Polarization Of Tumour-associated Macrophages Research Articles

Astragaloside IV inhibits colorectal cancer metastasis by reducing extracellular vesicles release and suppressing M2-type TAMs activation

Ethnopharmacological relevanceTumour-derived extracellular vesicles (TEVs) have been confirmed to facilitate colorectal cancer (CRC) metastasis by remodelling the tumour microenvironment (TME). Drugs targeted TEVs is considered as a promising therapeutic strategy for cancer treatment. Traditional Chinese medicine (TCM) plays a vital role in improving the prognosis of CRC patients and eventually CRC patients with distant metastasis. Although the anti-tumour effects of active compounds from TCM prescriptions are observed widely, the molecular mechanisms remain unknown. Aim of the studyThis study aims to investigate the effects of active compounds in our library of TCM on preventing CRC metastasis, and also explore the potential mechanisms from the perspective of TEVs. Materials and methods: The effects of active compounds on the proliferation of CRC cells were determined by CCK-8 assay. TEVs were extracted from MC38 cells by ultracentrifugation and characterized by electron microscopy, Nanosight NS300 and western blotting. The TEV particles were quantified by Nanosight NS300. The potential mechanism by which astragaloside IV (ASIV) reduced TEV secretion was determined by western blotting. RAW264.7 cells were cocultured with the conditioned medium (CM) of MC38 cells treated with or without ASIV, and the activation of tumour-associated macrophages (TAMs) was assessed by immunofluorescence and quantitative polymerase chain reaction (qPCR). The migration of CRC cells was measured by wound healing and Transwell assay. A spleen-to-liver metastasis model of colorectal cancer was used to confirm the efficiency of ASIV in vivo. Liver metastatic tumours of the mice were used for liver weight measures and H&E staining. Immunofluorescence was applied to observe the infiltration of TAMs, the expression of neutral sphingomyelinase 2 (nSMase2) and Rab27a. ResultsBy screening our TCM monomer library, we found that ASIV, which is mainly extracted from Radix Astragali, reduced the release of TEVs from CRC cells in a time- and concentration-dependent manner. Mechanistically, ASIV inhibited the production and secretion of TEVs by downregulating nSMase2 and Rab27a expression in CRC cells. CM from ASIV-treated CRC cells reshaped the polarization of TAMs by decreasing M2-type polarization, increasing M1-type polarization. Consequently, the repolarization of M2-type to M1-type macrophages led to reduced invasion and migration of CRC cells. Moreover, we confirmed that ASIV inhibited the liver metastasis of CRC, reduced M2-type macrophage infiltration and decreased the expression of nSMase2 and Rab27a in liver metastases. ConclusionsASIV inhibited CRC metastasis by reducing EVs release and suppressing M2-type TAMs activation. All these findings reveal a new insight into the mechanisms of ASIV in preventing CRC progression and provide a promising approach for anti-tumour therapy.

Hypoxia-induced ZEB1 promotes cervical cancer immune evasion by strengthening the CD47-SIRPα axis

BackgroundThe dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear.MethodsExosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed.ResultsOur previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα+ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα+ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression.ConclusionsOur results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

Nocardia rubra cell wall skeleton regulates tumour-associated macrophage polarization by reprogramming M2 macrophages into M1 macrophages via STAT1/STAT6 pathways.

Targeted therapy with tumour-associated macrophages (TAMs) has emerged as a new paradigm for immunotherapy of cervical cancer. Nocardia rubra cell wall skeleton (Nr-CWS) for external use is an immunotherapeutic agent. In this study, we aimed to explore the effects of Nr-CWS on TAMs and the potential mechanisms. Cervical tissue samples were collected before and after Nr-CWS treatment from patients with high-risk HPV infection and cervical intraepithelial neoplasia (CIN). The effect of Nr-CWS on macrophages in vivo was examined by immunohistochemistry and double-labeling immunofluorescence histochemistry. In vitro experiments were performed using a TAM model established by THP-1 cells under Nr-CWS treatment. We found that Nr-CWS treatment significantly reduced the numbers of total macrophages and M2 macrophages, increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages in cervical tissues. After Nr-CWS treatment in vitro, the expression levels of the M1 macrophage markers were increased, while the expression levels of the M2 macrophage markers were decreased. Nr-CWS treatment also activated STAT1 pathways but inhibited STAT6 pathways. These results indicated that Nr-CWS may improve local immune response and reverse immunosuppression by regulating the M2 to M1 polarization of TAMs via STAT1/STAT6 pathways.

