M2-polarized Tumor-associated Macrophages Research Articles

YAP/STAT3 inhibited CD8+ T cells activity in the breast cancer immune microenvironment by inducing M2 polarization of tumor-associated macrophages.

Breast cancer (BC) is the leading cause of cancer-related death among women. One of the hallmarks of cancer is sustained angiogenesis. YAP/STAT3 may promote angiogenesis and driving BC progression. This study aimed to investigate how YAP/STAT3 affects the immune microenvironment in BC and understand the underlying mechanism. To establish a tumor-associated macrophages (TAMs) model, macrophages were cultured in the 4T1 cell culture medium. A BC mouse model was created by injecting 4T1 cells. The expression of YAP, STAT3, p-STAT3, VEGF, VEGFR-2, and PD-L1 was analyzed using immunofluorescence, western blotting, and quantitative real-time PCR. Flow cytometry was used to identify M1 and M2 macrophages, CD4+ T, CD8+ T, and Treg cells. Levels of iNOS, IL-12, IL-10, TGF-β, Arg-1, and CCL-22 were measured using enzyme-linked immunosorbent assay. Co-IP was used to verify whether YAP binds to STAT3. Hematoxylin-eosin staining was used to observe tumor morphology. Cell counting kit-8 was selected to detect T-cell proliferation. YAP, STAT3, P-STAT3, VEGF, VEGFR-2, and PD-L1 were highly expressed in BC tissues. The M2/M1 macrophages ratio increased in the TAMs group compared with the control group. Inhibiting of YAP and STAT3 decreased the M2/M1 macrophages ratio. YAP was found to bind with STAT3. T-cell proliferation was enhanced after YAP inhibition, and overexpression of STAT3 reversed the regulation of YAP on T-cell proliferation. In animal studies, inhibiting YAP inhibited tumor weight and volume development. After YAP inhibition, inflammatory infiltration, M2/M1 macrophage ratio, and Treg cell ratio declined, while CD8+ and CD4+ T-cell ratio increased. In conclusion, this study suggested inhibition of YAP/STAT3 reversed M2 polarization of TAMs and suppressed CD8+ T-cell activity in the BC immune microenvironment. These findings open up new avenues for the development of innovative therapies in the treatment of BC.

Exosomal miR-6733-5p mediates cross-talk between glioblastoma stem cells and macrophages and promotes glioblastoma multiform progression synergistically.

Exosomal miRNAs derived from glioblastoma stem cells (GSCs) are important mediators of immunosuppressive microenvironment formation in glioblastoma multiform (GBM), especially in M2-like polarization of tumor-associated macrophages (TAMs). However, the exact mechanisms by which GSCs-derived exosomes (GSCs-exo) facilitate the remodeling of the immunosuppressive microenvironment of GBM have not been elucidated. Transmission electron microscopy (TME) and nanoparticle tracking analysis (NTA) were applied to verify the existence of GSCs-derived exosomes. Sphere formation assays, flow cytometry, and tumor xenograft transplantation assays were performed to identify the exact roles of exosomal miR-6733-5p. Then, the mechanisms of miR-6733-5p and its downstream target gene regulating crosstalk between GSCs cells and M2 macrophages were further investigated. GSCs-derived exosomal miR-6733-5p induce macrophage M2 polarization of TAMs by positively targeting IGF2BP3 to activate the AKT signaling pathway, which further facilitates the self-renewal and stemness of GSCs. GSCs secrete miR-6733-5p-rich exosomes to induce M2-like polarization of macrophages, as well as enhance GSCs stemness and promote malignant behaviors of GBM through IGF2BP3 activated AKT pathway. Targeting GSCs exosomal miR-6733-5p may provide a potential new strategy against GBM.

