FOXM1 derived from Triple negative breast cancer exosomes promotes cancer progression by activating IDO1 transcription in macrophages to suppress ferroptosis and induce M2 polarization of Tumor-associated macrophages

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

Exosomes are pivotal in triple-negative breast cancer (TNBC) development, and accumulating evidence underscores their potential as therapeutic targets and diagnostic indicators. In this study, we revealed a significant enrichment of the POU domain, class 5, transcription factor 1 (POU5F1) in TNBC cells-derived exosomes. Functionally, silencing endogenous POU5F1 in TNBC cells substantially inhibited their aggressive phenotypes. Moreover, exosomes derived from TNBC cells contributed to macrophage M2 polarization by transferring POU5F1 to the recipient macrophages. Mechanistically, POU5F1 within these exosomes prevented the tumor necrosis factor receptor-associated factor 6 (TRAF6) degradation in macrophages, thereby activating the protein kinase B (AKT) signaling cascade and driving M2 polarization. Furthermore, in vivo experiments provided evidence that POU5F1 knockdown significantly reduced tumor growth and macrophage M2 polarization in a mouse model of TNBC cells by modulating the TRAF6/AKT signaling axis. Our study concludes that POU5F1 in TNBC cells-derived exosomes is vital for promoting macrophage M2 polarization by inhibiting TRAF6 ubiquitination and activating AKT signaling, thereby contributing to TNBC progression.

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

Cetylpyridinium chloride inhibits the polarization of tumor-associated macrophages and the proliferation of triple-negative breast cancer cells.

<div>Abstract<p>The immunologic effects of chemotherapy-induced tumor cell death are not completely understood. Accumulating evidence suggests that phagocytic clearance of apoptotic tumor cells, also known as efferocytosis, is an immunologically silent process, thus maintaining an immunosuppressive tumor microenvironment (TME). Here we report that, in the breast tumor microenvironment, thymosin α-1 (Tα-1) significantly reverses M2 polarization of IL10-producing tumor-associated macrophages (TAM) during efferocytosis induced by apoptotic cells. Mechanistically, Tα-1, which bound to phosphatidylserine on the surface of apoptotic tumor cells and was internalized by macrophages, triggered the activation of SH2-containing inositol 5′-phosphatase 1 (SHIP1) through the lysosomal Toll-like receptor 7 (TLR7)/MyD88 pathway, subsequently resulting in dephosphorylation of efferocytosis-activated TBK1 and reduction of efferocytosis-induced IL10. Tα-1 combined with epirubicin chemotherapy markedly suppressed tumor growth in an <i>in vivo</i> breast cancer model by reducing macrophage-derived IL10 and enhancing the number and function of tumor-infiltrating CD4<sup>+</sup> and CD8<sup>+</sup> T cells. In conclusion, Tα-1 improved the curative effect of chemotherapy by reversing M2 polarization of efferocytosis-activated macrophages, suggesting that Tα-1 injection immediately after chemotherapy may contribute to highly synergistic antitumor effects in patients with breast cancer.</p>Significance:<p>Thymosin α-1 improves the curative effect of chemotherapy by reversing efferocytosis-induced M2 polarization of macrophages via activation of a TLR7/SHIP1 axis.</p></div>

