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.

This article is featured in Highlights of This Issue, p. 5491

Translational Relevance

Given the fact that inflammatory infiltration usually occurs with androgen deprivation therapy (ADT) and metformin is capable of repressing prostate cancer progression by inhibiting the infiltration of tumor-associated macrophages, we propose that a combination of ADT with metformin could be a more effective therapeutic strategy in prostate cancer treatment.

Tumor microenvironment (TME) is the overall cellular surroundings of tumor cells formed by blood vessels, extracellular matrix, and some nonmalignant cells, including mesenchymal stem/stromal cells, bone marrow–derived dendritic cells, fibroblasts, and immune cells (1). Tumor-associated macrophages (TAM), one of the most important constituents of the TME, are recruited by tumor cells and subsequently polarized into M2-like cells. Instead of interacting with cancer cell antigens and destroying the cancer cells, M2 macrophages facilitate a protumorigenic function by producing cytokines such as interleukins (IL4, IL6, IL10, and IL13) and TGFβ. This ultimately leads to subsequent disruption of the surrounding tissues and enhances survival, growth, invasion, migration, and dissemination of tumor cells (2). Multiple lines of evidence indicate that TME plays critical roles in not only the initiation, progression, and metastasis but also is implicated in the development of therapeutic resistance of different cancers (3, 4). In addition, stromal cells within the TME have been suggested as an attractive therapeutic target and, due to their genetically stable trait, targeting these cells is usually accompanied with reduced risk of resistance and recurrence (5).

Accumulating evidence implies that inflammatory diseases increase the risk of development of many types of cancers, including bladder, cervical, gastric, intestinal, esophageal, ovarian, colon, thyroid as well as prostate cancer (6). Inflammatory infiltration has been frequently found in prostate biopsies (7), especially the nearby zones of prostatic intraepithelial neoplasia (PIN; ref. 8). Moreover, prostate tissues with histologically confirmed inflammation were not only more likely to promote the development of prostate cancer but also accompanied with higher-grade cancers than those without (9). Two prospective studies found that prostate cancer patients with greater extent of intraprostatic inflammation had poorer outcome (10, 11). The prostatic inflammation can be induced by multiple causal factors, including bacterial infections, viruses, dietary factors, urine reflux as well as androgen deprivation therapy (ADT; ref. 8). It has been demonstrated that ADT-mediated inflammatory infiltration not only promotes tumor cell survival through regulating antiapoptotic signaling pathways (12) but also leads to the development of castration-resistant prostate cancer (CRPC; ref. 13). Consistently, administration of nonsteroidal anti-inflammatory drugs (NSAID) is able to reduce prostate cancer carcinogenesis and progression, presumably through anti-inflammation mechanisms (14). Therefore, the TME contributed by chronic inflammation with macrophage infiltration increases the risk of prostate carcinogenesis and CRPC development by affecting the levels of cytokines, chemokines, transcriptional factors, and reactive oxygen species (15).

Transgenic adenocarcinoma of the mouse prostate (TRAMP) has been serving as one of the best in vivo prostate cancer animal model for many decades. With the expression of viral SV40 oncoprotein in the prostatic epithelium (16), mice develop PIN at 10 to 12 weeks of age with rare prostate cancer. However, invasive prostate adenocarcinoma usually occurs by 18 to 20 weeks, and almost all TRAMP mice become prostate cancer positive at the age of 30 to 36 weeks, and some of the cancer cells may even metastasize to other organs such as lymph nodes, lungs, livers, and bone (17). Although this animal model has some inherent limitations because the viral antigens are not naturally associated with human prostate cancer and even some extensive neuroendocrine differentiation (18), it not only resembles human prostate cancer development and progression (19) but is also suitable for studying the development of resistance to antiandrogen therapy (20).

Although metformin was originally developed as the first-line agent for the treatment of type 2 diabetes, accumulating data suggest that it also possesses antitumor properties for different cancers including prostate cancer evidenced by inhibiting castration-induced prostate cancer progression (21), reducing the risk of cancer in diabetic patients (22) and sensitizing breast cancer cell to neoajuvant chemotherapy (23). Mechanistically, metformin can inhibit cancer cell proliferation, promote cancer cell and cancer stem cell apoptosis, repress epithelial–mesenchymal transition (EMT), and delay the development of therapeutic resistance (24). Data from in vitro experiments suggest that metformin can repress lipopolysaccharide (LPS)-induced inflammatory response in macrophages and endothelial cells (25, 26). Experiments in high-fat-diet-induced obese mice demonstrated that metformin can reduce the level of inflammatory cytokines (27). In this study, we demonstrated that in TRAMP model mice metformin is capable of repressing prostate cancer progression partially by inhibiting infiltration of TAMs via downregulating the levels of cyclooxygenase-2 (COX2) and subsequent production of prostaglandin E2 (PGE2). Moreover, we found that metformin can inhibit ADT-induced inflammatory infiltration in human prostate cancer tissues. These findings suggest that combination of ADT with metformin could be a more efficient therapeutic strategy for prostate cancer treatment.

Animals and experimental procedures

Experiments involving animals were performed in accordance with international laws (EEC Council Directive 86/609, O.J. L 358. 1, December 12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996) and approved by the Institutional Animal Care and Use Committee of Third Military Medical University. Male transgenic TRAMP (C57BL/6) and FVB mice at the age of 5 to 6 weeks were obtained from Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). They were housed and crossed in the Animal Experimental Center of Daping Hospital, Third Military Medical University (Chongqing, China), and their offspring were used for the different treatments. Mice were fed with standard laboratory mouse chow with free access to tap water. They were divided into three groups: normal C57 group (normal, n = 3 per time point), control TRAMP group (control), and TRAMP treated with metformin (5 g/L) group (metformin). The mice in the metformin group access tap water with metformin freely since 4 weeks of age. Pelvic magnetic resonance imaging (MRI, Biospec 70/20USR) were conducted to monitor the development of prostate cancer at week 12 (n = 5 per group), week 25 (n = 5 per group), and week 32 (n = 8 per group). The volumes of the prostates were estimated using the formula: length × width2/2 when the areas of the prostates were the largest revealed by MRI. For castration treatment, TRAMP mice were crossed with FVB mice. TRAMP mice with FVB background were obtained (C57BL/6 TRAMP × FVB) and divided into three groups: control group without treatment (Con, n = 4), castrated mice (Castration group, n = 4), and castrated mice with metformin treatment (castration + metformin group, n = 4). All castration procedure was conducted at age of 20 weeks, and the mice were sacrificed 1 week after castration.

