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Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1β production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The immune microenvironment is crucial for epithelial ovarian cancer (EOC) progression and consists of tumor-associated macrophages (TAM) and T lymphocytes, such as regulatory T cells (Treg) and T helper 17 (Th17) cells. In this study, the Treg/Th17 ratio was significantly higher in EOC in situ and in metastatic peritoneal tissues than in benign ovarian tumors and benign peritoneum. The Treg/Th17 ratio was associated with histologic grade and was an independent prognostic factor for overall survival of EOC patients. On the basis of microarray analysis of exosomes derived from TAMs, we identified miRNAs enriched in the exosomes, including miR-29a-3p and miR-21-5p. When the two miRNA mimics were transfected into CD4 + T cells, they directly suppressed STAT3 and regulated Treg/Th17 cells, inducing an imbalance, and they had a synergistic effect on STAT3 inhibition. Taken together, these results indicate that exosomes mediate the interaction between TAMs and T cells, generating an immune-suppressive microenvironment that facilitates EOC progression and metastasis. These findings suggest that targeting these exosomes or their associated miRNAs might pave the way for the development of novel treatments for EOC., (©2018 American Association for Cancer Research.)

Recently, cancer has been considered to be a complex system that includes the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are the most common immune-related stromal cells in the TME, and communication between cancer cells and TAMs is crucial for the progression of epithelial ovarian cancer (EOC). In this study, we revealed that exosomes derived from EOC cells remodel macrophages to a tumor-promoted phenotype, namely TAMs. In addition, hypoxic microenvironments have been postulated to facilitate this process in the TME, and hypoxia-inducible factors (HIFs) play an important role in this process. We found that TAMs educated by hypoxic exosomes derived from EOC cells promote tumor proliferation and migration in a feedback loop. Based on microarray analysis of normoxic and hypoxic exosomes, we discovered that a panel of miRNAs was enriched in hypoxic exosomes. And these three highly expressed miRNAs were induced by hypoxia via HIFs. In this study, we revealed that under hypoxic conditions, EOC cell-derived exosomes deliver miRNAs to induce M2 macrophage polarization, which promotes EOC cell proliferation and migration. This study suggests that these exosomes and associated miRNAs might serve as targets for novel treatments or diagnostic biomarkers for EOC., (Copyright © 2018. Published by Elsevier B.V.)

Ovarian cancer (OC), of which epithelial ovarian cancer (EOC) is the most common, is the deadliest gynecological tumor because of the difficulties in detection at early stages, and metastasis and chemoresistance at advanced stages. Tumor-associated macrophages (TAMs) differentiate through alternative pathways and play important roles in tumor growth and metastasis. However, the underlying mechanism remains unclear. Here, we established a mouse TAM model using bone marrow monocytes and conditioned medium (CM) of TAMs to culture ID8 mouse EOC cells. The results showed that TAM CM accelerated the proliferation and migration of ID8 cells. In a previous study, gene chip analysis showed that human TAMs expressed significantly higher levels of insulin-like growth factor‑1 (IGF1) than undifferentiated M0 myeloid cells. In the present study, we observed that the IGF1 level was higher in human EOC specimens than that in benign ovarian tumor specimens, and further analysis showed that a higher level of IGF1 was related to more advanced clinical stage and liver metastasis. Therefore, we hypothesized that TAMs may accelerate the proliferation and migration of EOC cells by upregulating IGF1. As expected, increased IGF1 expression at both the mRNA and protein levels was observed in ID8 cells cultured with TAM CM, whereas blockade of the IGF1 pathway in ID8 cells with an IGF1 neutralizing antibody effectively reversed the promotion of proliferation and migration. Finally, we inhibited the phosphorylation of insulin-like growth factor‑1 receptor (IGF1R) and its downstream molecules Akt and Erk with the IGF1R inhibitor linsitinib, and observed that the treatment effectively suppressed the proliferation and migration of ID8 cells exposed to TAM CM. Thus, we demonstrated that TAMs may promote the growth and metastasis of EOC via the activation of the IGF1 pathway; thus, targeting the IGF1 pathway may be promising for EOC therapy.