<div>Abstract<p>The immunologic effects of chemotherapy-induced tumor cell death are not completely understood. Accumulating evidence suggests that phagocytic clearance of apoptotic tumor cells, also known as efferocytosis, is an immunologically silent process, thus maintaining an immunosuppressive tumor microenvironment (TME). Here we report that, in the breast tumor microenvironment, thymosin α-1 (Tα-1) significantly reverses M2 polarization of IL10-producing tumor-associated macrophages (TAM) during efferocytosis induced by apoptotic cells. Mechanistically, Tα-1, which bound to phosphatidylserine on the surface of apoptotic tumor cells and was internalized by macrophages, triggered the activation of SH2-containing inositol 5′-phosphatase 1 (SHIP1) through the lysosomal Toll-like receptor 7 (TLR7)/MyD88 pathway, subsequently resulting in dephosphorylation of efferocytosis-activated TBK1 and reduction of efferocytosis-induced IL10. Tα-1 combined with epirubicin chemotherapy markedly suppressed tumor growth in an <i>in vivo</i> breast cancer model by reducing macrophage-derived IL10 and enhancing the number and function of tumor-infiltrating CD4<sup>+</sup> and CD8<sup>+</sup> T cells. In conclusion, Tα-1 improved the curative effect of chemotherapy by reversing M2 polarization of efferocytosis-activated macrophages, suggesting that Tα-1 injection immediately after chemotherapy may contribute to highly synergistic antitumor effects in patients with breast cancer.</p>Significance:<p>Thymosin α-1 improves the curative effect of chemotherapy by reversing efferocytosis-induced M2 polarization of macrophages via activation of a TLR7/SHIP1 axis.</p></div>

Background Tumor-associated macrophages (TAMs), particularly M2-polarized TAMs, are significant contributors to tumor progression, immune evasion, and therapy resistance in gastric cancer (GC). Despite efforts to target TAM recruitment or depletion, clinical efficacy remains limited. Consequently, the identification of targets that specifically inhibit or reprogram M2-polarized TAMs presents a promising therapeutic strategy. Objective This study aims to identify a dual-function target in GC cells that drives both malignant phenotypes and M2 macrophage polarization, revealing its molecular mechanisms to provide novel therapeutic targets for selectivly targeting M2-polarized TAMs in GC. Methods Transcriptomic and clinical data from GC and adjacent tissues were utilized to identify mRNAs associated with high M2 macrophage infiltration and poor prognosis. Single-cell sequencing elucidated cell types expressing the target gene. Transwell co-culture and exosome intervention experiments demonstrated its role in M2 polarization. Small RNA sequencing of exosomes, western blotting, and CoIP assays revealed the molecular mechanisms underlying exosome-mediated M2 polarization. Protein array, ChIP and dual-luciferase reporter assays clarified the molecular mechanisms by which the target gene regulated exosomal miRNA. In vivo validation was performed using xenograft tumor models. Results SERPINE1 was identified as a highly expressed mRNA in GC tissues and cells, significantly associated with advanced clinical stages, worse prognosis, and higher M2 macrophage infiltration in patients with GC. SERPINE1 overexpression in GC cells promoted tumor growth and M2 macrophage polarization. SERPINE1 facilitated the transfer of let-7 g-5p to macrophages via cancer-derived exosomes, inducing M2 polarization. Exosomal let-7 g-5p internalized by macrophages downregulated SOCS7 protein levels, disrupting its interaction with STAT3 and relieving the inhibition of STAT3 phosphorylation, thereby leading to STAT3 hyperactivation, which consequently drove M2 polarization. Additionally, in GC cells, elevated SERPINE1 expression activated JAK2, enhancing STAT3 binding to the let-7 g-5p promoter and promoting its transcription, thereby increasing let-7 g-5p levels in exosomes. Conclusion GC cell-derived SERPINE1, functioning as a primary driver of GC growth and TAM M2 polarization, promotes M2 polarization through the regulation of exosomal let-7 g-5p transfer via autocrine activation of the JAK2/STAT3 signaling pathway. These findings elucidate a novel mechanism of SERPINE1-induced M2 polarization and highlight SERPINE1 as a promising target for advancing immunotherapy and targeted treatments in GC.