Histologic analysis

Ventral prostate samples were collected from animals and fixed in 10% formalin for 24 hours, and then paraffin embedded for hematoxylin and eosin (H&E). The slides were photographed under a light microscope (Olympus, BX53). The histologic classification of different degrees of prostatic lesions in TRAMP mice was partially based on descriptions made by Roy-Burman P (20). The histologic features were classified according to the following specifications: (i) Normal tissue (NT); (ii) low-grade PIN (LGPIN); (iii) high-grade PIN (HGPIN); (iv) well-differentiated adenocarcinoma (WD-Adeno); and (v) undifferentiated adenocarcinoma (UD-Adeno). Quantitative analysis was conducted as described previously with some minor modifications (28). Briefly, for each animal, 10 random fields were captured at 10× magnification, which was further divided into 4 quadrants. In each quadrant, the most advanced histologic feature was classified as normal, LGPIN, HGPIN, WD-Adeno, or UD-Adeno. Thus, the numbers of each subtype of lesions in each experimental group were established, and the percentages of different subtypes of lesions were compared among different experimental groups.

Patients and tissue samples

All procedures involving human participants were carried out in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All patient samples were collected by the Department of Pathology with approval from the Research Ethics Committee of Daping Hospital, Third Military Medical University and written informed consent was obtained from each patient. A total of 60 prostate samples (range from 54 to 78 years old) collected during surgical radical prostatectomy were used for tissue microarray (TMA). Among them, 16 samples were excluded because no tumor cells were identified. Therefore, 44 specimens containing prostate cancer cells with different Gleason scores were further analyzed, including 7 specimens with Gleason score < 7, 13 with Gleason score = 7, and 24 with Gleason score > 7. To investigate the effect of metformin on ADT-induced inflammatory infiltration, another cohort of 12 patients (range from 54 to 73 years old) was carefully selected from our database. These patients were divided into three groups with 4 patients in each group. Patients underwent radical prostatectomy without ADT and metformin treatments were considered as controls. Those treated with bicalutamide before radical prostatectomy were considered as the Bic group. The Bic + Met group represents those treated with both bicalutamide and metformin before radical prostatectomy. All the clinical information of the patients was supplied in Supplementary Table S1.

Immunohistochemical staining

The embedded tumor specimens were sliced and mounted onto glass slides followed by immunostaining as described previously (21, 29). Primary antibodies against AR (1:200, Abcam), KI67 (1:200, Cell Signaling), CD68 (1:200, Abcam), CD163 (1:150, Origene), CD204 (1:150, Origene), COX2 (1:400 Origene), synaptophysin (1:400, Proteintech), and CD56 (1:600, Proteintech) were used. The staining intensities were scored by certified clinical pathologists (Dr. Qiang Ma and Hualiang Xiao) as described previously (30–32). Of note, the scores of inflammatory markers (CD68, CD163, and CD204) were obtained by counting the positive cells per area (30). Briefly, 10 random or 3 representative fields (for human tissue microarray and mouse tissues, respectively) were captured at 40× magnification. The positively stained cells in each image were counted. The IHC scores of AR and COX2 were computed by multiplying the intensity scores with the extent of staining scores (31). Measurement of Ki67 was reviewed using the IHC pictures at 40× magnification. Briefly, 10 random fields containing tumor epithelial cells each mouse were captured at 40× magnification and the labeling index was then evaluated by two certified pathologists (32). IHCs were photographed under a light microscope (Olympus, BX53).

Immunofluorescence staining

Immunofluorescence stainings for CD68/CD163, CD163/CD204, CD68/CD204, and CD68/CD163/CD204 were carried out on formalin-fixed, paraffin-embedded sections. The sections were incubated with primary antibodies against CD68 (rabbit, Abcam, 1:100), CD163 (mouse, Origene, 1:100), and CD204 (goat, Abcam, 1:2000). Secondary antibodies used for detection are rabbit anti-goat antibodies conjugated to fluorescein isothiocyanate (FITC, 1:200, DINGGUO CHANGSHENG BIOTECHNOLOGY, Beijing, China), goat anti-mouse antibodies conjugated to Alexa Flour 594 (1:200, Zhongshan Gold Bridge Biotechnology), goat anti-rabbit antibodies conjugated to Alexa Fluor 647 (1:200, Affinity Biosciences). The sections were counterstained with DAPI for 15 minutes (KGA215, KeyGen BioTECH) at 37°C, and images were obtained with confocal microscopy (LSM700; Zeiss).

Cell culture and viability assay (MTT or CCK8)

Cell lines (LNCaP, PC-3, DU145, THP1, RAW264.7, RM-1, and WPMY-1) were obtained from Cell Bank of Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences). These cells were cultured at 5% CO2 and 37°C in the corresponding media according to ATCC. Viability assays were conducted with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) dye (5 mg/mL, Sigma) or cell counting Kit-8 (CCK8). The density of the solubilized formazan was read at 490 nm spectrophotometrically (Bio-Rad).

Macrophage recruitment assay

Prostate cancer cells were treated as indicated, and the conditioned media (CM) or control media were collected, diluted with 10% fresh fetal bovine serum (FBS) media (1:1), plated into the lower chamber of transwell plates with a 5-mm pore polycarbonate membrane insert (Corning). THP1 (1 × 105) or RAW264.7 (1 × 105) cells were plated onto the upper chamber for macrophage migration assay. The migrated cells were collected from the lower media and counted or stained with crystal violet and five randomly selected views were counted. Each experiment was repeated at least three times.

Cell invasion and migration assay

Prostate cancer/macrophage cells were cocultured in 24-well transwell plates. Cells were treated as indicated and the CM or control media were collected, diluted with 10% fresh FBS media (1:1), and plated into the lower 24-well plates; and the prostate cancer cells (LNCaP, 1 × 105; RM-1, 3 × 104) were plated in the upper transwell chambers (Corning) with or without matrigel. After incubation at 37°C for 48 hours, cells attached to the upper surface of the membrane were carefully removed with cotton swabs, whereas cells that reached the underside of the chamber were fixed with 10% formalin and stained with crystal violet for 3 minutes at room temperature and counted. Each experiment was repeated at least twice.

Western blotting

LNCaP and RM-1 cells were seeded in 6-well plates, 2 × 105 cells/well, treated with different concentrations of metformin (0, 1, 5, 10, 20 mmol/L). Cell lysates were separated on SDS–PAGE followed by Western blotting assay as described previously (23, 33) with the following primary antibodies: COX2 (1:1,000, Origene) and β-actin (1:5,000, Cell Signaling).

