Foxp3+ Tregs Promote M2 Macrophage Polarization via Sirt1-ERK1/2-STAT3 Pathway in Ovarian Cancer Progression.

Activation of VDR pathway was a promising anti-tumor therapy strategy. However, numerous clinical studies have demonstrated the effect of activating VDR is limited, which indicates that VDR plays a complex role in vivos. We analyzed the TCGA database to examine the association between VDR expression and immune cell infiltration in pancreatic adenocarcinoma (PAAD). Western blot, ELISA, ChIP, and dual-luciferase reporter assays were performed to determine the mechanism of VDR regulating CCL20. Migration assay and immunofluorescence were used to investigate the role of CCL20 in M2 macrophage polarization and recruitment. We employed multiplexed immunohistochemical staining and mouse models to validate the correlation of VDR on macrophages infiltration in PAAD. Flow cytometry analysis of M2/M1 ratio in subcutaneous graft tumors. VDR is extensively expressed in PAAD, and patients with elevated VDR levels exhibited a significantly reduced overall survival. VDR expression in PAAD tissues was associated with increased M2 macrophages infiltration. PAAD cells overexpressing VDR promote macrophages polarization towards M2 phenotype and recruitment in vitro and vivo. Mechanistically, VDR binds to the CCL20 promoter and up-regulates its transcription. The effects of polarization and recruitment on macrophages can be rescued by blocking CCL20. Finally, the relationship between VDR and M2 macrophages infiltration was evaluated using clinical cohort and subcutaneous graft tumors. A positive correlation was demonstrated between VDR/CCL20/CD163 in PAAD tissues and mouse models. High expression of VDR in PAAD promotes M2 macrophage polarization and recruitment through the secretion of CCL20, which activates tumor progression. This finding suggests that the combination of anti-macrophage therapy may improve the efficacy of VDR activation therapy in PAAD.

Tongue squamous cell carcinoma-derived exosomes miR-21-5p affect tumor progression via promoting M2 macrophage polarization.

This study aimed to investigate whether CXCL8-CXCR2 axis in regulating M2 macrophage polarization via RASGRP4 related signaling in ovarian cancer. Data from The Cancer Genome Atlas (TCGA) database was used to assess the correlation between CXCR2 expression and M2 macrophage infiltration. THP-1 human monocytic cells were utilized to analyze the effects of CXCL8 on RASGRP4 expression and M2 polarization. In vivo experiments were conducted using xenograft models to evaluate the impact of CXCL8 and RASGRP4 on tumor growth and macrophage polarization. Among the CXCR2 co-expressed genes, RASGRP4 showed the highest positive correlation with M2 macrophage infiltration in ovarian cancer. Higher expression of RASGRP4 is associated with poorer progression-free survival in patients with serous ovarian cancer. CXCR2 knockdown or inhibition (using SB225002) reduced IL-8-induced upregulation of RASGRP4 mRNA and protein in THP-1 cells. Additionally, PLCβ2 silencing attenuated IL-8-induced RASGRP4 expression. Knockdown of RASGRP4 in THP-1 cells reduced M2 polarization, while overexpression restored it. The CXCL8-CXCR2 axis further enhances M2 polarization through RASGRP4-mediated mTOR-STAT3 signaling. In xenograft ovarian tumor models, knockdown of CXCL8, CXCR2, or RASGRP4 reduced tumor growth and M2 macrophage infiltration. In summary, the CXCL8-CXCR2 axis promotes M2 macrophage polarization via RASGRP4-mediated mTOR-STAT3 signaling in ovarian cancer. Targeting this pathway may be a promising therapeutic strategy to reprogram tumor-associated macrophages and enhance treatment efficacy.