A2aR on lung adenocarcinoma cells: A novel target for cancer therapy via recruiting and regulating tumor-associated macrophages

Adenosine 2a receptor (A2aR), a typical GPCR with a high affinity for adenosine, is widely expressed on immune cells, inhibiting anti-tumor immune response accordingly. Here, we identify that A2aR is specifically expressed on tumor cells from lung adenocarcinoma (LUAD) patients, closely related to their prognosis and positively correlated with tumor-associated macrophages (TAMs) infiltration. We hypothesize that blocking A2aR on LUAD cells will inhibit the role of TAMs and control tumor growth. Constructing models of TAMs and LUAD mice, we find that A2aR highly expressed on LUAD cells promotes the secretion of chemokines and polarizing factors through activating PI3K/AKT/NF-κB pathway, thereby promoting the migration and invasion of TAMs. Functionally, blocking A2aR significantly suppresses TAMs infiltration and attenuates tumor burden in LUAD mice. Notably, the M2 polarization of TAMs can also be prevented by inhibiting A2aR in vitro. Together, our studies demonstrate that A2aR on LUAD cells drives TAMs migration and polarization, and blockade of A2aR may support a novel and potent therapeutic option for LUAD.

Targeting of Annexin A1 in Tumor-associated Macrophages as a therapeutic strategy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common aggressive, malignant tumor with limited treatment options. Currently, immunotherapies have low success rates in the treatment of HCC. Annexin A1 (ANXA1) is a protein related to inflammation, immunity and tumorigenesis. However, the role of ANXA1 in liver tumorigenesis remains unknown. Therefore, we sought to explore the feasibility of ANXA1 as a therapeutic target for HCC. Here, we analyzed ANXA1 expression and localization by HCC microarray and immunofluorescence experiments. Using an in vitro culture system, monocytic cell lines and primary macrophages were employed to investigate the biological functions of cocultured HCC cells and cocultured T cells. In vivo, Ac2-26, human recombinant ANXA1 (hrANXA1), and cell depletion (macrophages or CD8 + T cells) experiments were further conducted to investigate the role of ANXA1 in the tumor microenvironment (TME). We found that ANXA1 was overexpressed in mesenchymal cells, especially macrophages, in human liver cancer. Moreover, the expression of ANXA1 in mesenchymal cells was positively correlated with programmed death-ligand 1 expression. Knockdown of ANXA1 expression inhibited HCC cell proliferation and migration by increasing the M1/M2 macrophage ratio and promoting T-cell activation. hrANXA1 promoted malignant growth and metastasis in mice by increasing the infiltration and M2 polarization of tumor-associated macrophages (TAMs), generating an immunosuppressive TME and suppressing the antitumor CD8 + T-cell response. Together, our findings reveal that ANXA1 may be an independent prognostic factor for HCC and demonstrate the clinical translational significance of ANXA1 for tumor immunotherapy in HCC.

Abstract 2781: Mechanism study of surufatinib on the optimized strategy of AG plus PD-1 antibody treatment in pancreatic cancer

Abstract Background: Gemcitabine combined with albumin-bound paclitaxel (AG) is the standard treatment for pancreatic cancer. However, it provides no benefit after combined AG with PD-1 antibody, which needs to be optimized urgently. Surufatinib (SUR) is a novel small-molecule inhibitor that observably inhibits VEGFR 1, 2, 3, fibroblast growth factor receptor 1 (FGFR 1), and colony stimulating factor-1 receptor (CSF-1R), which simultaneously shows anti-angiogenic activity and immune microenvironment activation. Herein, we aimed to explore the synergistic effect and related mechanism of SUR plus AG and PD-1 antibody (SAP) regimen. Methods: Surgical tumor specimens were collected and analyzed using single-cell sequencing and immunohistochemistry technology. Orthotopic transplantation mice model of pancreatic cancer and co-culture system of pancreatic cancer cells and tumor-associated macrophages (TAMs) were successively established to reveal the potential molecular mechanism of SUR in optimizing the effects of AG plus PD-1 antibody, by western blot, immunofluorescence and ubiquitination experiments. Results: Tumor CSF-1 levels were markedly promoted in pancreatic cancer patients after the treatment of gemcitabine, while an increased infiltration of M2 polarized TAMs (M2-TAMs) was induced. A time-dependent upregulation of M2-TAMs infiltration and PD-L1 expression were also observed after the treatment of AG regimen, which might interfere the therapeutic benefits of AG plus PD-1. In co-culture system, tumor PD-L1 expression levels were distinctly down-regulated after the treatment of SAP regimen, while the decreased infiltration of M2-TAMs was observed. In pancreatic cancer cell, PD-L1 protein synthesis were obviously inhibited by SUR, instead of PD-L1 mRNA expression. After the pancreatic cancer cells pretreated with cycloheximide to inhibit protein synthesis, the half-life period of PD-L1 was ulteriorly reduced by SUR. Inversely, the inhibitory action of SUR on PD-L1 expression was reversed by proteasome inhibitor PS-341, rather than lysosomal inhibitor chloroquine. As a result, it indicated that the protein stability of PD-L1 might interfered by SUR via ubiquitin-proteasome pathway. Particularly, when compared with AG plus PD-1 antibody treatment in tumor-bearing model, SAP regimen significantly down-regulated the expression levels of CSF1R, CD163 and PD-L1, and inhibited tumor growth by promoting tumor cell apoptosis and improving the tumor immune microenvironment. Conclusions: Forceful evidence supported that PD-L1 expression could be interfered by surufatinib via ubiquitin-proteasome pathway and M2-TAMs regulation. Herein, additional administration of surufatinib might improve the immunosuppression and drug resistance caused by AG, and increase the curative effect of AG plus PD-1 antibody treatment in clinical practice. Citation Format: Song Gao, Wenbo Zhu, Xiaofan Guo, Yuexiang Liang, Hongwei Wang, Peijun Xu, Yuning Song, Jihui Hao, Shaoshi Wen, Xing Lv, Jiancai Zhou. Mechanism study of surufatinib on the optimized strategy of AG plus PD-1 antibody treatment in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2781.