DACH1 regulates macrophage activation and tumour progression in hypopharyngeal squamous cell carcinoma.

Dachshund family transcription factor 1 (DACH1) has been shown to exhibit a tumour-suppressive role in a number of human cancers. However, the role of DACH1 in hypopharyngeal squamous cell carcinoma (HPSCC) and its function in the tumour microenvironment (TME) are still not clear. Crosstalk between cancer cells and tumour-associated macrophages (TAMs) mediates tumour progression in HPSCC. The expression of DACH1, CD86 and CD163 was detected in 71 matched HPSCC-non-cancerous tissue pairs using quantitative real-time polymerase chain reaction and IHC analysis. Cell proliferation, migration and invasion were monitored by colony formation, Transwell and EdU incorporation assays. ChIP-qPCR and dual-luciferase reporter assays were applied to verify the targeting relationships between DACH1 and IGF-1. Stably transfected HPSCC cells were co-cultured with MΦ macrophages to assess macrophage polarization and secretory signals. DACH1 was decreased in HPSCC tissues and was indicative of a poor prognosis for HPSCC patients. Decreased DACH1 expression in HPSCC was associated with fewer CD86+ TAMs and more CD163+ TAMs. Knockdown of DACH1 inhibited the proliferation, migration and invasion of FaDu cells via Akt/NF-κB/MMP2/9 signalling. Additionally, DACH1 was found to directly bind to the promoter region of IGF-1 to downregulate the secretion of IGF-1, which inhibited TAMs polarization through the IGF-1R/JAK1/STAT3 axis. Furthermore, in nude mice, the effects of DACH1 inhibition on tumour progression and M2-like TAMs polarization were confirmed. These findings suggest that IGF-1 is a critical downstream effector of DACH1 that suppresses cell migration and invasion and inhibits TAMs polarization. DACH1 could be a therapeutic target and prognostic marker for HPSCC.

Sialic acid-targeted cyclodextrin-based nanoparticles deliver CSF-1R siRNA and reprogram tumour-associated macrophages for immunotherapy of prostate cancer

Prostate cancer remains a serious condition threatening the health of men. Due to the complicated nature of the tumour microenvironment (TME), conventional treatments face challenges including poor prognosis and tumour resistance, therefore new therapeutic strategies are urgently needed. Small interfering RNA (siRNA), a double-stranded non-coding RNA, regulates specific gene expression through RNA interference. Tumour-associated macrophages (TAMs) are a potential therapeutic target in cancer immunotherapy. Colony stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) signaling pathway plays a crucial role in the polarization of the immunosuppressive TAMs, M2 macrophages. Downregulation of CSF-1R is known to reprogram the immunosuppressive TAMs, M2 macrophages, to the immunostimulatory phenotype, M1 macrophages. Sialic acid is a ligand for Siglec-1 (CD169) which is overexpressed on M2 macrophages with little expression in other phenotypes. Therefore, a sialic acid-targeted cyclodextrin-based nanoparticle was developed to specifically deliver CSF-1R siRNA to M2 macrophages. The nanoparticles were studied in vitro using both human and mouse prostate cancer cell lines. Results show that the targeted nanoparticles achieved cell specific delivery to M2 macrophages via the sialic acid-CD169 axis. The expression of CSF-1R was significantly downregulated in M2 macrophages (29.64% for targeted vs 19.31% for non-targeted nanoparticles in THP-1-derived M2 macrophages and 38.94% for targeted vs 18.51% for non-targeted nanoparticles in RAW 264.7-derived M2 macrophages, n = 4, p < 0.01). The resulting reprograming of M2 macrophages to M1 enhanced the level of apoptosis in the prostate cancer cells in a Transwell model (49.17% for targeted vs 37.68% for non-targeted nanoparticles in PC-3 cells and 69.15% for targeted vs 44.73% for non-targeted nanoparticles in TRAMP C1 cells, n = 3, p < 0.01). Thus, this targeted cyclodextrin-based siRNA drug delivery system provides a potential strategy for prostate cancer immunotherapy.

