CB2R activation enhances tumor-associated macrophages-mediated phagocytosis of glioma cell

Abstract

Background: Cannabinoid administration has demonstrated promising anti-tumor effects for glioblastoma (GBM) by inhibiting glioma cell proliferation and inducing glioma cell death. However, the impact of cannabinoids and endocannabinoid receptors on immune cells within the tumor microenvironment (TME) remains largely unexplored. Tumor-associated macrophages (TAMs), the most abundant immune cells in the TME, and their mediated phagocytosis of tumor cells have shown potential in preclinical xenografts of various human malignancies. This study aimed to investigate the effect and mechanism of endocannabinoid receptor 2 (CB2R) on TAMs-mediated phagocytosis in xenografted mice with GL261-GFP cell lines.

Methods: We measured the phagocytic activity using immunofluorescence and flow cytometry, and we used the IVIS Spectrum System for bioluminescent imaging to track the growth of the tumor.

Results: Our findings demonstrated that administering JWH133, a selective CB2R agonist, significantly boosted TAMs-mediated phagocytosis. However, administering AM630, a selective CB2R antagonist, significantly inhibited TAMs-mediated phagocytosis. Mechanistically, CB2R activation upregulated the expression of CD36 on TAMs, a scavenger receptor known to facilitate phagocytosis. Furthermore, sulfo-N-succinimidyl oleate (SSO), an irreversible CD36 inhibitor, could reverse the CB2R activation-induced enhancement of phagocytosis by TAMs. Additionally. JHW133 also effectively augmented the chemotherapeutic efficacy of temozolomide.

Conclusion: Overall, our findings show that CB2R activation promotes TAMs-mediated phagocytosis of tumor cells by enhancing CD36 expression, implying that JWH133 could be a useful therapeutic approach to improving chemotherapeutic efficacy against GBM.

Keywords: CD36; Cannabinoid receptor 2; JWH133; Phagocytosis; Tumor-associated macrophages.

Fig. 1

CB2R activation impairs GBM growth and improves the survival of xenografted mice. (A) Images of the bioluminescence intensity obtained on day 14 post-implantation of representative mice from each treatment group. (B) Tumor progression quantification for each mouse based on flux data obtained using bioluminescence intensity photometry. (C) Kaplan-Meier (K–M) analysis of the survival of each treatment group's mice carrying GL261 xenografts. The format of mean ± SEM is used to display the data. Each group contains n = 6–10 mice. One-way ANOVA with the Bonferroni correction was used to determine ∗P < 0.05 and ∗∗∗∗P < 0.0001 relative to the control group (B). The K-M method was used for survival analysis, and the log-rank test was used as a comparison (C).

Fig. 2

CB2R activation promotes TAMs-mediated phagocytosis of glioma cells. (A) Representative immunofluorescence images of the xenografted brain stained with GFP (green) and CD11b (red) in mice injected with Vehicle, JWH133 or AM630. White arrows indicated the phagocytes. Scale bar: 10 μm. (B) Representative flow cytometry plots depicting the phagocytosis of GFP-labeled glioma cells with CD11b-labeled TAMs in the presence of Vehicle, JWH133, or AM630. (C) The determination of the proportion of phagocytosed GFP cells in mice that received injections of Vehicle, JWH133, or AM630. (D) Quantification of the percentage of GFP and CD11b double-positive cells among all the sorted cells in mice injected with Vehicle, JWH133, or AM630. The format of mean ± SEM is used to display the data. Each group contains n = 6 mice. One-way ANOVA with the Bonferroni correction was used to determine ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗∗P < 0.0001 relative to the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

CB2R activation promotes TAMs-mediated phagocytosis of glioma cells via increasing the expression of CD36 in TAMs. (A) Representative western blot bands of CD36 and GAPDH from flow cytometry sorted CD11b positive cells in xenografted mice injected with Vehicle, JWH133, or AM630 at day 14 after implantation. (B) Quantification of CD36 and GAPDH from flow cytometry sorted CD11b positive cells with Vehicle, JWH133, or AM630 at day 14 after implantation. (C) Representative immunofluorescence images of xenografted brain tissue depict GFP (green) and CD11b (red) staining in mice injected in each group. White arrows indicated the phagocytes. Scale bar: 10 μm. (D) The determination of the proportion of phagocytosed GFP cells in mice in each group. (E) Representative flow cytometry plots depicting the phagocytosis of GFP-labeled glioma cells with CD11b-labeled TAMs in each group. (F) Quantification of the percentage of GFP and CD11b double-positive cells among all the sorted cells in each group. The format of mean ± SEM is used to display the data. Each group contains n = 6 mice. One-way ANOVA with the Bonferroni correction was used to determine ∗P < 0.05 and ∗∗∗P < 0.001 relative to the control group (B). Student t-test was used to determine ∗∗P < 0.01 relative to the JWH133 group (D and F). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

JWH133 enhances the therapeutic efficacy of temozolomide (TMZ). (A) Images of the bioluminescence intensity obtained on day 14 post-implantation of representative mice from each treatment group. (B) Tumor progression quantification for each mouse based on flux data obtained using bioluminescence intensity photometry. (C) Kaplan-Meier (K–M) analysis of the survival of each treatment group's mice carrying GL261 xenografts. Each group contains n = 6–10 mice. One-way ANOVA with the Bonferroni correction was used to determine ∗∗∗P < 0.001 relative to the control group and ^##P < 0.01 relative to the TMZ group (B). The K-M method was used for survival analysis, and the log-rank test was used as a comparison (C).