Enzyme-linked immunosorbent assay (ELISA)

As described previously (29), LNCaP, RM-1, and RAW264.7 cells were seeded in 6-well plates and serum starved for 24 hours. The indicated concentrations of metformin were added to the media and cultured for 48 hours. The supernatant was collected and prostaglandin E2 (PGE2) or IL6 and TNFα released to the culture media was measured using commercially available ELISA kits from Cloud-clone corp.

Knocking down COX-2 by siRNA

The sequences of the siRNA against human COX2 were as described previously (29). And the sequences of siRNA against mouse COX2 were obtained from Shanghai Genechem Co., Ltd (China). For siCOX2-1: sense oligo 5′-UAC CCG GAC UGG AUU CUA UTT-3′, antisense oligo 5′-AUA GAA UCC AGU CCG GGU ATT-3′; for siCOX2-2: sense oligo 5′-GCC AUG GAG UGG ACU UAA ATT-3′, antisense oligo 5′-UUU AAG UCC ACU CCA UGG CTT-3′; and for siCOX2-3: sense oligo 5′-GAG CAC CAU UCU CCU UGA ATT-3′, antisense oligo 5′-UUC AAG GAG AAU GGU GCU CTT-3′. Prostate cancer cells were plated at 80% confluence in 6-well plates and starved with serum deprivation for 24 hours before transfection. For transfection, 5 μL lipo2000 was added into diluted siRNA, and the mixture was added to the medium. The cells were transfected for 48 hours, and cell lysates were used for Western blot, and the media were used for macrophage migration assays.

Cell cycle and apoptosis

PC-3, DU145, or WPMY-1 cells were seeded in 75-cm2 flasks and serum starved for 24 hours. Metformin was added to the culture to final concentrations of 0, 1, 5, 10, and 20 mmol/L and incubated for 24 hours. Cells were washed with cold PBS for three times, resuspended in 70% ethanol for analysis of cell-cycle distribution or directly Annexin V binding buffer used for analysis of apoptosis.

Statistical analysis

GraphPad Prism 5.0 was used for all statistical analyses. Data were presented as means ± SD. If the data follow normal distribution, the statistical significance of differences between two groups of data was analyzed by t test and χ2 test, and differences among several groups were analyzed by one-way analysis of variance followed by the least significant difference procedure for comparison of means. For those do not fit normal distribution, nonparametric statistical tests were used. A P value of less than 0.05 was considered statistically significant.

Effects of metformin on prostate cancer development and metastasis

To explore the effect of metformin on prostate cancer initiation and progression, we conducted a series experiments in TRAMP model mice administered with or without metformin (5 g/L). MRI was used to estimate the sizes of the prostate at weeks 12, 25, and 32 (Fig. 1A). The volumes of the prostates were calculated using the formula: length × width2/2 when the areas of the prostates were the largest revealed by MRI, and then compared between the groups at each time point (Fig. 1B). At week 12, the sizes of the prostates in the control and metformin groups were comparable. However, compared with that in the control group, the sizes of the prostates in the metformin group were markedly smaller at weeks 25 and 32, although no statistical difference was observed between the control and metformin groups at week 32. Of note, the treatment did not affect the weight of the mice throughout the experimental period. Then, we compared the pathologic status of the control and metformin groups at week 12 (Fig. 1C), 25 (Fig. 1D), and 37 (Fig. 1E), respectively. As expected, at week 12, more HGPIN lesions were seen in the mice in the control group. However, in the metformin group, only fewer number of HGPIN lesions were seen, and most of them were either normal or considered as LGPIN (Fig. 1F), suggesting that metformin may be able to inhibit or delay prostate cancer progression from LGPIN to HGPIN. At week 25, more adenocarcinoma lesions than PIN were seen in the control group, whereas more PINs than adenocarcinoma lesions in the metformin group (Fig. 1G), indicating that metformin might also be able to inhibit the transformation from PIN to adenocarcinoma. At week 37, adenocarcinoma lesions developed in both groups, the control group contains predominantly UD-Adeno, and a majority of lesions in metformin group were WD-Adeno (Fig. 1H), suggesting that metformin is capable of delaying the progression from WD-Adeno to UD-Adeno. All the numbers of the lesions in each group at the indicated time points were supplied in Supplementary Table S2. As expected, the IHC results of KI67 showed that metformin could inhibit the proliferation of prostate cancer cells in the metformin group compared with those in the control group at each experimental time point (Supplementary Fig. S1A). It has been described previously that metformin might have some inhibitory effects on stroma cells (34). Therefore, we decided to investigate the effect of metformin on prostate stroma cells (Supplementary Fig. S2). We first assessed the ratio between the numbers of epithelial and stroma cells (Supplementary Fig. S2A) and found that the ratios between two groups are comparable at all the time points examined. This suggests that the decreased prostate sizes in the metformin group might be due to the inhibitory effects of metformin on both epithelial cells and stroma cells. We then examined the inhibitory effect of metformin on stroma cells in the cell line model (WPMY-1, a stroma cell line from the peripheral zone of the prostate). Results from CCK8 assays indicate that metformin (20 mmol/L) can inhibit the proliferation of WPMY-1 cells (Supplementary Fig. S2B). Moreover, by using flow cytometry, we found that metformin can arrest WPMY-1 cells in S-phase and promote apoptosis in a dose-dependent manner (Supplementary Fig. S2C and S2D). Interestingly, we discovered that the effects of metformin on the expression of AR were diverse at different time points. At weeks 12 and 25, metformin inhibited the expressions of AR significantly; however, at week 37, the expressions of AR were upregulated in the metformin group compared with those in the control group (Supplementary Fig. S1B), which might because the control group had more mice with neuroendocrine tumors than the metformin group. The TRAMP mice model, with the expression of SV40 T antigen in the prostatic epithelium (16), has been studied to be susceptible to the formation of neuroendocrine prostate cancer (NEPC), partially due to the inactivation of p53 and retinoblastoma protein (35). A previous study has shown that about 20% of the TRAMP mice (C57/BL6 background) developed NEPC in their lifetime (36). These findings prompted us to investigate the effect of metformin on neuroendocrine markers (Synaptophysin/Syn and CD56/NCAM-1). By staining AR, Syn, and CD56 in the serial slices, we found that metformin treatment significantly reduced the area occupied by the NEPC cells (AR-negative, Syn-, and CD56-positive) at week 37 (Supplementary Fig. S1C).

Figure 1.