Introduction: Intermittent hypoxia (IH), an important feature of obstructive sleep apnea, enhances the function of lung cancer cell-derived extracellular vesicles (EVs) to exacerbate the immunosuppressive properties of macrophages. Herein, we investigated the effects of EVs obtained from lung adenocarcinoma cells cultured under IH on macrophage polarization. Methods: The M1-type and M2-type tumor-associated macrophages (TAMs) in tissues from lung adenocarcinoma cases with (n = 10) or without (n = 12) OSA were assessed by immunohistochemical studies. EVs obtained from A549 cells grown under normoxia (EV-NA) or IH (EV-IH) were isolated and cocultured with macrophages. MicroRNA sequencing was used to determine discrepant miRNAs in EVs, selecting miR-20a-5p for subsequent experiments. Next, reverse transcription-quantitative polymerase chain reaction, flow cytometry, luciferase reporter assay, western blotting assay, and gain- and loss-of-function assays were used to explore the mechanism by which miR-20a-5p promotes M2 macrophage polarization by targeting phosphatase and Tensin homolog gene (PTEN). Results: Stromal M2 TAMs were highly abundant in patients with lung adenocarcinoma and obstructive sleep apnea. Macrophages treated with EV-IH that highly expressed miR-20a-5p showed the M2 phenotype. Luciferase reporter assay confirmed PTEN as a target of miR-20a-5p. Transfection of miR-20a-5p mimics decreased PTEN expression, upregulated M2 polarization markers, and promoted Akt phosphorylation in macrophages, while transfection with a miR-20a-5p inhibitor had the opposite effects. Furthermore, miR-20a-5p inhibition in macrophages eliminated the PTEN downregulation, Akt phosphorylation, and upregulation of M2 polarization markers induced by EV-IH transfection.Conclusion: These findings indicate that EVs obtained from lung adenocarcinoma cells cultured under IH deliver miR-20a-5p to promote M2 macrophage polarization by targeting PTEN.

lncRNAs are related to the progression of various diseases, including oral squamous cell carcinoma (OSCC), which is a common squamous cell carcinoma of the head and neck. Tumor-associated macrophages and tumor cells are significant components of tumor microenvironment. M2 polarization of tumor-associated macrophages is a crucial actor in tumor malignancy and metastasis. In this study, we studied the molecular mechanism of lncRNA DCST1-AS1 in OSCC. Here, we reported that DCST1-AS1 was significantly increased in OSCC cells. We found that loss of DCST1-AS1 obviously inhibited the proliferation, migration, and invasion of OSCC cells and xenograft tumor growth. Meanwhile, silencing of DCST1-AS1 also repressed the percentage of macrophages expressing M2 markers CD206 and CD11b. DCST1-AS1 shRNA enhanced the percentage of macrophages expressing M1 markers CD80 and CD11c. Then, we observed that loss of DCST1-AS1 suppressed OSCC progression via inactivating NF-κB signaling. As well established, NF-κB signaling exerts critical roles in tumor progression, and our study proved that DCST1-AS1 could regulate NF-κB signaling. We proved that blocking the NF-κB pathway using antagonists greatly downregulated OSCC progression and M2 macrophage polarization induced by the overexpression of DCST1-AS1. To sum up, we reported that DCST1-AS1 plays an important role in modulating OSCC tumorigenicity and M2 macrophage polarization through regulating the NF-κB pathway.

M2-polarized tumor-associated macrophage infiltration is a key risk factor for poor prognosis in triple-negative breast cancer (TNBC). This study investigated the role of transcription elongation factor B polypeptide 2 (TCEB2) in regulating M2 macrophage polarization during TNBC development. The expression of gene or protein was tested by qRT-PCR, western blot, and IHC. CCK8, colony formation, wound healing, and Transwell assays were used to evaluate TNBC cell malignant behaviors, including cell viability, proliferation, migration, and invasion. The secretion levels of cytokines were detected by ELISA. Ubiquitin-based IP assays were used to detect Slit2 ubiquitination. The combined relation between TCEB2, Slit2, and NEDD4 was investigated using Co-IP assay. Our results demonstrated that M2 macrophage polarization was activated in TNBC, which might be related to TCEB2 upregulation. TCEB2 knockdown reduced TNBC cell growth, migration, invasion, and their ability to induce M2 macrophage polarization. Mechanistically, TCEB2 mediated Slit2 K63 ubiquitination degradation in TNBC by interacting with NEDD4. As expected, the inhibitory effect of TCEB2 silencing on the ability of TNBC cells to induce M2 macrophage polarization was reversed by Slit2 knockdown. Finally, TCEB2 knockdown inhibited TNBC tumor growth and TNBC-induced M2 macrophage polarization in vivo. TCEB2 upregulation promoted TNBC-induced M2 macrophage polarization to accelerate TNBC development by mediating Slit2 K63-ubiquitination degradation through interacting with NEDD4.