BackgroundHepatocellular carcinoma (HCC) is an aggressive and lethal malignancy. Metabolic reprogramming dynamically remodels the tumor microenvironment (TME) and drives HCC progression. This study investigated the mechanism through which metabolic reprogramming remodels the TME in HCC.MethodsHCC patient transcriptome data were subjected to bioinformatics analysis to identify differentially expressed genes and immune infiltration status. Immunohistochemical analysis was performed to determine the correlation between succinate dehydrogenase complex subunit A (SDHA) expression and M2 macrophage infiltration. SDHA-knockdown or SDHA-overexpressing HCC cells were used for in vitro experiments, including co-culturing, flow cytometry, and enzyme-linked immunosorbent assay. Western blotting assay, functional assays, and subcutaneous tumor model mice were used to elucidate the molecular mechanisms underlying succinate-mediated HCC cell-macrophage interactions in the TME.ResultsHigher infiltration of M2 macrophages correlated with worse prognosis in HCC patients. SDHA was downregulated in HCC tumor tissues and showed a negative correlation with M2 macrophage infiltration. SDHA knockdown promoted M2 macrophage polarization, whereas SDHA overexpression reversed this effect. Mechanistically, SDHA deficiency in HCC cells induced succinate accumulation, which promoted M2 macrophage polarization by activating the G protein-coupled receptor 91 (GPR91)/signal transducer and activator of transcription 3 (STAT3) pathway. Concurrently, succinate stimulation enhanced mitochondrial oxidative phosphorylation in M2 macrophages, thereby promoting HCC progression. Serum succinate levels were elevated in HCC patients. The receiver operating characteristic curve analysis indicated that serum succinate is a promising diagnostic marker for HCC (area under the curve = 0.815).ConclusionSDHA deficiency leads to succinate accumulation, which promotes M2 macrophage polarization through the GPR91/STAT3 pathway, thereby facilitating HCC progression. Based on these findings, serum succinate could be a promising diagnostic biomarker for HCC.

SLC16A3 is considered to affect the malignant progression of lung adenocarcinoma (LUAD), but its mechanism remains elusive. Lactate secretion can facilitate the M2 polarization of macrophages, which are essential components of the tumor immune microenvironment (TIME). Based on the Cancer Genome Atlas (TCGA) database, differential expression analysis of SLC16A3 in LUAD was undertaken and the Pearson correlation analysis was on SLC16A3 and targets of M2 macrophages. Pathway enrichment analysis on SLC16A3 was achieved by utilizing the gene set enrichment analysis (GSEA). The expression of SLC16A3 in cells was examined by qPCR and Western blot (WB). The levels of glycolysis marker proteins in cells were tested by WB. The Glucose test kit, lactate test kit, Seahorse energy metabolism analyzer, and pHrodo™ Green AM intracellular indicator reagent kit were applied in assessing cellular glycolysis levels. CCK-8, scratch assay, Transwell assay, and flow cytometry were conducted to evaluate the malignant phenotype and apoptosis level of cancer cells. Flow cytometry and Enzyme-linked immunosorbent assay (ELISA) were utilized to assess the polarization of macrophages. Finally, a mouse model of allograft tumors was created, and the effects of SLC16A3 on glycolysis and M2 polarization of macrophages in vivo were evaluated by tracking tumor growth and detecting related protein distribution through Immunohistochemistry. SLC16A3 was greatly upregulated in LUAD. Knocking down SLC16A3 remarkably repressed the malignant phenotype of LUAD cells and reinforced apoptosis. The results derived from GSEA manifested that SLC16A3 had a higher enrichment in the glycolysis pathway. SLC16A3 positively modulated the extracellular and intracellular levels of lactate and glycolysis. Pearson correlation analysis uncovered a positive linkage between SLC16A3 and M2 macrophage markers. According to the rescue experiment, glycolysis inhibitors were observed to greatly reduce the enhancement in M2 polarization of macrophages caused by overexpression of SLC16A3. The final mouse experiment demonstrated that SLC16A3 boosted tumor growth in vivo and enhanced tumor glycolysis level and M2 macrophage infiltration in the TIME. SLC16A3 in LUAD modulates the glycolysis pathway to facilitate M2 polarization of macrophages.