Influencing Factors and Significance of Tumor-associated Macrophage Polarization in Tumor Microenvironment

Tumor-associated macrophages (TAMs), characterized by high plasticity, are the most abundant immune cells in the tumor microenvironment (TME). TAMs are recruited to tumor region in response to various TME components such as cytokines, chemokines as well as exosomes. Subsequently, these environmental cues induce a certain polarization state of TAMs, ranging from anti-tumor states (M1-like) to pro-tumor states (M2-like). Furthermore, the polarization process of TAMs is continuous and gradually inclines toward the M2-like state with malignant progression, forming a positive feedback loop that facilitates tumor growth and metastasis. Consequently, figuring out the factors and mechanisms affecting TAMs polarization is beneficial for developing novel therapeutic strategies that can be combined with other immunotherapies for lung cancer. In previous studies, many significant molecules and pathways which could promote the M2 polarization of TAMs were identified. However, the underlying mechanisms of this sophisticated cross-talk between tumor cells, stroma cells and TAMs remain still difficult to by fully understand. In this review, we summarize the comprehensive factors involved in triggering TAMs polarization towards the M2 phenotype and further explore the relevant molecular mechanisms. .

SHP-2-induced M2 polarization of tumor associated macrophages via IL-4 regulate colorectal cancer progression.

To explore the effect and molecular mechanism of Src homology region 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) in tumor-associated macrophages (TAMs) repressing the migration and invasion of colorectal cancer (CRC) cells. The relevant data sets of human colon specimens were obtained from GEO database, and then the performed correlation analysis, principal component analysis and differentially expressed gene (DEGs) analysis on the samples were conducted. GO and KEGG enrichment analysis were performed on the common DEGs, and then functional interaction prediction was performed to verify the gene regulatory circuit of SHP-2. Furthermore, western blot was used to detect the effect of low expression of SHP-2 on related proteins, including the markers of promoting M2 polarization and exosome secretion, and keys proteins of the PI3K pathway. The relationship between SHP-2 and PI3K pathway was further verified by adding PI3K inhibitor LY294002. Finally, the effect of SHP-2 on the function of colon cancer cells was confirmed by wound healing assay and Transwell assay. Through bioinformatics analysis, SHP-2 was screened as a possible key gene affecting CRC. The low expression of SHP-2 promoted the protein levels of Arginase-1 and IL-10 in IL-4 induced M2 macrophages, while inhibited the protein levels of IL-1β and TNF-α. Meanwhile, low expression of SHP-2 was found to similarly promote the expression of p-PI3K, p-AKT, and the release of exosomes. Interestingly, the promotion was suppressed after the addition of the PI3K inhibitor LY294002. In terms of cellular behavior, wound healing and transwell data showed that low expression of SHP-2 enhanced the migration and invasion abilities of CRC cells. The low expression of SHP-2 induced by PHPS1 may regulate M2 polarization of TAMs and release of exosomes through PI3K/AKT pathway, thereby enhancing the migration and invasion ability of CRC cells.