RNA-seq shows Angiopoietin-like 4 promotes hepatocellular carcinoma progression by inducing M2 polarization of tumor-associated macrophages.

Berberine (BBR), an isoquinoline alkaloid extracted from Coptis chinensis, is clinically used to treat chronic colitis, diabetes, and other diseases. Although BBR has antitumor effects, it is unclear whether it can inhibit hepatocellular carcinoma (HCC) by modulating the tumor inflammatory microenvironment. In this study, we demonstrated that BBR inhibits HCC development in mice by suppressing the M2 polarization of macrophages. Using an H22 tumor-bearing xenograft mouse model, we found that BBR significantly inhibited H22 tumor growth. Analysis of scRNA-seq results revealed reduced M2 macrophage infiltration and polarization in BBR-treated HCC tissues. Pharmacodynamic studies showed that BBR treatment markedly increased CD8+ T cell infiltration and attenuated M2 polarization. In vitro, BBR suppressed IL-4 or tumor cell supernatant-induced M2 polarization, as evidenced by decreased expression of M2 polarization marker genes (Arg1, Retnla, etc.) and reduced JAK1/STAT6 phosphorylation levels. Molecular docking and protein stability assays revealed that BBR directly binds to JAK1’s FERM domain, stabilizing it. Combination therapy with BBR and anti-PD-L1 antibody synergistically inhibited H22 tumor growth. These findings suggest that BBR can reduce the M2 polarization of tumor-associated macrophages (TAMs) by targeting the IL-4-JAK1-STAT6 axis, and combining with anti-PD-L1 antibody may represent a promising therapeutic strategy to enhance BBR’s antitumor efficacy.

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.

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.

Background Triple-negative breast cancer (TNBC) is an aggressive subtype with a poor prognosis and limited treatment options. Tumor-associated macrophages (TAMs), the predominant and abundant immune cells in the tumor immune microenvironment (TIME), critically drive TNBC progression. Consequently, TAM reprogramming has emerged as a promising therapeutic approach. However, a major barrier remains the incomplete understanding of the molecular mechanisms governing TAM reprogramming. Methods The role of CD44v5 in TAM polarization was evaluated with a CD44v5 monoclonal antibody and CD44v5-knockdown cell lines. Subsequently, cell functional assays, including wound healing, invasion, and colony formation assays, were performed to assess changes in the MDA-MB-468 cell line. Cytokine secretion levels (IL-4 and IL-6) were measured by electrochemiluminescence immunoassay (ECLIA). Results We found that M2 macrophages and tumor-associated macrophages (TAMs) polarized through the IL4/IL4R signaling pathway and exerted similar protumorigenic functions, and that IL4 is the key protumorigenic factor secreted by M2 macrophages. Interestingly, CD44v5 blockade effectively inhibited M2 polarization and promoted the phenotypic shift to M1 macrophages, which was supported by increased CD86 expression and decreased IL-4 secretion. Furthermore, molecular docking analysis and colocalization microscopy confirmed that CD44v5 colocalized with IL-4Rα, preventing its internalization. Conclusion CD44v5 promotes M2 macrophage polarization by stabilizing and enhancing the IL-4Rα/STAT6/IL-4 signaling pathway, thereby facilitating the progression of triple-negative breast cancer. CD44v5 serves as an important therapeutic target for the reprogramming of both TAMs and M2 macrophages, thereby providing a novel strategy for the treatment of TNBC.