**8. Neutrophils**

Neutrophils primarily work as an antibacterial immune response, but tumor-associated neutrophils (TANs) also play important roles in tumor microenvironments. Similar to M1 and M2 macrophages, terms for antitumoral N1 neutrophils and protumoral N2 neutrophils were proposed [54]. The chemokines CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8 secreted from tumor cells attract neutrophils in the blood to the tumor microenvironment via CXCR1 and CXCR2 on the surface of neutrophils [55]. TANs share a similar surface marker with PMN-MDSCs. Murine neutrophils are defined as CD11b+/GR1+/Ly6G+cells, whereas PMN-MDSCs are defined as CD11b+/GR1high/Ly6G+ cells. PMN-MDSCs were named based on the function of immunosuppression. However, neutrophils can work in immunosuppression, but also have the opposite function of anti-tumor activity. N1 TANs function in tumor cell cytotoxicity, CD8+ T cell recruitment, and antibody-dependent cell-mediated cytotoxicity. In contrast, N2 TANs play roles in angiogenesis, immunosuppression, and tumor growth via several cytokines or proteins released from TANs [55]. In a mouse model, obesity caused the increase of neutrophils in the lung and promoted the metastasis of breast cancer cells to the lung in a GM-CSF- and IL-5-dependent manner [56]. In HFD-fed mice, cholesterol metabolites promoted the metastasis of breast cancer via neutrophils and γδ-T cells [57]. In another mouse model, obesity promoted the progression of pancreatic cancer and resistance to chemotherapy via TANs recruited by adipocyte-secreted IL1β [58]. Murine neutrophils are different from human neutrophils. Thus, it is still unclear whether the TANs play roles in prostate cancer progression in the late stage. The administration of cabozantinib resulted in the clearance of prostate cancer in mice by recruiting neutrophils to the tumor [59].

In humans, no markers equivalent to the mouse Gr1 marker exist, and human neutrophils are defined as CD14-/CD15+/CD66b+/CD16+. The neutrophil-lymphocyte ratio in peripheral blood is associated with a high Gleason score and the poor prognosis of men with prostate cancer [60,61]. The ratio is also a prognostic factor of abiraterone and docetaxel treatment in men with castration-resistant prostate cancer [62,63]. Low serum neutrophil count is a predictor of positive prostate biopsy results [64]. The presence of neutrophils in the epithelial lining of the prostate gland indicate prostatic inflammation and is a predictive factor of benign biopsy [65]. The protumor roles of neutrophils in human prostate cancer have not been confirmed yet, and further studies are warranted.

### **9. B Cells and Complements**

In the mRNA microarray analysis of prostate tumors in CD- and HFD-fed double knockout mice, the expressions of immune-related genes including splice variants of immunoglobulins, complements (*Hc*, *C4b*), *Ccl8*, and *Cd52* were significantly higher in HFD-fed double knockout model mice compared with CD-fed double knockout model mice (Table 1). Gene ontology analysis revealed that humoral immune responses were key factors of HFD-induced tumor progression (Figure 3C). These results suggested that B cell-mediated and immunoglobulin-mediated immune responses could be key factors of HFD-induced prostate cancer growth. B cells play important roles in diet-induced obesity, chronic inflammation, and humoral immunity, the latter two of which are influenced by some kinds of fatty acids and lipid mediators [66]. B cells are also related with tumor progression in various types of cancer, including prostate cancer [67]. Tumor-infiltrating B cells produce lymphotoxin, a cytokine belonging to the TNF family, that leads to activation of IκB kinase α and STAT3, which promote the survival and proliferation of androgen-deprived prostate cancer cells that result in the development of a castration-resistant state in experiments using the TRAMP mice model [68]. It was reported that higher B cell infiltration was present within the intra-tumoral prostate cancer regions compared to the extra-tumoral benign prostate tissue regions in prostatectomy sections [69]. Immunoglobulins are expressed by B cells and a variety of tumor tissues and cancer cell lines [70,71]. Immunoglobulins are suggested to play important roles in promoting cancer progression. Immunoglobulin G silencing induced apoptosis and suppressed proliferation, migration, and invasion in LNCaP prostate cancer cells [72].




**Table 1.** *Cont*.

A complement system is also related to cancer progression [73,74]. Complement activation in the tumor microenvironment enhances tumor growth and increases metastasis. The hemolytic complement encoded by the *Hc* gene in mice, the expressions of which were increased in the prostatic tissues of HFD-fed mice in our results, corresponds to C5 in human. C5, one of the complements, was suggested to promote tumor progression controlling the tumor microenvironment [75,76]. A humoral immune response including B cells, immunoglobulins, and complements could be key factors of prostate cancer progression induced by inflammation in the late stage. The detailed mechanism of the phenomenon remains unclear, and further investigations are necessary to explore the causative mechanism.