LINC01004-SPI1 axis-activated SIGLEC9 in tumor-associated macrophages induces radioresistance and the formation of immunosuppressive tumor microenvironment in esophageal squamous cell carcinoma

Radioresistance and immunosuppression remain the major obstacles in the anti-cancer treatments. This work studies the functions of sialic acid binding Ig like lectin 9 (SIGLEC9) and its related molecules in radioresistance and immunosuppression in esophageal squamous cell carcinoma (ESCC). The single-cell analysis showed that SIGLEC9 was mainly expressed on tumor-associated macrophages (TAMs). Monocytes-derived macrophages were co-cultured with ESCC cells and subjected to radiotherapy. High or low doses of radiotherapy induced SIGLEC9 upregulation and M2 polarization of TAMs. Artificial inhibition of SIGLEC9 in TAMs suppressed the radioresistance and immunosuppressive tumor microenvironment (TME) in the co-cultured ESCC cells. Upstream molecules of SIGLEC9 were predicted via bioinformatics. LINC01004 recruited Spi-1 proto-oncogene (SPI1) in nucleus of TAMs to induce transcriptional activation of SIGLEC9. SIGLEC9 interacted with mucin 1 (MUC1). MUC1 overexpression in ESCCs induced M2 skewing of TAMs, enhanced radioresistance and immunosuppression, and promoted nuclear translocation of β-catenin to suppress radiotherapy-induced ferroptosis of ESCC cells. These effects were blocked upon SIGLEC9 suppression. In vitro results were reproduced in the animal models with xenograft tumors. Taken together, this study demonstrates that the LINC01004-SPI1 axis-activated SIGLEC9 in TAMs induces radioresistance and the formation of immunosuppressive TME in ESCC.

Selective COX-2 Inhibitor Etoricoxib’s Liposomal Formulation Attenuates M2 Polarization of TAMs and Enhances its Anti-metastatic Potential

COX-2 inhibition in pro-tumoral M2 polarization of Tumor-Associated Macrophages (TAMs) underscore the improved prognosis and response to cancer therapy. Thus, etoricoxib, a COX-2 inhibiting NSAID drug is highly effective against tumorigenesis, but its compromised solubility and associated hepatotoxicity, and cardiotoxicity limit its clinical translation. In view of the consequences, the proposed study entails the development of a liposomal formulation for etoricoxib and evaluates its anticancer potential. Etoricoxib loaded liposome was prepared by thin layer hydration method and characterized as a nearly monodisperse system with particle size (91.64nm), zeta potential (-44.5mV), drug loading (17.22%), and entrapment efficiency (94.76%). The developed formulation was administered subcutaneously into the orthotopic 4T1/Balb/c mice model. Its treatment significantly reduced tumor size and skewed M2 polarization of TAMs to a greater extent against free etoricoxib. Furthermore, Tumor tissues analyzed through immunoblotting study confirmed the reduction in Akt phosphorylation at Thr308 residue and pro-tumoral VEGF, MMP-9, and MMP-2 proteins; Moreover, histology studies and microCT analysis of bones revealed the enhanced anti-metastatic potential of etoricoxib delivered through developed formulation against free etoricoxib. As an epilogue, the developed formulation efficiently delivers poorly soluble etoricoxib, enhances its therapeutic potential as an anti-tumor and anti-metastatic agent, and directs explorative research for clinical translation.