Hypoxia is a common feature of solid tumors. It is closely related to tumor progression. Exosomal microRNAs derived from cancers are considered to be mediators between cancer cells and the tumor microenvironment. In addition, the number of tumor-associated macrophages (TAMs) in the tumor microenvironment has also been demonstrated to correlate with tumor development. However, the relationship between tumor-secreted exosomes and TAM polarization under hypoxic conditions during tumor progression is not clear. Herein, we demonstrated that hypoxia induces the high expression of microRNA-940 (miR‑940) in exosomes derived from epithelial ovarian cancer (EOC). We also found that miR‑940 is highly expressed in exosomes isolated from ascites of EOC patients. Moreover, the overexpression of miR‑940 in macrophages delivered by exosomes stimulated M2 phenotype polarization, while the M2 subtype macrophages promoted EOC proliferation and migration. These results highlight the function of hypoxia in enhancing the high level of expression of miR‑940 in tumor exosomes taken up by macrophages. We also showed that the tumor-promoting function of miR‑940 is mediated by TAM polarization in EOC. These findings show that tumor-derived exosomal miR‑940 induced by hypoxia plays an important role in stimulating TAM polarization in the progression of EOC.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. Inflammatory cells in the EOC microenvironment play a key role in tumor progression. In the present study, we investigated the mechanism of the accumulation of regulatory T cells (Tregs) induced by interleukin-10 (IL-10) derived from tumor-associated macrophages (TAMs) in the EOC microenvironment. The frequency of Tregs and TAMs was detected by immunofluorescence in 40 EOC tissues and 20 benign ovarian tumors, as well as the expression of IL-10 which was assessed by immunohistochemistry. It was found that the frequency of Treg cells and TAMs was significantly higher in the EOC than those in the benign ovarian tumors. The expression of IL-10 was also found to be higher in the EOC than that in the benign tumors. EOC patients with a high frequency of Tregs exhibited a significantly shorter overall survival time compared to those with a low frequency of Tregs. In addition, the expression of IL-10 in ascites and blood serum and the IL-10 released in the co-cultured system supernatants were detected by ELISA. Following CD4+ T-cell co-culturing with macrophages and IL-10, it was observed by flow cytometric analysis that the frequency of Treg cells was increased in the presence of IL-10. It was also established that IL-10 released in the co-cultured supernatants was increased. We also detected the mechanism of Treg cells induced by IL-10 in vivo. The SKOV3 cell tumor volume and weight were much higher in the presence of IL-10 in a mouse subcutaneous model. These data suggest that IL-10 secreted by TAMs increase the frequency of Treg cells through the activation of Foxp3 during T-cell differentiation and promotes tumor progression.

Cancer secreted exosomal miRNAs are emerging as mediators between tumor-stoma crosstalk. Here, we show epithelial ovarian cancer (EOC)-derived exosomes activated macrophages to a tumor-associated macrophage (TAM)-like phenotype with SOCS3/STAT3 pathway involvement, which could facilitate the progression of cancer. MiR-222-3p was enrichment in exosomes released from EOC cells and it could be transferred to macrophages. Overexpression of miR-222-3p in macrophages induced polarization of the M2 phenotype. Luciferase assay verified miR-222-3p targeted SOCS3 genes and expression of SOCS3 was decreased after transfection with a miR-222-3p mimic. Down-regulation of SOCS3 correlated with an increased expression of STAT3 activation. MiR-222-3p could be detected in the exosomes from serum and its levels were related to EOC. These observations propose tumor-derived exosomal miR-222-3p is an effective regulator in the polarization of tumor-promoting M2 macrophages and may be a biomarker of EOC., Competing Interests: None.

It has been established that macrophages and endothelial cells infiltrate peritoneum in the vicinity of tumor implants of epithelial ovarian cancer (EOC). This study investigates whether the interaction of ovarian cancer cells and tumor-associated macrophages could promote the involvement of endothelial cells in angiogenesis. Macrophage phenotypes were detected by fluorescence-activated cell sorting, and cytokine/chemokine secretion was measured by enzyme linked immunosorbent assay. The effect of co-culture of ovarian cancer cells and tumor-associated macrophage (TAM) cells on endothelial cell migration and tube formation was investigated. Signaling pathway mediators were also evaluated for their potential roles in endothelial cell activation by ovarian cancer cells co-cultured with TAM cells. Our results showed that higher expression of interleukin-8 (IL-8) expression associated with 54.26 ± 34.46% of TAM infiltration of peritoneum was significantly higher than 16.58 ± 17.74% of CD3(+) T-cell by immunofluorescence co-staining and confocal microscopy. THP-1 cells exhibited M2-polarized phenotype markers with high proportion of CD68(+) , CD206(+) and CD204(+) markers after phorbol 12-myristate 13-acetate (PMA) treatment, After co-culturing with TAM cells in a transwell chamber system, EOC cells (SKOV3) increased their IL-8 expression at the level of mRNA and protein. After exposure to the conditioned medium obtained by co-culturing TAM and SKOV3 cells, the migration and tube formation of endothelial cells were enhanced significantly. Furthermore, the upregulation of IL-8 expression in ovarian cancer cells induced by macrophages could be inhibited by pyrollidine dithiocarbamate, an inhibitor of nuclear factor (NF)- κB signal pathway. We suggest that the interaction of ovarian cancer cells and tumor-associated macrophages enhances the ability of endothelial cells to promote the progression of ovarian cancer., (© 2013 Japanese Cancer Association.)