To explore the oncogenic mechanism of FOXM1 in the tumor microenvironment (TME) regarding triple-negative breast cancer (TNBC) promotion, the mRNA and protein levels of target genes in TNBC cells and their exosomes were detected by RT-qPCR and western blot. A co-culture model of TNBC cells and THP-1/M0 macrophages was established to detect the impact of co-culture on FOXM1 expression and the direction of macrophage polarization. A bioinformatics website was used to predict FOXM1 binding sites in the IDO1 promoter, which were further validated using dual-luciferase reporter and chromatin immunoprecipitation assays. Next, after erastin-induced ferroptosis, we conducted cell viability assays, apoptosis assays and other experiments to investigate whether the FOXM1/IDO1 axis regulates M2 macrophage polarization through ferroptosis. We found that FOXM1 was abundant in exosomes derived from TNBC cells, and that TNBC cells upregulated FOXM1 expression in THP-1 cells through exosomes to promote M2 macrophage polarization. Furthermore, FOXM1 upregulated IDO1 in M2-type tumor-associated macrophages (TAMs) by stimulating its transcription. Finally, FOXM1/IDO1 inhibited ferroptosis, promoting M2 macrophage polarization, thereby advancing TNBC progression. In conclusion, FOXM1 carried by TNBC cell-derived exosomes activated IDO1 transcription in TAMs to inhibit ferroptosis, promoting M2 polarization of TAMs and exerting carcinogenic effects.

Epithelial Ovarian Cancer (EOC) is complex and heterogeneous, making accurate prognosis and treatment prediction difficult. New therapeutic targets and their mechanisms are urgently needed. This study explored the role of PTTG1 in ovarian cancer via the cGMP-PKG signaling pathway, focusing on its effects on M2 macrophage polarization and EMT progression in EOC cells. Using the GSE135886 database, we performed differential gene expression, pathway enrichment, and immune infiltration analyses to identify key targets influencing EMT and macrophage polarization. We then constructed PTTG1 knockdown and overexpression cell lines to assess the impact of PTTG1 on cell proliferation, migration, invasion, EMT, and macrophage polarization in vitro. Analysis revealed that differentially expressed genes were enriched in the cGMP-PKG pathway and correlated with M2 macrophages. PTTG1 overexpression in A2780 and SK-OV-3 ovarian cancer cells promoted proliferation, invasion, and migration, while enhancing sGC, PKG1, and PKG2 expression to activate the cGMP-PKG pathway and induce M2 macrophage polarization. PTTG1 knockdown produced opposite results, reinforcing our conclusions. This study uncovers a novel mechanism of PTTG1 in ovarian cancer development and suggests it as a potential therapeutic target.

Norepinephrine stimulates M2 macrophage polarization via β2-adrenergic receptor-mediated IL-6 production in breast cancer cells

It has been documented that M2 macrophage polarization plays a suppressive role in atherosclerosis in diabetes mellitus (DM). In addition, prostaglandin E2 (PGE2) is implicated in the development of M2 macrophage polarization. Therefore, the study aimed to investigate the specific mechanism of PGE2 in M2 macrophage polarization in diabetic coronary atherosclerosis (DMAS). Initially, clinical samples were obtained and DMAS mouse model was established. The expression of BDNF was determined, and M1 and M2 macrophage polarizations were evaluated. Then, the levels of BDNF and PGE2 were modified in DMAS mice and the serum indicator, atherosclerotic plaque, lipid uptake by PBMCs, as well as M1 and M2 macrophage polarization were determined. Macrophages were isolated and the effects of PGE2 and the CREB/BDNF/TrkB signaling pathway on M2 macrophage polarization were explored. BDNF was downregulated and macrophages were differentiated into M1 in DMAS patients and mice. BDNF and PGE2 were observed to promote M2 macrophage polarization, where atherosclerotic plaque and lipid uptake by PBMCs were reduced, and DMAS was alleviated in mice. Overexpression of BDNF activated the CREB/BDNF/TrkB signaling pathway and stimulated M2 macrophage polarization in macrophages. PGE2 stimulated M2 macrophage polarization by inducing KLF4 via the activation of the CREB/BDNF/TrkB signaling pathway. This study demonstrates that PGE2 promotes M2 macrophage polarization by activating the CREB/BDNF/TrkB signaling pathway, thus alleviating DMAS.