Effects of metformin on prostate cancer development and metastasis. A, Magnetic resonance images of the mice pelvic cavities at weeks 12, 25, and 32 in the control and metformin groups. B, The calculated volumes of the prostates at weeks 12, 25, and 32 in the control and metformin groups. C–E, The predominant histologic classifications of the prostatic lesions of the mice at weeks 12 (C), 25 (D), and 37 (E). F–H, The statistical results of the histologic analysis between control and metformin groups at weeks 12 (F), 25 (G), and 37 (H). I–P, H&E staining confirmed the multiple sites of the metastasis in the TRAMP mice, including metastasis in the kidney (I and M), lung (J and N), liver (K and O), and abdominal cavity (L and P). *, P < 0.05.

Figure 1.

Effects of metformin on prostate cancer development and metastasis. A, Magnetic resonance images of the mice pelvic cavities at weeks 12, 25, and 32 in the control and metformin groups. B, The calculated volumes of the prostates at weeks 12, 25, and 32 in the control and metformin groups. C–E, The predominant histologic classifications of the prostatic lesions of the mice at weeks 12 (C), 25 (D), and 37 (E). F–H, The statistical results of the histologic analysis between control and metformin groups at weeks 12 (F), 25 (G), and 37 (H). I–P, H&E staining confirmed the multiple sites of the metastasis in the TRAMP mice, including metastasis in the kidney (I and M), lung (J and N), liver (K and O), and abdominal cavity (L and P). *, P < 0.05.

Close modal

In addition, more metastases were seen in the control group than those in the metformin group at both weeks 25 and 37. At week 25, one metastasis in the abdominal cavity occurred in the control group and no metastasis in the metformin group (P > 0.05). At week 37, nine metastases happened in three mice in the control group, with three to the kidneys (Fig. 1I and M), one to the lung (Fig. 1J and N), two to the liver (Fig. 1K and O), two to the abdominal cavity (Fig. 1L and P), and one to the testis. Of note, only one metastasis to the lung in the metformin group was observed. These results suggested that metformin tends to inhibit prostate cancer metastasis in the TRAMP model. However, it does not reach statistically significance, which is likely due to the small sample size used.

Metformin inhibits the inflammatory infiltration during prostate cancer development

Inflammatory cells, chemokines, and cytokines contribute part of the microenvironment of all tumors and TME plays pivotal roles in cancer initiation, progression as well as metastasis (6). To determine if metformin represses prostate cancer progression through inhibiting inflammatory macrophage recruitment, we first conducted immunohistochemistry (IHC) staining against CD68, CD163, and CD204 on prostate cancer tissues from patients with different Gleason scores. More infiltrated macrophages were found in the samples with higher Gleason scores evidenced by increased CD68-, CD163-, and CD204-positive cells (Fig. 2A–D). Then, we conducted coculturing prostate cancer cells with macrophages (LNCaP/THP1 and RM-1/RAW264.7) with or without the presence of metformin (20 mmol/L; Supplementary Fig. S3A). As expected, macrophages were capable of enhancing prostate cancer cell invasion and migration, and metformin is capable of counteracting the macrophage-enhanced processes (Supplementary Fig. S3B and S3C). Based on these findings, we hypothesized that the effect of metformin on prostate cancer development in TRAMP mice could be through inhibition of macrophage recruitment. To test this hypothesis, we conducted IHC staining with the prostates from TRAMP mice treated with or without metformin against the macrophage markers (CD68, CD163, and CD204). Figure 2E–J shows that positive staining in the metformin treatment group reduced significantly at all time points. The negative control staining without corresponding primary antibodies was supplied in the Supplementary Fig. S4A.

Figure 2.

Metformin inhibits the inflammatory infiltration during tumor development. A–D, The relationship between inflammatory infiltrations and Gleason score. Representative H&E staining and IHC results for CD68, CD163, and CD204 of the tissue microarray from the patients with different Gleason scores, including Gleason score <7 (A), 7 (B), and >7 (C). D, The statistical results of the tissue microarray. E–J, Prostate tissues from TRAMP mice in both control (Con) and metformin (MET) groups at weeks 12 (A), 25 (B), and 37 (C) were stained for CD63, CD163, and CD204. Magnifications: 40×. *, P < 0.05.

Figure 2.

Metformin inhibits the inflammatory infiltration during tumor development. A–D, The relationship between inflammatory infiltrations and Gleason score. Representative H&E staining and IHC results for CD68, CD163, and CD204 of the tissue microarray from the patients with different Gleason scores, including Gleason score <7 (A), 7 (B), and >7 (C). D, The statistical results of the tissue microarray. E–J, Prostate tissues from TRAMP mice in both control (Con) and metformin (MET) groups at weeks 12 (A), 25 (B), and 37 (C) were stained for CD63, CD163, and CD204. Magnifications: 40×. *, P < 0.05.

Close modal

Metformin inhibits castration-induced inflammatory infiltration

We have reported that castration can cause local hypoxia in the prostate cancer tissues (33), an important inducer of inflammatory infiltration. In fact, it has been reported that the inflammatory infiltration induced by ADT might play crucial roles in the development of castration resistance (37). We wondered if metformin could inhibit castration-induced inflammatory infiltration. Surgically castrated mice were treated with or without metformin (5 g/L) and their prostates were IHC stained against CD68, CD163, and CD204. As expected, we found more macrophage infiltration in castrated TRAMP mice (Fig. 3A, middle) than that in the intact TRAMP mice (Fig. 3A, left), while positively stained cells in castrated mice administrated with metformin (Fig. 3A, right) were significantly reduced than those without metformin treatment (Fig. 3A, middle). To determine if metformin can inhibit inflammatory infiltration to cancer tissues in prostate cancer patients, the macrophage infiltrations were compared between the prostate cancer patients administered with or without bicalutamide and metformin. Similarly, we found that more inflammatory infiltration, evidenced by CD68, CD163, and CD204 positive staining, in the tissues of prostate cancer patients treated with bicalutamide (Bic group) than those without bicalutamide (Fig. 3B). However, in the patients treated with both bicalutamide and metformin (Met group), much fewer positively stained cells (Fig. 3B, right) were seen when compared with those treated with bicalutamide alone (Fig. 3B, middle). Altogether, data from both animal experiment and human patients demonstrated that metformin is capable of repressing castration-induced inflammatory infiltration.

Figure 3.

Metformin inhibits the inflammatory infiltration after castration in vivo. A, Prostate tissues from TRAMP × FVB mice in control, castration, and castration + metformin groups were stained for CD63, CD163, and CD204. B, Prostate cancer tissues from the patients in control, Bic, and Bic + Met groups were stained for CD63, CD163, and CD204. Magnifications: 40×. *, P < 0.05.