BDNF is a prognostic biomarker involved in immune infiltration of lung adenocarcinoma and is associated with brain metastasis.

Non-small cell lung cancer (NSCLC) is one of the leading causes of death worldwide. Brain metastases are a common complication of a wide range of human malignancies, particularly lung adenocarcinoma (LUAD). Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has been linked to several human malignancies and has been shown to promote LUAD tumorigenesis. However, its function in the tumour immune microenvironment (TIME) remains largely unexplored, especially in complex brain tissue environments. In this study, BDNF was found to be particularly increased in patients with advanced tumour stage, lymphatic metastasis, and distant metastasis, indicating a correlation with LUAD progression. We characterized the prognostic value of BDNF and defined BDNF as an unfavourable prognostic indicator through a common driver gene-independent mechanism in LUAD. Furthermore, patients with increased BDNF levels in primary LUAD might have a higher risk of developing brain metastasis (BM), and central nervous system (CNS) metastasis showed an elevated expression of BDNF compared to their matched primary lesions. Additionally, we investigated the interaction between BDNF and infiltrating immune cells in both primary lesions and paired BM using multiplex immunostaining. The results showed that BDNF might drive an immunosuppressive tumour microenvironment (TME) by re-education of tumour-associated macrophages (TAMs) toward a pro-tumorigenic M2 phenotype, particularly in BM. Our findings demonstrate that BDNF serves as an independent potential prognostic marker and correlates with BM in LUAD. As it is closely related to TAM polarization, BDNF may be a promising immune-related biomarker and molecular target in patients with LUAD.

Facile construction of tumour spheroids with induced M2 macrophage polarization for anticancer drug screening

Tumour-associated macrophages (TAMs) are involved in cancer progression and drug resistance in the tumour microenvironment (TME). Consequently, macrophages as therapeutic targets have garnered increased attention; however, there are hurdles to screening interactions between cancer and macrophages owing to technical difficulties in recapitulating in vitro physiological systems. In this study, we propose a simple strategy to construct tumour spheroids with induced M2 macrophage polarization for anticancer drug screening. We observed that cytokine expression related to the TME in three-dimensional (3D) cancer spheroids was enhanced compared with that in two-dimensional conventional cancer cell cultures. We also demonstrated that the 3D breast tumour spheroids promote M2-like TAM polarization via granulocyte–macrophage colony-stimulating factor and granulocyte colony-stimulating factor. Furthermore, adipose tissue-derived stem cells, an abundant stromal cell population in the breast cancer TME, further enhanced the M2 phenotype in the in vitro tumour spheroids. Therefore, we propose the tumour spheroids as a drug screening platform to evaluate drug efficacy in cancers. Overall, the simple strategy to form tumour spheroids developed in this study will broaden the understanding of communication between cancer cells and macrophages and contribute to the evaluation of cancers and the development of better strategies for their therapy and management.

CDK5RAP3 acts as a tumour suppressor in gastric cancer through the infiltration and polarization of tumour-associated macrophages

We have demonstrated that CDK5RAP3 exerts a tumour suppressor effect in gastric cancer, but its role in regulating tumour-associated macrophages (TAMs) has not yet been reported. Here, we show that CDK5RAP3 is related to the infiltration and polarization of macrophages. It inhibits the polarization of TAMs to M2 macrophages and promotes the polarization of the M1 phenotype. CDK5RAP3 reduces the recruitment of circulating monocytes to infiltrate tumour tissue by inhibiting the CCL2/CCR2 axis in gastric cancer. Blocking CCR2 reduces the growth of xenograft tumours and the infiltration of monocytes. CDK5RAP3 inhibits the nuclear transcription of NF-κB, thereby reducing the secretion of the cytokines IL4 and IL10 and blocking the polarization of M2 macrophages. In addition, the absence of CDK5RAP3 in gastric cancer cells allows macrophages to secrete more MMP2 to promote the epithelial-mesenchymal transition (EMT) process of gastric cancer cells, thereby enhancing the invasion and migration ability. Our results imply that CDK5RAP3 may be involved in the regulation of immune activity in the tumour microenvironment and is expected to become a potential immunotherapy target for gastric cancer.