LncRNA NRON promotes M2 macrophage polarization and alleviates atrial fibrosis through suppressing exosomal miR-23a derived from atrial myocytes

1-Ethoxycarbonyl-beta-carboline inhibits the M2 polarization of tumor-associated macrophages: A study based on network pharmacology and molecular docking analyses

IntroductionHypoxia and tumor-associated macrophage (TAM) are key regulators in remodeling the microenvironment of esophageal squamous cell carcinoma (ESCC). Hypoxia could stimulate tumor cells to secrete more exosomes and activate TAMs to M2 type. Here, we investigated the function and the underlying mechanism of tumor-derived exosomal hsa-circ-0048117 in TAM polarization in ESCC. Collectively, these data indicate that PC cells generate miR-301a-3p-rich exosomes in a hypoxic microenvironment, which then polarize macrophages to promote malignant behaviors of PC cells.MethodsTransmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to analyze the physical characteristics of exosomes. High-throughput sequencing and bioinformatic analysis were performed to screen the potential exosomal circRNA. FISH, Ago2 RIP, pull-down and dual-luciferase reporter assay were conducted to figure out the correlation among hsa-circ-0048117, miR-140 and toll-like receptor 4 (TLR4). Flow cytometry and Western blot were used to evaluate their joint effect in macrophages polarization. Then, the invasion and migration ability were evaluated by transwell experiment. At last, serum exo-hsa-circ-0048117 in ESCC patients was compared and the correlation between its expression and T stage, N stage and TNM grades was analyzed.ResultsHsa-circ-0048117 was significantly upregulated and enriched in exosomes secreted by hypoxia pre-challenged tumor cells and contributed to M2 macrophage polarization. Hsa-circ-0048117 depletion in macrophage led to inhibition of M2 polarization while restoration of hsa-circ-0048117 could rescue the process. Moreover, hsa-circ-0048117 could act as sponge of miR-140 by competing with TLR4 to facilitate the M2 macrophage polarization. Exo-hsa-circ-0048117 could be transmitted to macrophages to promote M2 polarization and M2 macrophages could enhance the ability of invasion and migration of tumor cells by secreting Arg1, IL-10 and TGF-β. Higher serum exo-hsa-circ-0048117 predicted an advanced T and N stage and positively correlated with TNM grade.ConclusionOur findings indicated that ESCC cells generate hsa-circ-0048117-rich exosomes in a hypoxic microenvironment; hsa-circ-0048117 was believed to promote M2 macrophage polarization which favors the malignant behaviors of ESCC cells. These results reminded us that exosomal hsa-circ-0048117 may play a key role in remodeling the microenvironment and modulating progression in ESCC.

PAARH promotes M2 macrophage polarization and immune evasion of liver cancer cells through VEGF protein