Glioma-derived LRIG3 interacts with NETO2 in tumor-associated macrophages to modulate microenvironment and suppress tumor growth

Tumor-associated macrophages (TAMs) account for 30–50% of glioma microenvironment. The interaction between glioma tumor cells and TAMs can promote tumor progression, but the intrinsic mechanisms remain unclear. Herein, we reported that soluble LRIG3 (sLRIG3) derived from glioma tumor cells can block the M2 polarization of TAMs via interacting with NETO2, thus suppressing GBM malignant progression. The expression or activity of ADAM17 in glioma cells was positively correlated with the expression of sLRIG3 in cell supernatant. Soluble LRIG3 can suppress the M2-like polarity transformation of TAMs and inhibit the growth of tumor. High expression of LRIG3 predicts a good prognosis in patients with glioma. Mass spectrometry and Co-immunoprecipitation showed that sLRIG3 interacts with the CUB1 domain of NETO2 in TAMs. Silencing or knockout of NETO2 could block the effect of sLRIG3, which inhibited the M2-like polarity transformation of TAMs and promoted GBM tumor growth. However, overexpressing His-target NETO2 with CUB1 deletion mutation does not fully recover the suppressive effects of sLRIG3 on the TAM M2-polarization in NETO2-Knockout TAMs. Our study revealed vital molecular crosstalk between GBM tumor cells and TAMs. Glioma cells mediated the M2 polarization of TAM through the sLRIG3-NETO2 pathway and inhibited the progression of GBM, suggesting that sLRIG3-NETO2 may be a potential target for GBM treatment.

PROS1 shapes the immune-suppressive tumor microenvironment and predicts poor prognosis in glioma.

Glioma is the most malignant cancer in the brain. As a major vitamin-K-dependent protein in the central nervous system, PROS1 not only plays a vital role in blood coagulation, and some studies have found that it was associated with tumor immune infiltration. However, the prognostic significance of PROS1 in glioma and the underlying mechanism of PROS1 in shaping the tumor immune microenvironment (TIME) remains unclear. The raw data (including RNA-seq, sgRNA-seq, clinicopathological variables and prognosis, and survival data) were acquired from public databases, including TCGA, GEPIA, CGGA, TIMER, GEO, UALCAN, and CancerSEA. GO enrichment and KEGG pathway analyses were performed using "cluster profiler" package and visualized by the "ggplot2" package. GSEA was conducted using R package "cluster profiler". Tumor immune estimation resource (TIMER) and spearman correlation analysis were applied to evaluate the associations between infiltration levels of immune cells and the expression of PROS1. qRT-PCR and WB were used to assay the expression of PROS1. Wound-healing assay, transwell chambers assays, and CCK-8 assays, were performed to assess migration and proliferation. ROC and KM curves were constructed to determine prognostic significance of PROS1 in glioma. The level of PROS1 expression was significantly increased in glioma in comparison to normal tissue, which was further certificated by qRT-PCR and WB in LN-229 and U-87MG glioma cells. High expression of PROS1 positively correlated with inflammation, EMT, and invasion identified by CancerSEA, which was also proved by downregulation of PROS1 could suppress cells migration, and proliferation in LN-229 and U-87MG glioma cells. GO and KEGG analysis suggested that PROS1 was involved in disease of immune system and T cell antigen receptor pathway. Immune cell infiltration analysis showed that expression of PROS1 was negatively associated with pDC and NK CD56 bright cells while positively correlated with Macrophages, Neutrophils in glioma. Immune and stromal scores analysis indicated that PROS1 was positively associated with immune score. The high level of PROS1 resulted in an immune suppressive TIME via the recruitment of immunosuppressive molecules. In addition, Increased expression of PROS1 was correlated with T-cell exhaustion, M2 polarization, poor Overall-Survival (OS) in glioma. And it was significantly related to tumor histological level, age, primary therapy outcome. The results of our experiment and various bioinformatics approaches validated that PROS1 was a valuable poor prognostic marker. Increased expression of PROS1 was correlated with malignant phenotype and associated with poor prognosis in glioma. Besides, PROS1 could be a possible biomarker and potential immunotherapeutic target through promoting the glioma immunosuppressive microenvironment and inducing tumor-associated macrophages M2 polarization.

Prognostic and immunological role of acetaldehyde dehydrogenase 1B1 in human tumors: A pan-cancer analysis.