The CTCF/LncRNA-PACERR complex recruits E1A binding protein p300 to induce pro-tumour macrophages in pancreatic ductal adenocarcinoma via directly regulating PTGS2 expression.

BackgroundTumour‐associated macrophages (TAMs) play an important role in promoting the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to study the epigenetic mechanisms in regulating pro‐tumour M2‐polarised TAMs in the PDAC tumour microenvironment.MethodsThis study was conducted based on ex vivo TAMs isolated from PDAC tissues and in vitro THP1‐derived TAM model. RNA‐sequencing (RNA‐seq), assay for transposase‐accessible chromatin with sequencing and chromatin immunoprecipitation sequencing were performed to investigate gene expression, chromatin accessibility, transcription factor binding sites and histone modifications. Gene knockdown in THP1‐derived TAMs was performed with lentivirus, and the impact of THP1‐derived TAMs on invasion and metastasis ability of PDAC cells were investigated with in vitro and in vivo functional assays. RNA‐chromatin interaction was analysed by chromatin isolation through RNA purification with sequencing. RNA‐protein interaction was studied by RNA immunoprecipitation and RNA pull‐down.ResultsOur data showed that the transcription factor CTCF (CCCTC‐binding factor) was highly expressed in TAMs and predicted to be significantly enriched in hyper‐accessible chromatin regions when compared to monocytes. High infiltration of CTCF+ TAMs was significantly associated with poor prognosis in PDAC patients. Knockdown of CTCF in THP1‐derived TAMs led to the down‐regulation of specific markers for M2‐polarised TAMs, including CD206 and CD163. When THP1‐derived TAMs with CTCF knockdown, they showed a decreased ability of invasion and metastasis. Further integrative analysis of multi‐omics data revealed that prostaglandin‐endoperoxide synthase 2 (PTGS2) and PTGS2 antisense NF‐κB1 complex‐mediated expression regulator RNA (PACERR) were critical downstream targets of CTCF and positively correlated with each other, which are closely situated on a chromosome. Knockdown of PACERR exhibited a similar phenotype as observed in CTCF knockdown THP1‐derived TAMs. Moreover, PACERR could directly bind to CTCF and recruit histone acetyltransferase E1A binding protein p300 to the promoter regions of PACERR and PTGS2, thereby enhancing histone acetylation and gene transcription, promoting the M2 polarization of TAMs in PDAC.ConclusionsOur study demonstrated a novel epigenetic regulation mechanism of promoting pro‐tumour M2‐polarised TAMs in the PDAC tumour microenvironment.

EMP3 mediates glioblastoma\u2010associated macrophage infiltration to drive T cell exclusion

BackgroundThe immunosuppressive tumour microenvironment is a critical factor in the initiation and progression of glioblastoma (GBM), which is characterized by an abundance of tumour-associated macrophages (TAMs) but a paucity of infiltrating T cells. In this research, we studied whether epithelial membrane protein 3 (EMP3) plays a crucial role in immune modulation in GBM.MethodsTCGA and CGGA transcriptomic profiles of wild-type IDH1 GBM were used for bioinformatic analysis. The role of EMP3 in GBM was validated through in vivo and in vitro experiments. Human GBM specimens were collected and evaluated using immunofluorescence analysis.ResultsEMP3 was associated with immunosuppression in GBM. Elevated EMP3 in GBM areas was accompanied by high expression of PD-L1 and abundant M2 TAM recruitment but a lake of T cell infiltration. We found that EMP3 was a potent protein in M2 TAM polarization and recruitment that impaired the ability of GBM cells to secrete CCL2 and TGF-β1. Furthermore, EMP3 suppressed T cell infiltration into GBM tumours by inhibiting the secretion of CXCL9 and CXCL10 by macrophages and led to an effective response to anti-PD1 therapy.ConclusionsEMP3 is thus a critical immunosuppressive factor for recruiting TAMs in GBM and suppressing intratumoural T cell infiltration to facilitate tumour progression and is a potential therapeutic target.