Figure 3.

Metformin inhibits the inflammatory infiltration after castration in vivo. A, Prostate tissues from TRAMP × FVB mice in control, castration, and castration + metformin groups were stained for CD63, CD163, and CD204. B, Prostate cancer tissues from the patients in control, Bic, and Bic + Met groups were stained for CD63, CD163, and CD204. Magnifications: 40×. *, P < 0.05.

Close modal

Metformin represses macrophage migration/recruitment by prostate cancer cells

To determine the inhibitory effect of metformin on inflammatory infiltration through blocking the recruitment of macrophages to the TME, we conducted a series of experiments with both human and mouse prostate cancer cells and their corresponding macrophages. First, macrophage migration assays were conducted with conditional media collected from human (LNCaP) and mouse (RM-1) prostate cancer cells treated with or without metformin (20 mmol/L) for 48 hours (Fig. 4A). Figure 4B and C showed that metformin is capable of inhibiting human (LNCaP) and mouse (RM-1) prostate cancer cell–mediated human (THP1) and mouse (RAW264.7) macrophage migration/recruitment. Next, we want to further substantiate the inhibitory effects of metformin on castration-enhanced inflammatory infiltration. LNCaP cells cultured in media with charcoal-stripped FBS (CS-FBS) were treated with or without dihydrotestosterone (DHT) and metformin (20 mmol/L), and CM was collected and used in macrophage (THP1) migration assays. Figure 4D showed that CM from cells cultured in CS-FBS without DHT mimicking castration treatment significantly enhanced macrophage migration; Figure 4E showed that metformin can block castration-enhanced macrophage migration significantly.

Figure 4.

Metformin represses the migration of macrophages in vitro. A, The schematic mode of the in vitro macrophage (Mϕ) migration with/without metformin. The prostate cancer cells were treated with/without metformin (20 mmol/L) for 48 hours. The CM was collected, diluted with 10% fresh FBS medium (1:1), and then placed into the lower 24-well plates. B, THP1 cells that migrated to the LNCaP cell CM were counted. C, The RAW264.7 cells that migrated to the RM-1 cell CM were stained with crustal violet, and five randomly selected views (magnification: 40×) were counted. D, The schematic mode of the in vitro macrophage (Mϕ) migration after castration with/without metformin. The LNCaP cells were cultured with charcoal-stripped FBS (CS-FBS) with or without dihydrotestosterone (DHT) and metformin (20 mmol/L) for 48 hours. The CM was collected, diluted with 10% fresh FBS medium (1:1), and then placed into the lower 24-well plates. E, The THP1 cells that migrated to the CM of D were counted. *, P < 0.05.

Figure 4.

Metformin represses the migration of macrophages in vitro. A, The schematic mode of the in vitro macrophage (Mϕ) migration with/without metformin. The prostate cancer cells were treated with/without metformin (20 mmol/L) for 48 hours. The CM was collected, diluted with 10% fresh FBS medium (1:1), and then placed into the lower 24-well plates. B, THP1 cells that migrated to the LNCaP cell CM were counted. C, The RAW264.7 cells that migrated to the RM-1 cell CM were stained with crustal violet, and five randomly selected views (magnification: 40×) were counted. D, The schematic mode of the in vitro macrophage (Mϕ) migration after castration with/without metformin. The LNCaP cells were cultured with charcoal-stripped FBS (CS-FBS) with or without dihydrotestosterone (DHT) and metformin (20 mmol/L) for 48 hours. The CM was collected, diluted with 10% fresh FBS medium (1:1), and then placed into the lower 24-well plates. E, The THP1 cells that migrated to the CM of D were counted. *, P < 0.05.

Close modal

Metformin inhibits inflammatory infiltration by targeting the COX2/PGE2 axis

Multiple lines of evidence suggest that COX2 plays an important role in the recruitment of macrophages to TME (6) and we have observed previously that metformin can reduce the levels of COX2 in cancer tissues of the prostate cancer patients (21). To dissect the molecular mechanism of metformin-repressed inflammatory infiltration, we first estimated the effect of metformin on the expression of COX2 in prostate cancer cells. Figure 5A showed that metformin can inhibit the levels of COX2 in the TRAMP model at all time points. In addition, metformin is capable of counteracting castration-induced COX2 upregulation with concurrently reduced inflammatory infiltration in castrated TRAMP-FVB mice (Fig. 5B). Consistently, the levels of COX2 in LNCaP and RM-1 cells were downregulated by metformin dose dependently (Fig. 6A and B). Because PGE2, a major product of the enzyme COX2, has been suggested to be involved in the recruitment of macrophage to the TME (6), we hypothesized that metformin represses macrophage recruitment by downregulating COX2 and subsequently PGE2. To test this hypothesis, we treated prostate cancer cells (LNCaP and RM-1) with different concentrations of metformin (1, 5, 10, 20 mmol/L) and estimated the levels of PGE2 by ELISA assays. Figure 6C and D showed that metformin inhibits the levels of PGE2 in a dose-dependent manner.

Figure 5.

Effects of metformin on the expression of COX2 during the development of prostate cancer and after castration. A, Prostate tissues from TRAMP mice in both control (Con) and metformin (MET) groups at weeks 12, 25, and 37 were stained for COX2. Magnification: 40×. B, Prostate tissues from TRAMP × FVB mice in control, castration, and castration + metformin groups were stained for COX2. Magnifications: 20×, top; 40×, bottom. *, P < 0.05.

Figure 5.

Effects of metformin on the expression of COX2 during the development of prostate cancer and after castration. A, Prostate tissues from TRAMP mice in both control (Con) and metformin (MET) groups at weeks 12, 25, and 37 were stained for COX2. Magnification: 40×. B, Prostate tissues from TRAMP × FVB mice in control, castration, and castration + metformin groups were stained for COX2. Magnifications: 20×, top; 40×, bottom. *, P < 0.05.

Close modal
Figure 6.

Effects of metformin on the COX2/PGE2 axis. A and B, LNCaP (A) and RM-1 (B) cells were seeded in 6-well plates and treated as indicated for 48 hours. The levels of COX2 were estimated by Western blot assays. C and D, The culture media were collected for ELISA assays of PGE2. E and F, LNCaP and RM-1 cells were transfected with three siRNAs against COX2 and one for negative control, and the cells were cultured for 72 hours. The lysates of the cells were collected for Western blot of COX2. G and H, The CMs were used for the migration assays of THP1 and RAW264.7, the migrated cells were counted. I and J, PGE2 was added into the CMs of the cells transfected with siCOX2, the migration assays of THP1 and RAW264.7 were conducted. K and L, PGE2 was added to the CMs of the cells treated with metformin (20 mmol/L), the migration assays of THP1 and RAW264.7 were conducted. M, Schematic model of the hypothesized mechanism by which metformin inhibits the recruitment of the macrophage. *, P < 0.05.

