I'd like to point out that a mouse study should not be rejected out of hand because it is not a human study.
"Purpose: Inflammatory infiltration plays important roles in both carcinogenesis and metastasis. We are interested in understanding the inhibitory mechanism of metformin on tumor-associated inflammation in prostate cancer.
"Experimental Design: By using a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model, in vitro macrophage migration assays, and patient samples, we examined the effect of metformin on tumor-associated inflammation during the initiation and after androgen deprivation therapy of prostate cancer.
"Results: Treating TRAMP mice with metformin delays prostate cancer progression from low-grade prostatic intraepithelial neoplasia to high-grade PIN, undifferentiated to well-differentiated, and PIN to adenocarcinoma with concurrent inhibition of inflammatory infiltration evidenced by reduced recruitment of macrophages. Furthermore, metformin is capable of inhibiting the following processes: inflammatory infiltration after androgen deprivation therapy (ADT) induced by surgically castration in mice, bicalutamide treatment in patients, and hormone deprivation in LNCaP cells. Mechanistically, metformin represses inflammatory infiltration by downregulating both COX2 and PGE2 in tumor cells.
"Conclusions: Metformin is capable of repressing prostate cancer progression by inhibiting infiltration of tumor-associated macrophages, especially those induced by ADT, by inhibiting the COX2/PGE2 axis, suggesting that a combination of ADT with metformin could be a more efficient therapeutic strategy for prostate cancer treatment. Clin Cancer Res; 24(22); 5622–34. ©2018 AACR."
"We demonstrated that in the TRAMP model, metformin is capable of delaying multiple processes in prostate cancer progression including from LGPIN to HGPIN, from WD to UD, and from PIN to adenocarcinoma as well as from adenocarcinoma to NEPC. These effects were accompanied by repressed levels of COX2 and its product PGE2 in tumor cells as well as inhibited inflammatory infiltration. These findings were further substantiated by the results from both prostate cancer patient samples and prostate cancer cell model. Altogether, these findings suggest that administration of metformin alone or in combination with ADT could be more beneficial to prostate cancer patients.
"Inflammatory infiltration has been considered as a double-edged sword in tumor biology because it can either aid or fight tumors depending on specific tumor microenvironment. Although some studies found that in certain circumstances inflammatory infiltration could be inhibitive in tumor progression by maintaining organ homeostasis and ensuring stable tissue structure, more evidence supports the conclusion that chronic inflammation contributes to tumor initiation, metastasis, and progression (3). A meta-analysis by Martel and colleagues found that 15% cancers could be directly attributed to the infection of viruses, bacteria, and parasites (38); and individuals with chronic inflammation generally have high cancer incidence (39). Furthermore, the number of infiltrated inflammatory cells has been suggested as a hallmark of a tumor (40). Our tissue microarray data showed that the numbers of TAM infiltrated in the TME were positively correlated with Gleason scores, suggesting TAM infiltration is also associated with the malignancy of prostate cancer, and these findings were in line with the number of TAMs and the severity of tumors in our mouse model.
"Due to their plasticity and flexibility, monocytes differentiate into macrophages with distinct phenotypes depending on their microenvironment (41). There are two main subtypes of macrophages. The M1-like macrophages promote Th1 response with strong microbicidal and tumoricidal activity, and the M2-like macrophages usually promote Th2 response, tissue remodeling, immune tolerance, and tumor progression (42). Under certain circumstances, the subtypes are interchangeable depending on their local microenvironment (2, 43). In addition, M2-like macrophages can be further divided into four subgroups (M2a, M2b, M2c, and M2d). More recent evidence suggests that TAMs and M2d subtype share more characteristics such as promoting tumor growth, metastasis, and angiogenesis (44). Similarly, as shown by Joyce and Pollard, multiple cell surface markers specific for M2-like macrophage have been identified. However, not all markers were found on the surface of every M2-like cell. This finding is consistent with that not all M2-like markers are necessarily required for every cell (3). IHC staining of the consecutive sections of human lymphoma (positive control) using three markers (CD68, CD163, and CD204) commonly used for identification of M2-like macrophages (3, 45) showed that these markers were not completely colocated (Supplementary Fig. S4B). In addition, by using immunofluorescent double and triple labeled staining, we found that most of the macrophages express either two (CD163 and CD204) or three (CD68, CD163, and CD204) markers simultaneously, although a few cells only expressed one of them (Supplementary Fig. S4C). Although the intensities of immunostaining of these markers varied noticeably in our results, the overall intensities of these markers were significantly reduced in the metformin-treated group, indicating that metformin is capable of inhibiting the recruitment of TAMs in the TME.