Grounded on the bioinformatics insights, this study explores the role of flotillin 1 (FLOT1) in modulating macrophage phenotype and immune evasion in lung cancer cells. The bioinformatics analyses revealed positive correlations between FLOT1 expression and infiltration of M2 macrophages, neutrophils, dendritic cells, and CD4 memory T cells. Furthermore, elevated FLOT1 expression was associated with a poor prognosis in lung cancer patients. Analysis of tumor and adjacent non-tumor tissues from 53 lung cancer patients revealed significantly higher immunohistochemical staining of FLOT1 in tumor tissues, showing positive correlation with the staining intensity of PD-L1. Additionally, staining intensities for markers of M2 macrophages (Arg1), CD4 memory T cells (CD4), dendritic cells (CD83), and neutrophils (CD177) were significantly higher in tumor tissues with high FLOT1 levels. Silencing of FLOT1 was induced in two lung cancer cell lines. Co-culturing in conditioned media of the FLOT1-silenced cancer cells led to reduced chemotactic migration and M2 skewing of macrophages in vitro. Using xenograft models, we observed that FLOT1 silencing weakened tumorigenic activity of A549 cells in mice and reduced M2 macrophage infiltration in tumors. SWI/SNF related BAF chromatin remodeling complex subunit C1 (SMARCC1) was identified as a transcription factor that activated FLOT1 transcription by binding to its promoter. Knockdown of SMARCC1 in lung cancer cells similarly reduced the migration and M2 polarization of macrophages as well as weakened tumorigenesis in mice. However, these effects were counteracted by FLOT1 overexpression. Further analysis of the downstream effectors of the SMARCC1/FLOT1 cascade revealed the enrichment of these factors in ferroptosis-related pathways. Mechanistically, SMARCC1 knockdown led to a decreased GSH:GSSG ratio and increased lipid peroxidation in macrophages, while FLOT1 overexpression restored these changes. Transmission electron microscopic observation revealed typical features of ferroptosis-resistant mitochondria following SMARCC1 knockdown, including fragmented or reduced cristae and increased outer membrane integrity. These mitochondrial changes were mitigated by FLOT1 overexpression. In conclusion, SMARCC1 promotes immune evasion in lung cancer by activating FLOT1 transcription. This activation enhances recruitment and M2 polarization of macrophages, and increases PD-L1 expression, reduces ferroptosis. These findings provide valuable insights into the molecular mechanisms of immune evasion and suggest potential therapeutic targets for lung cancer treatment. KEY MESSAGES: • FLOT1 is associated with poor prognosis in lung cancer patients. • Association between FLOT1 and immune cell infiltration in lung cancer. • Silencing FLOT1 inhibits the recruitment of macrophages by lung cancer cells. • SMARCC1 is highly expressed in lung cancer and promotes the transcription of FLOT1. • FLOT1 overexpression rescues the inhibitory effect of SMARCC1 knockdown on M2 macrophage infiltration and activation of Ferroptosis.

Circular RNAs (circRNAs) have been shown to mediate tumor-associated macrophages (TAMs) to regulate the development of many cancers, including lung adenocarcinoma (LUAD). However, whether circ_0001715 regulates LUAD progression by mediating TAMs polarization remains uncertain. Monocytes (THP-1) were treated with PMA to induce M0 macrophages. M0 macrophages were incubated with LUAD cells-derived exosomes and then cocultured with LUAD cells. The levels of circ_0001715, M2 macrophage markers, microRNA (miR)-205-5p, and triggering receptor expressed on myeloid cells-2 (TREM2) were examined using quantitative real-time PCR. Flow cytometry was performed to assess M2 macrophage surface marker CD206. Cell proliferation, migration and invasion were determined using cell counting kit 8, EdU, colony formation and transwell assays. Dual-luciferase reporter assay was used to investigate the interactions between miR-205-5p and circ_0001715 or TREM2. Circ_0001715 knockdown inhibited M2 macrophage polarization and its overexpression had an opposite effect. After M0 macrophages transfected with si-circ_0001715 were cocultured with LUAD cells, the proliferation and metastasis of LUAD cells were markedly reduced. Exosomes transferred circ_0001715 between M0 macrophages and LUAD cells. Exosomal circ_0001715 promoted M2 macrophage polarization to increase LUAD cell proliferation and metastasis. In terms of mechanism, circ_0001715 sponged miR-205-5p to positively regulate TREM2. TREM2 upregulation also could promote LUAD cell proliferation and metastasis via increasing M2 macrophage polarization. In addition, TREM2 knockdown reversed the effect of exosomal circ_0001715 on M2 macrophage polarization and LUAD cell progression. Exosomal circ_0001715 led to LUAD cell proliferation and metastasis by promoting M2 macrophage polarization via the miR-205-5p/TREM2 axis.