Peritoneal metastasis is a distinct pathologic characteristic of advanced epithelial ovarian cancer (EOC), which is the most deadly disease of the female reproductive tract. The inflammatory environment of the peritoneum in EOC contains abundant macrophages, activated thrombin, and thrombin-associated receptors. However, little is known about the mechanism by which the thrombin-macrophages interaction contributes to tumor invasion and metastasis. We investigated the phenotype and cytokine/chemokine expression of thrombin-treated peripheral blood monocytes (MOs)/macrophages, it was found that the phenotype of MOs was altered toward a TAM-like macrophage CD163(high)IL-10(high)CCL18(high)IL-8(high) after thrombin stimulation. By Matrigel invasion assay, the conditioned medium of thrombin-stimulated MOs accelerated remarkable invasion of ES-2, SKOV3, and HO-8910, which was similar to invasive cell numbers of ascites stimuli (P < 0.05) and higher than MOs medium alone (P < 0.05). IL-8 was proposed as the major chemoattractant mediating EOC invasion based on MOs mRNA and protein expression profiling. It was observed that anti IL-8 monoclonal neutralizing antibody attenuated EOC cell invasion in a concentration-dependent manner. Increased transcriptional activation of NF-kappaB p50/p65 was identified in thrombin-treated MOs. This study provided insight the role of thrombin in the regulation of EOC peritoneal invasion via "educating" MOs.

Objectives: Pathological studies have indicated that the peritoneum of epithelial ovarian cancer (EOC) patients exhibits characteristics of chronic inflammation like peritonitis. Abundant macrophage infiltration and increased expression of coagulation factor XII (FXII) have been observed in the peritoneum of EOC patients. The aim of this study is to determine how the interaction between FXII and monocyte/macrophages (MO/MAs) contributes to EOC cell invasion and metastasis of the peritoneum., Methods: MO/MAs from the peripheral blood of healthy female donors and tumor-associated macrophages (TAMs) from EOC ascites were collected and cultured. We assessed phenotypes, cytokine/chemokine production, and phagocytic function of FXII-treated MO/MAs. The effects of the FXII-MO/MAs interaction on EOC cell invasion were determined by the Matrigel in vitro invasion assay. In addition, signaling pathway mediators were evaluated for their potential roles in MO/MA activation., Results: MO/MAs exhibited M2-polarized phenotypes after FXII treatment, which was CD163(high)IL-10(high)CCL18(high)IL-8(high)CCR2(high)CXCR2(high). The phagocytic potential of MO/MAs was also upregulated. Matrigel results indicated that invasion of EOC cells was enhanced when exposed to conditioned medium from FXII-stimulated MO/MAs. Transcription factors found to be upregulated in FXII-stimulated MO/MAs included Fra-1, Fra-2, Fos-B in the AP-1 family, oncogenes HIF-1 and Oct, and STAT-5A in the STAT family., Conclusions: FXII may facilitate EOC cell metastasis by transforming MO/MAs toward tumor-associated macrophage-like cells., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

Problem: To investigate the role of TSP-1 on decidual macrophages (DMPhi) in unexplained recurrent spontaneous abortion (RSA)., Method of Study: A total of 20 women undergoing artificial abortion in the first trimester and 10 patients with RSA were selected. (i) The expression of TSP-1 mRNA in deciduas was detected by real-time polymerase chain reaction; (ii) Flow cytometry was used to detect the percentage of positive TSP-1, CD36, CD47 markers on macrophages. (iii) Cytokine expression was measured by enzyme-linked immunosorbent spot-forming (ELISPOT) cell assay., Results: (i) The expression of TSP-1 mRNA on DMPhi in RSA was significantly decreased (P < 0.05). (ii) The increased expression of CD36 (P < 0.01) and the decreased expression of TSP1 (P < 0.01) were found on DMPhi of RSA. No different CD47 expression level on DMPhi was observed between two groups. (iii)The expression of IL-10 in DMPhi of RSA patients was decreased significantly compared with that of controls (P < 0.05). When adding TSP1 into culture medium, there was no change in IFN-gamma expression, but increased IL-10 (P < 0.05) expression in DMPhi of RSA patients was observed. The expression of IL-10 was decreased significantly in DMPhi from controls when adding anti-TSP-1 antibody to culture medium (P < 0.05)., Conclusion: TSP-1 on DMPhi could influence IL-10 expression as Th2 cytokines. The abnormal expression of TSP-1 could make some women undergo pregnancy loss.

Tumor-associated macrophages (TAMs) are a heterogeneous population that play diverse functions in tumors. Their identity is determined not only by intrinsic factors, such as origins and transcription factors, but also by external signals from the tumor microenvironment (TME), such as inflammatory signals and metabolic reprogramming. Metabolic reprogramming has rendered TAM to exhibit a spectrum of activities ranging from pro-tumorigenic to anti-tumorigenic, closely associated with tumor progression and clinical prognosis. This review implicates the diversity of TAM phenotypes and functions, how this heterogeneity has been re-evaluated with the advent of single-cell technologies, and the impact of TME metabolic reprogramming on TAMs. We also review current therapies targeting TAM metabolism and offer new insights for TAM-dependent anti-tumor immunotherapy by focusing on the critical role of different metabolic programs in TAMs. Highlights: 1. Single-cell sequencing has advanced the phenotypic and functional diversity in TAMs compared with the traditional M1/M2 polarization paradigm. 2. The heterogeneity in origins, phenotypes, and functions of TAMs dictates their diverse roles in tumor progression. 3. The metabolic reprogramming of TAMs triggered by stimuli in the TME constitutes one of the driving factors influencing their diversity. 4. Targeting specific metabolic subsets of TAMs leads to novel tumor immunotherapeutic strategies and improves patient outcomes. [ABSTRACT FROM AUTHOR]