Identification of ANXA1 as a potential prognostic biomarker and correlating with immune infiltrates in colorectal cancer

Background ANXA1 is a calcium-dependent phospholipid-binding protein and is frequently associated with inflammation, cell proliferation and apoptosis. However, the relationship between ANXA1 and the prognosis of multiple tumours and tumour infiltrating immune cells remains unclear. Methods Multivariate Cox proportional regression analysis was used for signature genes exploration in the basic of colon adenocarcinoma (COAD) RNA-sequence dataset obtained from TCGA, following the identification of 267 common differentially expressed genes, including ANXA1, among three expression profile datasets (GSE41328, GSE110224, and GSE113513). The differential expression of ANXA1 in different tumours and their corresponding normal tissues were evaluated through the Tumour Immune Estimation Resource (TIMER) and Oncomine database. Subsequently, we investigated the correlation between the expression level of ANXA1 and diverse panel of infiltrating immune cells and their related gene markers in colorectal cancer using correlation analysis in TIMER and GEPIA database. Results The high expression of ANXA1 was demonstrated to be closely correlated with poor survival in patients with colorectal cancer. More importantly, we found that changes in ANXA1 expression showed a moderate to strong, and statistically significant, correlation with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells. By contrast, there are only weak correlations between ANXA1 expression and immune cell infiltration in ESCA and STAD. ANXA1 expression was considerably associated with various immune markers involving immune cell recruitment, polarization of tumour-associated macrophages, and T cell exhaustion. Conclusion ANXA1 is not only an independent risk factor in the prediction of the prognosis of colorectal cancer, but also a crucial regulator in immune cell infiltration. This study may shed light on the clinical value of ANXA1, especially in the areas of early diagnosis of colorectal cancer and therapeutic target discovery.

Astragalus polysacharin inhibits hepatocellular carcinoma-like phenotypes in a murine HCC model through repression of M2 polarization of tumour-associated macrophages

Context Astragalus polysaccharin (APS), an extract of Astragalus propinquus Schischk, exerts antitumor effects in hepatocellular carcinoma (HCC). Objective This study investigated the mechanism of action of APS in HCC. Materials and methods Tumour-associated macrophages (TAMs) were treated with APS (0, 8, 16 mg/mL) for 24 h. APS (16 mg/mL)-treated TAMs were co-cultured with MHCC97H/Huh7 cells for 24 h. Finally, BALB/c nude mice were divided into PBS, APS (50 mg/kg), APS (100 mg/kg), APS (200 mg/kg) groups: mice were inoculated with Huh7 cells to construct tumour xenograft model, followed by administration of APS (50, 100, 200 mg/kg) or PBS daily for 30 days. Cell proliferation, migration, invasion, tumour growth, macrophage markers and proportions were measured. Results APS 16 mg/mL treatment enhanced the expression of M1 macrophage markers (iNOS, IL-1β and TNF-α) and M1 macrophage proportions, while reducing the expression of M2 macrophage markers (IL-10, Arg-1) and M2 macrophage proportions in TAMs. Moreover, the APS-mediated M1 phenotype of TAMs significantly repressed cell proliferation, migration and invasion of MHCC97H and Huh7 cells. Moreover, APS (50, 100, 200 mg/kg) enhanced M1 macrophage proportions and reduced M2 macrophage proportions in the tumour tissues, and thus inhibited tumour growth of HCC. Discussion and conclusions APS inhibits HCC-like phenotypes in a murine HCC model through repression of M2 polarization of TAMs. This work provides a novel theoretical basis for the application of APS in the clinical treatment of HCC.

CtBP1 promotes tumour-associated macrophage infiltration and progression in non-small-cell lung cancer.

The progression of lung cancer is majorly facilitated by TAMs (tumour‐associated macrophages). However, how the TAMs infiltrate the NSCLC microenvironment and the associated biochemical are not fully elaborated. Research has revealed that changes in CtBP1 modulates innate immunity. Here, we investigated if CtBP1 facilitates infiltration of TAM and the subsequent progression of NSCLC. Immunohistochemical analysis was carried out in 96 NSCLC patients to estimate the clinicopathological importance of CtBP1 in the disease. CtBP1 overexpression and knockdown were carried out to assess the activity of CtBP1 in NSCLC cells. Elevated expression of CtBP1 correlated positively with TAMs infiltration into NSCLC tissues, induced EMT (epithelial‐mesenchymal transition) in NSCLC cells and modulated the activated NF‐κB signalling pathway leading to increase in CCL2 secretion from NSCLC cells, thus promoting TAM recruitment and polarization. TAM induction and polarization reduced significantly on exhausting p65 in NSCLC cells with CtBP1. Moreover, infiltration of TMAs was reduced remarkably on antagonist‐mediated blocking of CCR2 and impeded the progression of NSCLC in a mouse model. These findings thus show a novel insight into the process of CtBP1‐regulated TAM infiltration in NSCLC.

