-CAR-PMs, oxaliplatin, and combo therapy (HF-CAR-PMs & oxaliplatin). b–d Quantification analysis of tumour weight and tumour inhibition rate in each treatment group. e Number of tumour nodules in the abdominal cavity of nude mice (nodules <3 mm). f Quantification analysis of spleen weight for each treatment group. g Tumour growth during treatment. h Representative bioluminescence imaging and survival curve of each treatment group.

Fig. 6. Safety evaluation of CAR-modified PMs in combination with oxaliplatin for solid tumours.

a Hematoxylin–eosin (H&E) staining of the heart, liver, spleen, lung, kidney, and tumour in each group of mice. b Expression of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (Cr) in peripheral blood of mice serum. c Body-weight changes in mice receiving different treatment. d Representative in vivo images (IVIS) and quantification of luciferase in mice organs.