Figure 6.

Effects of metformin on the COX2/PGE2 axis. A and B, LNCaP (A) and RM-1 (B) cells were seeded in 6-well plates and treated as indicated for 48 hours. The levels of COX2 were estimated by Western blot assays. C and D, The culture media were collected for ELISA assays of PGE2. E and F, LNCaP and RM-1 cells were transfected with three siRNAs against COX2 and one for negative control, and the cells were cultured for 72 hours. The lysates of the cells were collected for Western blot of COX2. G and H, The CMs were used for the migration assays of THP1 and RAW264.7, the migrated cells were counted. I and J, PGE2 was added into the CMs of the cells transfected with siCOX2, the migration assays of THP1 and RAW264.7 were conducted. K and L, PGE2 was added to the CMs of the cells treated with metformin (20 mmol/L), the migration assays of THP1 and RAW264.7 were conducted. M, Schematic model of the hypothesized mechanism by which metformin inhibits the recruitment of the macrophage. *, P < 0.05.

Close modal

Next, we knocked down COX2 in prostate cancer cells by siRNA technique and CMs were used for macrophage migration assays. In LNCaP cells, both siCOX2-1 and siCOX2-3 knocked down COX2 efficiently but siCOX2-2 showed minimum effect (Fig. 6E). In RM-1 cells, all three siRNAs knocked down COX2 efficiently (Fig. 6F). As expected, the capacity of macrophage recruitment is inversely proportional to the efficiency of COX2 knockdown (Fig. 6G and H), and exogenously added PGE2 into the CMs is capable of rescuing macrophage migration resulted from the COX2 knockdown (Fig. 6I and J). Moreover, we used metformin (20 mmol/L) combined with or without PGE2 (10 μmol/L) to treat prostate cancer cells and found that the inhibitory effects of metformin on macrophage migration could be reversed by the addition of PGE2 (Fig. 6K and L), suggesting that metformin downregulates COX2 and subsequently depresses the level of PGE2. These results altogether demonstrated that metformin, by targeting the COX2/PGE2 axis, is capable of inhibiting tumor-associated inflammatory infiltration in prostate cancer development.

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.

No potential conflicts of interest were disclosed.

Conception and design: Y. He, D. Zhang, W. Lan, J. Jiang

Development of methodology: Q. Liu, D. Tong, G. Liu, L.-a. Wang, J. Xu, X. Yang, J. Pang, D. Zhang, Q. Ma, W. Lan, J. Jiang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Tong, G. Liu, J. Gao, L.-a. Wang, J. Xu, X. Yang, Q. Xie, Y. Huang, J. Pang, L. Wang, W. Lan, J. Jiang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Q. Liu, D. Tong, G. Liu, J. Gao, Q. Xie, Y. Huang, L. Wang, Q. Ma, W. Lan, J. Jiang

Writing, review, and/or revision of the manuscript: Q. Liu, D. Zhang, W. Lan, J. Jiang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Q. Liu, D. Tong, G. Liu, J. Gao, L.-a. Wang, J. Xu, X. Yang, Q. Xie, Y. Huang, J. Pang, L. Wang, Q. Ma

Study supervision: Q. Liu, L. Wang, Y. He, W. Lan, J. Jiang

The authors thank Dr. Hualiang Xiao and Jianghong Mu (Department of Pathology, Institute of Surgery Research, Daping Hospital, Third Military Medical University) for their assistance in pathologic analysis and Prof. Ling Liu (Department of Health Statistics, Third Military Medical University) for her assistance in statistical analysis.