Acetaldehyde dehydrogenases (ALDH) 1B1 is associated with a poor prognosis in pancreatic cancer, colorectal cancer, and osteosarcoma. Overexpression of ALDH also impairs tumor immunity. However, it is unclear how ALDH1B1 is associated with patient prognosis and immune infiltration in different cancer types. This is an original research based on bioinformatics analysis. In this study, we investigated the expression and prognostic value of ALDH1B1 in pan-cancer specimens using several databases, including GEPIA2 and Kaplan-Meier Plotter. The GEPIA2 and TIMER2 databases were used to explore correlations between ALDH1B1 expression and immune infiltration in cancers, especially head and neck squamous cell carcinoma (HNSC) and stomach adenocarcinoma (STAD). Finally, the expression of ALDH1B1 was validated by qPCR and immunohistochemistry. The expression of ALDH1B1 differed in most cancers compared to normal tissue controls. ALDH1B1 has an important impact on the prognosis different cancer types, and the high expression of ALDH1B1 is inversely associated with survival in patients with HNSC. A significant positive correlation was identified between ALDH1B1 expression in HNSC and immune infiltration. The poor prognosis associated with high expression of ALDH1B1 may be related to the promotion of M2 polarization of tumor-associated macrophages. Furthermore, markers of immune cell infiltration, such as exhausted T cells and regulatory T cells showed different patterns of ALDH1B1-associated immune infiltration. ALDH1B1 can serve as a prognostic biomarker in pan-cancer types and is correlated with immune infiltration.

SLC1A5 enhances malignant phenotypes through modulating ferroptosis status and immune microenvironment in glioma

Glioma is the most common type of primary malignant tumor in the central nervous system with limited treatment satisfaction. Finding new therapeutic targets has remained a major challenge. Ferroptosis is a novel and distinct type of programmed cell death, playing a regulatory role in the progression of tumors. However, the role of ferroptosis or ferroptosis-related genes (FRGs) in glioma progression has not been extensively studied. In our study, a novel ferroptosis-related prognostic model, including 7 genes, was established, in which patients classified into the high-risk group had more immuno-suppressive status and worse prognosis. Among these 7 genes, we screened solute carrier family 1 member 5 (SLC1A5), an FRG, as a possible new target for glioma treatment. Our results showed that the expression of SLC1A5 was significantly upregulated in glioblastoma tissues compared with the low-grade gliomas. In addition, SLC1A5 knockdown could significantly inhibit glioma cell proliferation and invasion, and reduce the sensitivity of ferroptosis via the GPX4-dependent pathway. Furthermore, SLC1A5 was found to be related to immune response and SLC1A5 knockdown decreased the infiltration and M2 polarization of tumor-associated macrophages. Pharmacological inhibition of SLC1A5 by V9302 was confirmed to promote the efficacy of anti-PD-1 therapy. Overall, we developed a novel prognostic model for glioma based on the seven-FRGs signature, which could apply to glioma prognostic and immune status prediction. Besides, SLC1A5 in the model could regulate the proliferation, invasion, ferroptosis and immune state in glioma, and be applied as a prognostic biomarker and potential therapeutic target for glioma.

Erastin enhances metastatic potential of ferroptosis-resistant ovarian cancer cells by M2 polarization through STAT3/IL-8 axis.

Erastin is a small molecule identified in chemical screen that is capable of inducing ferropotosis. There is collective evidence proving that erastin-induced ferroptosis exhibits anti-tumor potential within diverse caners, such as ovarian cancer (OC). However, most OC cells show relative resistance to ferroptosis induced by erastin. M2-polarized tumor-associated macrophages (TAMs) have an important effect on the OC tumor microenvironment (TME), which makes M2 polarization a noticeable part in the context of OC therapy. The immunomodulatory effects of erastin on ferroptosis-resistant OC cells remain poorly understood. Here, we found that low concentration of erastin greatly promoted ferroptosis-resistant OC cell invasion and migration via STAT3-mediated M2 polarization of macrophages. As revealed by in-vitro experimental results, erastin significantly increased metastases of ferroptosis-resistant OC, and the percentage of M2 macrophage infiltration was also raised after erastin treatment. Furthermore, erastin augmented IL-8 production of macrophages, and pharmacological blockage of IL-8 partially abrogated the stimulatory effect of erastin on ferroptosis-resistant OC cells. This study demonstrates a new mechanism undering the tumor-promoting activity of erastin and has implications for the STAT3/IL-8 axis as a potential target for ferroptosis-resistant OC cells to improve overall anti-tumor efficacy.

