Previous research on CBD has focused on the nervous system as it is easy to pass through the blood-brain barrier. In addition, the anti-proliferative effects of CBD have long been explored to treat malignant tumors. Studies have shown that CBD could inhibit the proliferation of human glioma cells effectively both in vivo and in vitro. CBD could also inhibit tumor growth significantly in mouse orthotopic models, while further mechanism studies have shown that CBD can induce autophagy and cause tumor cell death. It has also been revealed that CBD can not only inhibit ERK signaling in human glioma cells, but also inhibit the expression of hypoxia-inducible factor HIF1a, thereby dually inhibiting tumor proliferation as well as tumor angiogenesis. In addition to its role in nervous system tumors, many studies have reported the anti-tumor effect of CBD on colorectal cancer. Increasing evidence has shown that CBD activates ROS production in colorectal cancer cells, causing mitochondrial dysfunction and cell death. Moreover, CBD may be used as an adjuvant to chemotherapy in treating colorectal cancer, and the combination of CBD with chemotherapy drugs such as 5-Fu and oxaliplatin may enhance its anti-tumor effect. Despite the impacts on the tumor cells, whether CBD would inhibit tumor progression through remodeling TME remains unclear. Our study provided a new insight that CBD would exert anti-tumor effects, at least in part by modulating immune responses.

Single-cell transcriptome sequencing, as a rapidly developing frontier technology in life sciences, has dramatically advanced disease research. Here, using single-cell transcriptome sequencing technology, we detected cellular components in the TME after CBD treatment and found that CBD significantly altered the number and proportion of myeloid cells in the TME. It is well known that myeloid cells play a critical role in tumor progression. Further, we divided myeloid cells into five populations, including classically activated M1 and alternatively activated M2 macrophages. CBD significantly decreased the number of M2 macrophages and, correspondingly, the number of M1 macrophages was increased, which significantly promoted anti-tumor immunity. This result was further confirmed by immunofluorescence staining. Accordingly, we hypothesized that CBD may affect the alternative activation of macrophages in the tumor microenvironment and therefore examined the effect of CBD on macrophage polarization in vitro. CBD was found to inhibit IL-4-induced expression of anti-inflammatory genes, which was largely dependent on the inhibitory effect of CBD on PI3K-Akt signaling. Macrophage activation involves extensive metabolic reprogramming, glycolytic switching, and phospholipid remodeling. Recent studies have shown macrophage polarization signals affect metabolic signaling pathways. As a metabolic regulatory signal, mTOR-PI3K has been identified as a key regulator of the macrophage switch between immune stimulation and suppression. This is consistent with our research that CBD indeed affects the metabolic shift of macrophages. Under IL-4-induced anti-inflammatory polarization conditions, CBD inhibited oxidative phosphorylation and fatty acid oxidation levels while promoting glycolysis in macrophages. Most importantly, CBD treatment substantially enhanced the efficacy of immune checkpoint inhibitor anti-PD-1 in a mouse model. Consistent with previous results, CBD decreased the number of pro-tumor macrophages and increased the number of anti-tumor macrophages in tumors. These findings suggest that CBD rewires TME through inhibiting the alternative activation of macrophages, resulting in the inhibition of tumor-bearing mice.
Studies have reported that THC could impair the function of tumor-specific T cells through cannabinoid receptor 2, and thereby, reduce the therapeutic effect of PD-1 blockade. We did not observe such results with CBD in vitro. Consequently, further research focusing on the regulation of CBD on the anti-tumor immune system is needed. Clarification regarding the specific targets of CBD regulating macrophages should also be conducted. To sum up, this study demonstrated that CBD inhibits the alternative activation of macrophages to modulate the suppressive tumor immune microenvironment and improves the therapeutic outcome of cancer immune therapies. Our study provides new insights into the anti-tumor effects of CBD, which might be a theoretical basis for the future application of cannabinoids in the anti-tumor market.