This work was supported by the National Natural Science Foundation of China (grant no. 81772704 to J. Jiang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Turley
SJ
,
Cremasco
V
,
Astarita
JL
. 
Immunological hallmarks of stromal cells in the tumour microenvironment
.
Nat Rev Immunol
2015
;
15
:
669
82
.
2.
Sica
A
,
Larghi
P
,
Mancino
A
,
Rubino
L
,
Porta
C
,
Totaro
MG
, et al
Macrophage polarization in tumour progression
.
Semin Cancer Biol
2008
;
18
:
349
55
.
3.
Joyce
JA
,
Pollard
JW
. 
Microenvironmental regulation of metastasis
.
Nat Rev Cancer
2009
;
9
:
239
52
.
4.
Junttila
MR
,
de Sauvage
FJ
. 
Influence of tumour micro-environment heterogeneity on therapeutic response
.
Nature
2013
;
501
:
346
54
.
5.
Quail
DF
,
Joyce
JA
. 
Microenvironmental regulation of tumor progression and metastasis
.
Nat Med
2013
;
19
:
1423
37
.
6.
Mantovani
A
,
Allavena
P
,
Sica
A
,
Balkwill
F
. 
Cancer-related inflammation
.
Nature
2008
;
454
:
436
44
.
7.
Di Silverio
F
,
Gentile
V
,
De Matteis
A
,
Mariotti
G
,
Giuseppe
V
,
Luigi
PA
, et al
Distribution of inflammation, pre-malignant lesions, incidental carcinoma in histologically confirmed benign prostatic hyperplasia: a retrospective analysis
.
Eur Urol
2003
;
43
:
164
75
.
8.
De Marzo
AM
,
Platz
EA
,
Sutcliffe
S
,
Xu
J
,
Gronberg
H
,
Drake
CG
, et al
Inflammation in prostate carcinogenesis
.
Nat Rev Cancer
2007
;
7
:
256
69
.
9.
Gurel
B
,
Lucia
MS
,
Thompson
IM
 Jr.
,
Goodman
PJ
,
Tangen
CM
,
Kristal
AR
, et al
Chronic inflammation in benign prostate tissue is associated with high-grade prostate cancer in the placebo arm of the prostate cancer prevention trial
.
Cancer Epidemiol Biomarkers Prev
2014
;
23
:
847
56
.
10.
Irani
J
,
Goujon
JM
,
Ragni
E
,
Peyrat
L
,
Hubert
J
,
Saint
F
, et al
High-grade inflammation in prostate cancer as a prognostic factor for biochemical recurrence after radical prostatectomy. Pathologist Multicenter Study Group
.
Urology
1999
;
54
:
467
72
.
11.
Davidsson
S
,
Fiorentino
M
,
Andren
O
,
Fang
F
,
Mucci
LA
,
Varenhorst
E
, et al
Inflammation, focal atrophic lesions, and prostatic intraepithelial neoplasia with respect to risk of lethal prostate cancer
.
Cancer Epidemiol Biomarkers Prev
2011
;
20
:
2280
7
.
12.
Ammirante
M
,
Luo
JL
,
Grivennikov
S
,
Nedospasov
S
,
Karin
M
. 
B-cell-derived lymphotoxin promotes castration-resistant prostate cancer
.
Nature
2010
;
464
:
302
5
.
13.
Nguyen
DP
,
Li
J
,
Yadav
SS
,
Tewari
AK
. 
Recent insights into NF-kappaB signalling pathways and the link between inflammation and prostate cancer
.
BJU Int
2014
;
114
:
168
76
.
14.
Thun
MJ
,
Henley
SJ
,
Patrono
C
. 
Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues
.
J Natl Cancer Inst
2002
;
94
:
252
66
.
15.
Nguyen
DP
,
Li
J
,
Tewari
AK
. 
Inflammation and prostate cancer: the role of interleukin 6 (IL-6)
.
BJU Int
2014
;
113
:
986
92
.
16.
Gingrich
JR
,
Greenberg
NM
. 
A transgenic mouse prostate cancer model
.
Toxicol Pathol
1996
;
24
:
502
4
.
17.
Gingrich
JR
,
Barrios
RJ
,
Morton
RA
,
Boyce
BF
,
DeMayo
FJ
,
Finegold
MJ
, et al
Metastatic prostate cancer in a transgenic mouse
.
Cancer Res
1996
;
56
:
4096
102
.
18.
Garabedian
EM
,
Humphrey
PA
,
Gordon
JI
. 
A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells
.
PNAS
1998
;
95
:
15382
7
.
19.
Bodmer
WF
. 
Prostate cancer 2000
.
Prostate Cancer Prostatic Dis
2000
;
3
:
218
23
.
20.
Roy-Burman
P
,
Wu
H
,
Powell
WC
,
Hagenkord
J
,
Cohen
MB
. 
Genetically defined mouse models that mimic natural aspects of human prostate cancer development
.
Endocr Relat Cancer
2004
;
11
:
225
54
.
21.
Tong
D
,
Liu
Q
,
Liu
G
,
Xu
J
,
Lan
W
,
Jiang
Y
, et al
Metformin inhibits castration-induced EMT in prostate cancer by repressing COX2/PGE2/STAT3 axis
.
Cancer Lett
2017
;
389
:
23
32
.
22.
Evans
JM
,
Donnelly
LA
,
Emslie-Smith
AM
,
Alessi
DR
,
Morris
AD
. 
Metformin and reduced risk of cancer in diabetic patients
.
BMJ
2005
;
330
:
1304
5
.
23.
Jiralerspong
S
,
Palla
SL
,
Giordano
SH
,
Meric-Bernstam
F
,
Liedtke
C
,
Barnett
CM
, et al
Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer
.
J Clin Oncol
2009
;
27
:
3297
302
.
24.
Del Barco
S
,
Vazquez-Martin
A
,
Cufi
S
,
Oliveras-Ferraros
C
,
Bosch-Barrera
J
,
Joven
J
, et al
Metformin: multi-faceted protection against cancer
.
Oncotarget
2011
;
2
:
896
917
.
25.
Kim
J
,
Kwak
HJ
,
Cha
JY
,
Jeong
YS
,
Rhee
SD
,
Kim
KR
, et al
Metformin suppresses lipopolysaccharide (LPS)-induced inflammatory response in murine macrophages via activating transcription factor-3 (ATF-3) induction
.
J Biol Chem
2014
;
289
:
23246
55
.
26.
Huang
NL
,
Chiang
SH
,
Hsueh
CH
,
Liang
YJ
,
Chen
YJ
,
Lai
LP
. 
Metformin inhibits TNF-alpha-induced IkappaB kinase phosphorylation, IkappaB-alpha degradation and IL-6 production in endothelial cells through PI3K-dependent AMPK phosphorylation
.
Int J Cardiol
2009
;
134
:
169
75
.
27.
Kim
D
,
Lee
JE
,
Jung
YJ
,
Lee
AS
,
Lee
S
,
Park
SK
, et al
Metformin decreases high-fat diet-induced renal injury by regulating the expression of adipokines and the renal AMP-activated protein kinase/acetyl-CoA carboxylase pathway in mice
.
Int J Mol Med
2013
;
32
:
1293
302
.
28.
Kido
LA
,
Montico
F
,
Sauce
R
,
Macedo
AB
,
Minatel
E
,
Costa
DB
, et al
Anti-inflammatory therapies in TRAMP mice: delay in PCa progression
.
Endocr Relat Cancer
2016
;
23
:
235
50
.
29.
Liu
Q
,
Yuan
W
,
Tong
D
,
Liu
G
,
Lan
W
,
Zhang
D
, et al
Metformin represses bladder cancer progression by inhibiting stem cell repopulation via COX2/PGE2/STAT3 axis
.
Oncotarget
2016
;
7
:
28235
46
.
30.
Guo
L
,
Akahori
H
,
Harari
E
,
Smith
SL
,
Polavarapu
R
,
Karmali
V
, et al
CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis
.
J Clin Invest
2018
;
128
:
1106
24
.
31.
Balasubramaniam
S
,
Comstock
CE
,
Ertel
A
,
Jeong
KW
,
Stallcup
MR
,
Addya
S
, et al
Aberrant BAF57 signaling facilitates prometastatic phenotypes
.
Clin Cancer Res
2013
;
19
:
2657
67
.
32.
Hijioka
S
,
Hosoda
W
,
Matsuo
K
,
Ueno
M
,
Furukawa
M
,
Yoshitomi
H
, et al
Rb Loss and KRAS mutation are predictors of the response to platinum-based chemotherapy in pancreatic neuroendocrine neoplasm with grade 3: a Japanese multicenter pancreatic NEN-G3 Study
.
Clin Cancer Res
2017
;
23
:
4625
32
.
33.
Tong
D
,
Liu
Q
,
Liu
G
,
Yuan
W
,
Wang
L
,
Guo
Y
, et al
The HIF/PHF8/AR axis promotes prostate cancer progression
.
Oncogenesis
2016
;
5
:
e283
.
34.
Xu
S
,
Yang
ZY
,
Jin
P
,
Yang
X
,
Li
X
,
Wei
X
, et al
Metformin suppresses tumor progression by inactivating stromal fibroblasts in ovarian cancer
.
Mol Cancer Ther
2018
;
17
:
1291
302
.
35.
Zhou
Z
,
Flesken-Nikitin
A
,
Corney
DC
,
Wang
W
,
Goodrich
DW
,
Roy-Burman
P
, et al
Synergy of p53 and Rb deficiency in a conditional mouse model for metastatic prostate cancer
.
Cancer Res
2006
;
66
:
7889
98
.
36.
Chiaverotti
T
,
Couto
SS
,
Donjacour
A
,
Mao
JH
,
Nagase
H
,
Cardiff
RD
, et al
Dissociation of epithelial and neuroendocrine carcinoma lineages in the transgenic adenocarcinoma of mouse prostate model of prostate cancer
.
Am J Pathol
2008
;
172
:
236
46
.
37.
Saylor
PJ
,
Kozak
KR
,
Smith
MR
,
Ancukiewicz
MA
,
Efstathiou
JA
,
Zietman
AL
, et al
Changes in biomarkers of inflammation and angiogenesis during androgen deprivation therapy for prostate cancer
.
Oncologist
2012
;
17
:
212
9
.
38.
de Martel
C
,
Ferlay
J
,
Franceschi
S
,
Vignat
J
,
Bray
F
,
Forman
D
, et al
Global burden of cancers attributable to infections in 2008: a review and synthetic analysis
.
Lancet Oncol
2012
;
13
:
607
15
.
39.
Grivennikov
SI
,
Greten
FR
,
Karin
M
. 
Immunity, inflammation, and cancer
.
Cell
2010
;
140
:
883
99
.
40.
Balkwill
F
,
Mantovani
A
. 
Inflammation and cancer: back to Virchow?
Lancet
2001
;
357
:
539
45
.
41.
Sica
A
,
Mantovani
A
. 
Macrophage plasticity and polarization: in vivo veritas
.
J Clin Invest
2012
;
122
:
787
95
.
42.
Mantovani
A
,
Germano
G
,
Marchesi
F
,
Locatelli
M
,
Biswas
SK
. 
Cancer-promoting tumor-associated macrophages: new vistas and open questions
.
Eur J Immunol
2011
;
41
:
2522
5
.
43.
Suganami
T
,
Ogawa
Y
. 
Adipose tissue macrophages: their role in adipose tissue remodeling
.
J Leukoc Biol
2010
;
88
:
33
9
.
44.
Chanmee
T
,
Ontong
P
,
Konno
K
,
Itano
N
. 
Tumor-associated macrophages as major players in the tumor microenvironment
.
Cancers
2014
;
6
:
1670
90
.
45.
Becht
E
,
Giraldo
NA
,
Germain
C
,
de Reynies
A
,
Laurent-Puig
P
,
Zucman-Rossi
J
, et al
Immune contexture, immunoscore, and malignant cell molecular subgroups for prognostic and theranostic classifications of cancers
.
Adv Immunol
2016
;
130
:
95
190
.
46.
Gupta
S
,
Srivastava
M
,
Ahmad
N
,
Bostwick
DG
,
Mukhtar
H
. 
Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma
.
Prostate
2000
;
42
:
73
8
.
47.
Foretz
M
,
Guigas
B
,
Bertrand
L
,
Pollak
M
,
Viollet
B
. 
Metformin: from mechanisms of action to therapies
.
Cell Metab
2014
;
20
:
953
66
.
48.
Rothermundt
C
,
Hayoz
S
,
Templeton
AJ
,
Winterhalder
R
,
Strebel
RT
,
Bartschi
D
, et al
Metformin in chemotherapy-naive castration-resistant prostate cancer: a multicenter phase 2 trial (SAKK 08/09)
.
European Urol
2014
;
66
:
468
74
.
49.
Raval
AD
,
Thakker
D
,
Vyas
A
,
Salkini
M
,
Madhavan
S
,
Sambamoorthi
U
. 
Impact of metformin on clinical outcomes among men with prostate cancer: a systematic review and meta-analysis
.
Prostate Cancer Prostatic Dis
2015
;
18
:
110
21
.
50.
Elia
EM
,
Quintana
R
,
Carrere
C
,
Bazzano
MV
,
Rey-Valzacchi
G
,
Paz
DA
, et al
Metformin decreases the incidence of ovarian hyperstimulation syndrome: an experimental study
.
Journal of ovarian research
2013
;
6
:
62
.
51.
Kim
SA
,
Choi
HC
. 
Metformin inhibits inflammatory response via AMPK-PTEN pathway in vascular smooth muscle cells
.
Biochem Biophys Res Commun
2012
;
425
:
866
72
.
52.
Yue
W
,
Yang
CS
,
DiPaola
RS
,
Tan
XL
. 
Repurposing of metformin and aspirin by targeting AMPK-mTOR and inflammation for pancreatic cancer prevention and treatment
.
Cancer Prev Res
2014
;
7
:
388
97
.
53.
Pecikoza
UB
,
Tomic
MA
,
Micov
AM
,
Stepanovic-Petrovic
RM
. 
Metformin synergizes with conventional and adjuvant analgesic drugs to reduce inflammatory hyperalgesia in rats
.
Anesth Analg
2017
;
124
:
1317
29
.
54.
Huang
Q
,
Chen
Y
,
Gong
N
,
Wang
YX
. 
Methylglyoxal mediates streptozotocin-induced diabetic neuropathic pain via activation of the peripheral TRPA1 and Nav1.8 channels
.
Metabolism
2016
;
65
:
463
74
.
55.
Kialka
M
,
Milewicz
T
,
Sztefko
K
,
Rogatko
I
,
Majewska
R
. 
Metformin increases pressure pain threshold in lean women with polycystic ovary syndrome
.
Drug Des Devel Ther
2016
;
10
:
2483
90
.
56.
Guzman-Priego
CG
,
Mendez-Mena
R
,
Banos-Gonzalez
MA
,
Araiza-Saldana
CI
,
Castaneda-Corral
G
,
Torres-Lopez
JE
. 
Antihyperalgesic effects of indomethacin, ketorolac, and metamizole in rats: effects of metformin
.
Drug Dev Res
2017
;
78
:
98
104
.
57.
Liu
Q
,
Tong
D
,
Liu
G
,
Xu
J
,
Do
K
,
Geary
K
, et al
Metformin reverses prostate cancer resistance to enzalutamide by targeting TGF-beta1/STAT3 axis-regulated EMT
.
Cell Death Dis
2017
;
8
:
e3007
.

Supplementary data