Functional Study Of UDP/P2Y₆ Regulated Macrophages In Tumor Formation And Progression

Tumor-associated macrophages have been regarded as the most abundant infiltrating immune cells in tumors. They play key roles in the process of angiogenesis, immune suppression, tumor invasion and metastasis, which makes them as an important target for cancer immunotherapy. More and more evidences demonstrate that cells and active substances are also involved in TAMs to regulate tumor formation and progression.Recently, many studies have shown that cells can release nucleotides to regulate the immune system in the case of stress and damage. Our previous work also indicated that immune cells, especially macrophages, can release UDP. The released UDP binds to their specific receptor P2Y6, which significantly enhance the antibacterial and antiviral ability of hosts. Meanwhile, we also found tumor cells release UDP in the case of stress. However, the function of UDP and its receptor P2Y6 in the tumor immune microenvironment is little deomnstrated. Therefore, in this study, we will use P2Y6 knockout mice injected with lung cancer cells or melanoma to explore the function of UDP/P2Y6 in the regulation of tumor microenvironment.To determine the correlation between UDP/P2Y6 and tumor progression, we used cBioPortal, GENT, Oncomine, Kaplan Meier plotter to make a comprehensive analysis of the corelation between P2Y6 and clinical tumor tissues. These data showed that P2Y6 is highly expressed in many tumor tissues, including lung cancer, melanoma, breast cancer and so on. The data from the clinical databases also suggests the releationship of high expression of P2Y6 with poor prognosis of lung cancer patients. Besides, IHC is used to evaluate the protein expression level of P2Y6.Which is consistent with bioinformatics analysis, the expression of P2Y6 is also dramatically increased in lung tumor tissues. Interestingly, high expression of P2Y6 does not only exist in cancer cells but also exist in monocytes infiltrating in the tumor tissues, which suggest the potential role of UDP/P2Y6 in the regulation of tumor immune microenvironment.To explore the function of P2Y6 in regulation of microenvironment, we generate P2Y6 knock-out mice and use Lewis lung cancer cells or melanoma cells to generate tumor-bearing mice model. Results showed that tumors in P2Y6 knock-out mice are obviously smaller than in wild-type mice. Both tumor models with LLC and B16 have the same phenotype. Compared with T cells and granulocyes, the ratio of M1 macrophages from P2Y6 knock-out mice obviously increased in tumors tissues, which suggests the important role of P2Y6 in regulation of TAMs. We further co-inject LLC cells with P2Y6 wild-tpye or knock-out BMDMs to generate another tumor-bearing mice model. Accordingly, LLC cells co-injected with P2Y6 knock-out BMDM generate smaller tumors and a higher ratio of M1 macrophages.In order to discuss the function of P2Y6 in regulation of macrophages, we investigate P2Y6 expression and UDP release during the macrophages differentiation. Results show that both the expression of P2Y6 and UDP release increased during macrophages differentiation. However, there is no significant difference of Ly6C+ monocytes in bone marrow and F4/80+ macrophages in spleen between P2Y6 wild-type and knock-out mice.To further determine the role of UDP/P2Y6 in regulation of macrophages, we use Q-PCR and ELISA to detect various cytokines and chemokines in UDP treated cells These results indicated that UDP can significantly up-regulate the expression of monocyte chemotactic protein-1 (MCP-1). Then we further proved that UDP/P2Y6 inreased MCP-1 could be rescused by MRS2578 and P2Y6 knock-out mice. Moreover, Western Blotting data shows that ERK assiciate signaling can be activated by UDP.In summary, deficiency of P2Y6 in tumor microenvironment can dramatically restrain the progression of tumor with increasing ratio of M1 macrophages and restrict the prouction of MCP-1 which paly key roles in both M2 like macrophage polarization and recruiting TAMs. Our findings further enrich the understanding of important function of macrophages in tumor immune environments and lay a foundation for the exploration of anti-tumor drugs based on purinergic signaling.