Cordyceps militaris polysaccharide converts immunosuppressive macrophages into M1-like phenotype and activates T lymphocytes by inhibiting the PD-L1/PD-1 axis between TAMs and T lymphocytes

Tumour-associated macrophages (TAMs) inhibit the killing effect of T lymphocytes on tumour cells through the immunocheckpoint programmed death ligand-1 (PD-L1)/programmed death-1 (PD-1) axis. TAMs-targeted therapy is a promising approach that could be used to reverse the immunosuppressive tumour microenvironment. Here, we further report CMPB90-1, a novel natural polysaccharide from Cordyceps militaris, could function as an anti-tumour modulator that resets TAMs from a tumour-promoting M2 phenotype to a tumour-killing M1 phenotype. This process involves reversing the functional inhibition of T lymphocytes by inhibiting the PD-L1/PD-1 axis between TAMs and T lymphocytes. Mechanistically, the membrane receptor of CMPB90-1 binding to M2 macrophages was identified by tandem mass spectrometry. CMPB90-1 converts immunosuppressive TAMs via binding to toll-like receptor 2 (TLR2), which causes the release of Ca2+ and the activation of p38, Akt and NF-κB, or ERK. This process then leads to the polarization of TAMs from M2 phenotype to the M1 phenotype. In vivo experiment shows that CMPB90-1 is able to polarize TAMs into the M1 phenotype and has anti-tumour effects with improved safety. Additionally, the anti-tumour effects of CMPB90-1 in vivo depend on the phenotypic conversion of TAMs. The results demonstrated that CMPB90-1 could be developed as a potential immune-oncology treatment reagent.

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer

ABSTRACT Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones – CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell–cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90β and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90β was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment. Abbreviations CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage

Diffuse large B cell lymphoma-derived extracellular vesicles educate macrophages to promote tumours progression by increasing PGC-1β.

Tumour-associated macrophages (TAMs) comprise an important part of the tumour microenvironment and play a key role in malignant tumours progression. Tumour-derived extracellular vesicles (EVs) modulated TAMs polarization and reprogrammed TAMs to influence the progression of various tumours. Here, we hypothesized that diffuse large B cell lymphoma (DLBCL)-derived EVs can regulate macrophages polarization and thus contribute to tumour progression. Our results demonstrated that EVs, released from DLBCL, augment the M2 polarization effects of macrophages. Moreover, conditional medium derived from macrophages by DLBCL-derived EVs stimulation revealed the superior effects of promoting tumour proliferation, migration and invasion. Further analysis demonstrated that DLBCL-derived EVs regulated the metabolism of macrophages by increasing the expression of PGC-1β protein, thereby reprogramming the macrophage phenotype of promoting tumour progression. In conclusion, our findings signify that the DLBCL-derived EVs mediated the increasing of functional PGC-1β protein in macrophages to promote the tumour progression.

Immunobiology of cholangiocarcinoma.

SummaryCholangiocarcinoma (CCA) represents a heterogeneous group of epithelial tumours that are classified according to anatomical location as intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA). Although surgical resection and liver transplantation following neoadjuvant therapy are potentially curative options for a subset of patients with early-stage disease, the currently available medical therapies for CCA have limited efficacy. Immunotherapeutic strategies such as immune checkpoint blockade (ICB) harness the host immune system to unleash an effective and durable antitumour response in a subset of patients with a variety of malignancies. However, response to ICB monotherapy has been relatively disappointing in CCA. CCAs are desmoplastic tumours with an abundant tumour immune microenvironment (TIME) that contains immunosuppressive innate immune cells such as tumour-associated macrophages and myeloid-derived suppressor cells. A subset of CCAs may be classified as immune ‘hot’ tumours with a high density of CD8+ T cells and enhanced expression of immune checkpoint molecules. Immune ‘hot’ tumour types are associated with higher response rates to ICB. However, the suboptimal response rates to ICB monotherapy in human clinical trials of CCA imply that the preponderance of CCAs are immune ‘cold’ tumours with a non-T cell infiltrated TIME. An enhanced comprehension of the immunobiology of CCA, particularly the innate immune response to CCA, is essential in the effort to develop effective combination immunotherapeutic strategies that can target a larger subset of CCAs.