Extracellular vesicles derived from M2-polarized tumor-associated macrophages promote immune escape in ovarian cancer through NEAT1/miR-101-3p/ZEB1/PD-L1 axis.

Evidence has been presented demonstrating that CD8+ T cells confer anti-cancer effects, which offers a promising approach to enhance immunotherapy. M2-polarized tumor-associated macrophages (TAMs) could transfer RNA to cancer cells by secreting extracellular vesicles (EVs) and stimulate immune escape of cancer cells. Thus, the current study aimed at exploring how EVs derived from M2-polarized TAMs (M2-TAMs) affected the proliferation of ovarian cancer (OC) cells and apoptosis of CD8+ T cells. M2-TAMs were observed in OC tissues, which promoted proliferation of OC cells and CD8+ T cell apoptosis by secreting EVs. OC-associated differentially expressed gene NEAT1 was screened by bioinformatics analysis. The in vitro and in vivo effects of TAM-EVs-NEAT1 and its regulatory mechanism were assessed using gain- and loss-of-function assays in co-culture systems of TAMs-derived EVs, OC cells, and CD8+ T cells and in tumor-bearing mice. NEAT1 was highly expressed in M2-derived EVs and OC cells co-cultured with M2-derived EVs. NEAT1 sponged miR-101-3p to increase ZEB1 and PD-L1 expression. In vitro and in vivo assays confirmed the tumor-supporting effects of NEAT1 delivered by M2-derived EVs on OC cell proliferation and CD8+ T cell apoptosis as well as tumor growth. Collectively, M2-derived EVs containing NEAT1 exerted a tumor-promoting role in OC via the miR-101-3p/ZEB1/PD-L1 axis.

Extracellular vesicle-packaged miR-181c-5p from epithelial ovarian cancer cells promotes M2 polarization of tumor-associated macrophages via the KAT2B/HOXA10 axis.

The molecular mechanistic actions of tumor-derived extracellular vesicles (EVs) in modulating macrophage polarization in the tumor microenvironment of epithelial ovarian cancer (EOC) is largely unknown. The study was performed to clarify the effect and downstream mechanism of microRNA-181c-5p (miR-181c-5p)-containing EVs from EOC cells in the M2 polarization of tumor-associated macrophages (TAMs). EVs were isolated from normoxic and hypoxic human EOC cells SKOV3. Human mononuclear cell THP-1 was induced by phorbol-12-myristate-13-acetate to differentiate into TAMs. The targeting relationship between miR-181c-5p and KAT2B was verified by dual luciferase reporter gene assay. The interaction between KAT2B and HOXA10 was detected by immunofluorescence, Co-IP and ChIP assays. EdU staining, the scratch test and Transwell assay were used to assess the resultant cell proliferation, migration and invasion. The mouse xenograft model and the pulmonary metastasis model were developed through intraperitoneal injection of SKOV3 cells and tail vein injection of THP-1 cells, respectively. Hypoxic SKOV3 cell-derived EVs could be internalized by TAMs. SKOV3 cell-derived EVs induced by hypoxia (H-EVs) promoted M2 polarization of TAMs and facilitated the proliferation, migration and invasion of SKOV3 cells. miR-181c-5p was highly expressed in H-EVs and promoted the M2 polarization of TAMs. Further, miR-181c-5p targeted KAT2B, upregulated HOXA10 and activated the JAK1/STAT3 pathway, thereby promoting the M2 polarization of TAMs. In both mouse models, H-EV-derived miR-181c-5p promoted growth and metastasis of EOC cells. The miR-181c-5p-containing EVs from hypoxic EOC cells may upregulate HOXA10 by targeting KAT2B and activate the JAK1/STAT3 pathway to promote the M2 polarization of TAMs, ultimately promoting growth and metastasis of EOC cells in vitro and in vivo.

Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer

Tumor associated macrophages (TAMs) play an important role in tumorigenesis, development and anti-cancer drug therapy. However, very few epigenetic compounds have been elucidated to affect tumor growth by educating TAMs in the tumor microenvironment (TME). Herein, we identified that EZH2 performs a crucial role in the regulation of TAMs infiltration and protumoral polarization by interacting with human breast cancer (BC) cells. We showed that EZH2 inhibitors-treated BC cells induced M2 macrophage polarization in vitro and in vivo, while EZH2 knockdown exhibited the opposite effect. Mechanistically, inhibition of EZH2 histone methyltransferase alone by EZH2 inhibitors in breast cancer cells could reduce the enrichment of H3K27me3 on CCL2 gene promoter, elevate CCL2 transcription and secretion, contributing to the induction of M2 macrophage polarization and recruitment in TME, which reveal a potential explanation behind the frustrating results of EZH2 inhibitors against breast cancer. On the contrary, EZH2 depletion led to DNA demethylation and subsequent upregulation of miR-124-3p level, which inhibited its target CCL2 expression in the tumor cells, causing arrest of TAMs M2 polarization. Taken together, these data suggested that EZH2 can exert opposite regulatory effects on TAMs polarization through its enzymatic or non-enzymatic activities. Our results also imply that the effect of antitumor drugs on TAMs may affect its therapeutic efficacy, and the combined application with TAMs modifiers should be warranted to achieve great clinical success.

Macrophage Distribution Affected by Virus-Encoded Granulocyte Macrophage Colony Stimulating Factor Combined with Lactate Oxidase.

Oncolytic virotherapy was approved as a localized treatment for advanced melanoma by the US Food and Drug Administration (FDA) in 2015. Granulocyte macrophage colony stimulating factor (GM-CSF) encoded by clinical virus-infected tumor cells, acting as a pro-inflammatory cytokine or growth factor, increases tumor antigen presentation, leading to the activation of macrophages and T cells. Notably, tumor-secreted lactate can promote the suppressive functions of M2-polarized tumor-associated macrophages and subsequently promote tumor growth. Furthermore, the consumption of tumor-secreted lactate has been implicated in the beneficial polarization of macrophages. Here, we report that GM-CSF-encoded recombinant adeno-associated virus (AAV2-GM-CSF) infection in B16-F10 mouse melanoma cells combined with lactate oxidase (LOX) leads to the recruitment of M1 macrophages for the inhibition of cancer cell growth. This study suggests that GM-CSF combined with LOX has potential as cancer virotherapy.

LINC00665 interacts with BACH1 to activate Wnt1 and mediates the M2 polarization of tumor-associated macrophages in GC

Gastric cancer (GC) remains one of the prevalent causes of cancer-related deaths globally. Long non-coding RNAs (lncRNAs) have been associated with different cancers. The polarization of macrophages towards the M2 (alternatively activated) phenotype promotes immunologic tolerance and can induce gastric tumorigenesis. Thus far, lncRNAs have been shown to modulate the differentiation of immune cells. Here, we investigated the biological effects of LINC00665 on the progression of GC and explored the mechanisms underlying its ability to mediate the polarization of macrophages towards the M2 phenotype. We report that the levels of LINC00665 were increased in GC tissues. Furthermore, this increase in LINC00665 expression could be associated with decreased overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS). Using cell-based macrophage polarization models, we demonstrated that LINC00665 upregulation in GC cells facilitated the polarization of macrophages towards the M2 but not M1 (classically activated) phenotype. Furthermore, the loss of LINC00665 prevented the M2 polarization of macrophages. Mechanically, we identified that Wnt1 was the downstream target of LINC00665. Additionally, LINC00665 could directly interact with the transcription factor BTB domain and CNC homology 1 (BACH1). The interaction between LINC00665 and BACH1 resulted in the activation and binding of BACH1 to the Wnt1 promoters. Furthermore, BACH1 silencing could inhibit GC progression, which highlighted a crucial role for BACH1 in LINC00665-mediated Wnt1 activation. In addition, genetic Wnt1 overexpression effectively abolished the repression of Wnt signaling after BACH1 depletion and mediated GC development by supporting M2 macrophage polarization. In conclusion, we report that LINC00665 modulates M2 macrophage polarization and suggest that it may facilitate macrophage-dependent GC progression.

Thymosin α-1 Reverses M2 Polarization of Tumor-Associated Macrophages during Efferocytosis.

Thymosin α-1 improves the curative effect of chemotherapy by reversing efferocytosis-induced M2 polarization of macrophages via activation of a TLR7/SHIP1 axis.