Cannabinoid Receptor Type-2 in B Cells Is Associated with Tumor Immunity in Melanoma

Abstract

Agents targeting the endocannabinoid system (ECS) have gained attention as potential cancer treatments. Given recent evidence that cannabinoid receptor 2 (CB2R) regulates lymphocyte development and inflammation, we performed studies on CB2R in the immune response against melanoma. Analysis of The Cancer Genome Atlas (TCGA) data revealed a strong positive correlation between CB2R expression and survival, as well as B cell infiltration in human melanoma. In a murine melanoma model, CB2R expression reduced the growth of melanoma as well as the B cell frequencies in the tumor microenvironment (TME), compared to CB2R-deficient mice. In depth analysis of tumor-infiltrating B cells using single-cell RNA sequencing suggested a less differentiated phenotype in tumors from Cb2r^-/- mice. Thus, in this study, we demonstrate for the first time a protective, B cell-mediated role of CB2R in melanoma. This gained insight might assist in the development of novel, CB2R-targeted cancer therapies.

Keywords: CB2R; cannabinoid receptor type-2; endocannabinoid system; melanoma; regulatory B cells.

Figure 1

CNR2 is positively associated with increased overall survival (OS) in human melanoma patients and is primarily expressed in B cells. (A) Survival curves of melanoma patients with high vs. low CNR2 expression (top vs. bottom quartile) up to 5 years OS; log-rank test. Median survival for CNR2 high (n = 114) and low (n = 117) was 3818 and 730 days, respectively. (B) Top 20 genes predictive for CNR2 expression in melanoma as determined by lasso regression. Red bars represent B-cell-related genes; n = 472. (C) Gene set enrichment analysis of genes with lasso coefficient > 0 (n = 73) using Human Gene Atlas library. (D) CNR2 expression in various cell types as determined by single-cell RNA sequencing; Kruskal–Wallis test followed by Dunn’s multiple comparisons testing. Bar graphs are shown as mean +/− SEM. * p ≤ 0.05; **** p ≤ 0.0001. (E) Representative RNAscope-derived immunofluorescence images on human melanoma tissue sections.

Figure 2

CB2R regulates tumor growth and B cell infiltration in murine melanoma. (A) Tumor growth shown as mean ± SEM. Statistical significance was determined by two-way ANOVA followed by Šidák’s multiple comparisons test, n = 10. (B–F) Frequencies of indicated immune cell types at day 14 post-tumor inoculation as determined by flow cytometry, gated on live CD45⁺ cells. n = 17–18; two-tailed, unpaired Student’s t-test. * p ≤ 0.05; *** p ≤ 0.001.

Figure 3

Tumor-infiltrating B cells from Cnr2^−/− mice have reduced metabolic activity, along with impaired differentiation and activation. Tumor-infiltrating B cells from wt and Cnr2^−/− mice were purified using flow cytometry at day 14 post-tumor inoculation and analyzed by means of scRNA-seq. (A–C) UMAP dimensionality reduction of 6381 combined B cells. (D) Heatmap showing the top 10 upregulated genes (highest fold change) for each cluster. (E) Gene ontology analysis for biological processes of downregulated genes with log fold change threshold of 0.25 and minimum prevalence of 0.2 from cluster 0 (n = 37) and (F) cluster 1 (n = 56).

Figure 4

CB2R mediates tumor growth control by regulating Breg infiltration and Treg induction. Tumors from wt and Cnr2^−/− mice were isolated at day 14 post-tumor inoculation and analyzed by means of flow cytometry. (A) Representative flow cytometry plots and (B) frequencies of intratumoral Foxp3⁺ cells as percentages of CD4⁺ T cells in wt and Cnr2^−/− mice (n = 6). (C) Intratumoral Foxp3⁺ cell frequencies in Cnr2^−/− mice with or without B cell depletion using anti-CD197 and anti-B220 monoclonal antibodies (mAbs). Statistical analyses were performed using two-tailed, unpaired Student’s t-tests. Error bars show mean ± SEM. * p ≤ 0.05; *** p ≤ 0.001.