Molecular model linking Th2 polarized M2 tumour-associated macrophages with deaminase-mediated cancer progression mutation signatures.

A new and diverse range of somatic mutation signatures are observed in late‐stage cancers, but the underlying reasons are not fully understood. We advance a “combinatorial association model” for deaminase binding domain (DBD) diversification to explain the generation of previously observed cancer‐progression associated mutation signatures. We also propose that changes in the polarization of tumour‐associated macrophages (TAMs) are accompanied by the expression of deaminases with a new and diverse range of DBDs, and thus accounting for the generation of new somatic mutation signatures. The mechanism proposed is molecularly reminiscent of combinatorial association of heavy (H) and light (L) protein chains following V(D)J recombination of immunoglobulin molecules (and similarly for protein chains in heterodimers α/β and γ/δ of V(D)Js of T Cell Receptors) required for pathogen antigen recognition by B cells and T cells, respectively. We also discuss whether extracellular vesicles (EVs) emanating from tumour enhancing M2‐polarized macrophages represent a likely source of the de novo deaminase DBDs. We conclude that M2‐polarized macrophages extruding EVs loaded with deaminase proteins or deaminase‐specific transcription/translation regulatory factors and like information may directly trigger deaminase diversification within cancer cells, and thus account for the many new somatic mutation signatures that are indicative of cancer progression. This hypothesis now has a plausible evidentiary base, and it is worth direct testing in future investigations. A long‐term objective would be to identify molecular biomarkers predicting cancer progression (or metastatic disease) and to support the development of new drug targets before metastatic pathways are activated.

Metformin's antitumour and anti-angiogenic activities are mediated by skewing macrophage polarization.

Beneficial effects of metformin on cancer risk and mortality have been proved by epidemiological and clinical studies, thus attracting research interest in elucidating the underlying mechanisms. Recently, tumour‐associated macrophages (TAMs) appeared to be implicated in metformin‐induced antitumour activities. However, how metformin inhibits TAMs‐induced tumour progression remains ill‐defined. Here, we report that metformin‐induced antitumour and anti‐angiogenic activities were not or only partially contributed by its direct inhibition of functions of tumour and endothelial cells. By skewing TAM polarization from M2‐ to M1‐like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2‐TAMs‐induced angiogenic promotion, while also abrogating M1‐TAMs‐mediated anti‐angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs‐conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2‐polarized RAW264.7 macrophages. Based on these results, metformin‐induced inhibition of tumour growth and angiogenesis is greatly contributed by skewing of TAMs polarization in microenvironment, thus offering therapeutic opportunities for metformin in cancer treatment.

Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages.

It is well known that tumour-associated macrophages (TAMs) play an important role in tumour development by modulating the tumour microenvironment, and targeting of protumour activation or the M2 polarization of TAMs is expected to be an effective therapy for cancer patients. We previously demonstrated that onionin A (ONA), a natural low molecular weight compound isolated from onions, has an inhibitory effect on M2 macrophage polarization. In the present study, we investigated whether ONA had a therapeutic anti-ovarian cancer effect using in vitro and in vivo studies. We found that ONA reduced the extent of ovarian cancer cell proliferation induced by co-culture with human macrophages. In addition, we also found that ONA directly suppressed cancer cell proliferation. A combinatorial effect with ONA and anti-cancer drugs was also observed. The activation of signal transducer and activator of transcription 3 (STAT3), which is involved in cell proliferation and chemo-resistance, was significantly abrogated by ONA in ovarian cancer cells. Furthermore, the administration of ONA suppressed cancer progression and prolonged the survival time in a murine ovarian cancer model under single and combined treatment conditions. Thus, ONA is considered useful for the additional treatment of patients with ovarian cancer owing to its suppression of the protumour activation of TAMs and direct cytotoxicity against cancer cells.