"Multiple lines of evidence imply that infiltrated TAMs interact with tumor cells and TAMs play crucial roles in most, if not all, processes of tumor development. Activation of transcription factors such as NF-κB, STAT3, and HIF1α in tumor cells by either inflammation or infection leads to the secretion of wide spectrum factors including cytokines, chemokines, and prostaglandins. These factors collectively result in the recruitment of TAMs, and inflammatory mediators secreted by the TAMs lead to further recruitment of more TAMs. Through some ill-defined mechanisms, the TAMs enhance different processes in cancer initiation and development including proliferation, survival, EMT, angiogenesis, migration, invasion and metastasis, as well as the development of resistance to various treatments (6). In this study, we demonstrated that metformin is capable of inhibiting TAM recruitment both in vivo and in vitro and reduced TAM recruitment concurrently accompanied by less tumor cell metastasis. Elevated levels of COX2 in prostate cancer cells were seen in both the TRAMP model (28) and human prostate adenocarcinoma (46). We demonstrated in this study that metformin treatment not only downregulated COX2 and its product PGE2 in prostate cancer cells but also inhibited the recruitment of TAMs (Fig. 6M). On the other hand, exogenously added PGE2 was able to counteract metformin-mediated downregulation of COX2 and rescue the recruitment of TAMs as well as cancer cell migration, suggesting PGE2 plays a crucial role in TAM recruitment. Of note, fewer TAMs in the TME were accompanied by reduced cytokines and chemokines. These lines of evidence are consistent with the inhibitory role of metformin in prostate cancer cell migration (21) and macrophage recruitment (25). Therefore, we conclude that the inhibitory effect of metformin on the recruitment of TAMs and cancer cell migration is at least in part by directly downregulating COX2, which subsequently reduced the levels of PEG2. In addition, by using PC-3 and DU145 prostate cancer cell lines, we demonstrated that metformin might have some direct inhibitory effects on proliferation and cell cycle, as well as acceleration of the apoptosis (Supplementary Fig. S5). Moreover, we found that metformin could also inhibit the functions of macrophages, such as producing cytokines IL6 and TNFα induced by LPS (Supplementary Fig. S6).
"Multiple lines of evidence showed that metformin possesses anticancer effects on various tumors including prostate cancer (47). Recent clinical trials have found that treatment with metformin yielded objective prostate-specific antigen response and slowed down progression of chemotherapy-naïve CRPC (48). Moreover, a systemic review and meta-analysis found that metformin use was associated with reduction of biochemical recurrence risk although only marginally (49). However, the exact underlying mechanisms are not completely understood. Here, in this study, we found that metformin exerts its anticancer effects by inhibiting the COX2/PGE2 axis. In fact, the inhibitory effects of metformin on the COX2 expression have been described previously in ovarian hyperstimulation syndrome (50), vascular smooth muscle cells (51), as well as a variety of cancers including bladder cancer (29) and pancreatic cancer (52). It has been suggested that metformin might regulate COX2 via activating AMP-activated protein kinase (AMPK) evidenced by that compound C, a specific AMPK inhibitor, and AMPK siRNA could rescue metformin-mediated COX-2 expression (51). Metformin may also exert its inhibitory effect on COX2 by inhibiting inflammatory mediators NF-κB and STAT3 (52).
"It is well established that coxibs can serve as an analgesic drug due to its inhibitory effect on COX-2. In this study, we found that metformin is also capable of inhibiting prostate cancer progression partially through repressing COX-2. Therefore, this represents a potential for using metformin as an analgesic drug. However, results from different studies are kind of controversial. Multiple lines of evidence indicate that metformin possesses antinociceptive properties in different models of inflammatory pain (53) and diabetic neuropathic pain (54), as well as in humans (55). However, a more recent study using carrageenan-induced thermal hyperalgesia animal model did not observe any antihyperalgesic effect when metformin is either locally (800 μg/paw) or systemically administered (200 mg/kg; ref. 56). Therefore, whether metformin possesses any algesic effect as coxibs does still need to be further clarified clinically. However, these controversies do not prevent the potential application of metformin as an anticancer reagent.
"In conclusion, our in vitro and in vivo data demonstrated that metformin is capable of inhibiting prostate cancer initiation and progression by repressing TAM infiltration partially through targeting the COX2/PGE2 axis. It appears that metformin can also reverse ADT-induced inflammatory infiltration through a similar mechanism. Together with our previous findings that metformin is capable of inhibiting castration-induced EMT (21, 57), the data from this study strongly suggest that combined treatment of ADT and metformin possesses a potential to be developed as an effective treatment for prostate cancer at different stages. However, more clinical trials are needed before combination of ADT and metformin in prostate cancer treatment can be considered clinically."