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M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma.

ABSTRACT: BACKGROUND:Treatment strategies targeting tumor-associated macrophages (TAMs) have been proposed in cancer areas. The functional alterations of macrophages in the microenvironment during the tumorigenesis of human epithelial cancer remain poorly understood. Here, we explored phenotypic alteration of macrophages during the development of oral squamous cell carcinoma (OSCC). METHODS:Conditioned media (CM) and exosome supernatants were harvested from normal oral epithelium, oral leukoplakia cells and OSCC cells. We measured phenotypic alteration of macrophages using flow cytometry, luminex assays, and quantitative real-time PCR assay. Intracellular signaling pathway analysis, mass spectrometry proteomics, western blotting, enzyme-linked immunosorbent assay, immunohistochemical staining, and bioinformatics analysis were performed to uncover the underlying mechanisms. RESULTS:THP-1-derived and PBMCs derived macrophages exhibited an M1-like phenotype but not M2-like phenotype, when treated with CM from OSCC cells but not with the CM from normal epithelium or leukoplakia cells. Further investigations revealed that macrophages were activated by taking up exosomes released from OSCC cells through p38, Akt, and SAPK/JNK signaling at the early phase. We further provided evidences that THBS1 derived from OSCC exosomes participated in the polarization of macrophages to an M1-like phenotype. Reciprocally, CM from exosomes induced M1-like TAMs and significantly promoted migration of OSCC cells. CONCLUSIONS:We proposed a novel paracrine loop between cancer cells and macrophages based on exosomes from OSCC. Therefore, target management of M1-like TAMs polarized by exosomes shows great potential as a therapeutic target for the control of cancerous migration in OSCC.

SUBMITTER: Xiao M

PROVIDER: S-EPMC6038304 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma.

Xiao Meng M Zhang Jianjun J Chen Wanjun W Chen Wantao W

Journal of experimental & clinical cancer research : CR 20180709 1

<h4>Background</h4>Treatment strategies targeting tumor-associated macrophages (TAMs) have been proposed in cancer areas. The functional alterations of macrophages in the microenvironment during the tumorigenesis of human epithelial cancer remain poorly understood. Here, we explored phenotypic alteration of macrophages during the development of oral squamous cell carcinoma (OSCC).<h4>Methods</h4>Conditioned media (CM) and exosome supernatants were harvested from normal oral epithelium, oral leuk ...[more]

PMID: 29986759

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Project description:BackgroundTumor-associated macrophages (TAMs) have a leading position in the tumor microenvironment. Previously, we have demonstrated that M1-like TAMs activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma (OSCC). However, the functional roles and associated molecular mechanisms of the activated M1-like TAMs need to be further clarified in OSCC.MethodsConditioned Media (CM) were harvested from the exosome activated M1-like TAMs. We measured the malignant behaviors of OSCC under the treatment of CM from M1-like TAMs by performing colony forming assays, invasion assays, wound-healing assays, spheroid forming assays and in vivo xenograft experiments. The underlying mechanisms were investigated by RNA-seq, cytokines analysis, intracellular signaling pathway analysis, ChIP assays, bioinformatics analysis and validation.ResultsM1-like TAMs significantly promoted the epithelial-mesenchymal transition (EMT) process, and induced the cancer-stem like cells (CSCs) by upregulating the expression of MME and MMP14 in OSCC cells. Cytokine analysis revealed a shark increase of IL6 secretion from M1-like TAMs. Blocking IL6 in the CM from M1-like TAMs could significantly weaken its effects on the colony forming, invasion, migration, microsphere forming and xenograft forming abilities of OSCC cells. Cellular signaling assays indicated the activation of Jak/Stat3 pathway in the OSCC cells treated by the CM from M1-like TAMs. Blocking the activation of the Jak/Stat3 pathway could significantly weaken the effects of M1-like TAMs on the colony forming, invasion, migration, microsphere forming and xenograft forming abilities of OSCC cells. Further RNA-seq analysis and bioinformatics analysis revealed an increased expression of THBS1 in the OSCC cells treated by M1-like TAMs. Bioinformatics prediction and ChIP assays revealed the activation of Stat3 by CM from M1-like TAMs could directly promote the transcription of THBS1 in OSCC cells.ConclusionsWe proposed that M1-like TAMs could cascade a mesenchymal/stem-like phenotype of OSCC via the IL6/Stat3/THBS1 feedback loop. A better understanding on the functional roles and associated molecular mechanisms of M1-like TAMs might facilitate the development of novel therapies for supplementing the current treatment strategies for OSCC patients.

Project description:Oral cancers, primarily squamous cell carcinomas (SCCs), progress either slowly or aggressively. Here we assessed the role of macrophages in SCC behavior. We used mouse SCC cells derived from tumors harboring a KrasG12D activation mutation and Smad4 deletion in keratin 15-positive stem cells and a human oral SCC cell line, FaDu, which has NRAS amplification and SMAD4 deletion. SCC cells were transplanted into immune-compromised or immune-competent (syngeneic) recipients. After tumors were established, we used clodronate liposomes to ablate macrophages. We found that the number of tumor-associated macrophages (TAMs) was not affected by the presence of T cells but differed considerably among tumors derived from different SCC lines. Clodronate significantly reduced TAMs and splenic macrophages, resulting in reduced SCC volumes. Tumors with clodronate treatment did not show decreased proliferation but did exhibit increased apoptosis and reduced vascular density. FLIP (Fas-associated via death domain-like interleukin 1?-converting enzyme inhibitory protein), an apoptosis inhibitor abundantly produced in tumor cells and TAMs, was reduced in tumor cells of clodronate-treated mice. Reduced FLIP levels correlated with reductions in phosphorylated nuclear NF?B p65 and NF?B inhibitor attenuated FLIP protein levels in SCC cells. Furthermore, TGF?1 serum levels and pSmad3 were reduced in clodronate-treated mice, but their reductions were insufficient to reverse epithelial-mesenchymal transition or TGF?-mediated angiogenesis in endothelial cells. Consequently, metastasis was not significantly reduced by macrophage reduction. However, reduced pSmad3 correlated with reduction of its transcriptional target, vascular endothelial growth factor A, in clodronate-treated tumor cells, which correlated with reduced vascular density in clodronate-treated tumors. Taken together, our study revealed that macrophages contribute to SCC expansion through interactions with tumor cells but are dispensable for SCC metastasis. Our study provides novel insights into understanding the contributions and limitations of TAMs in SCC progression.

Project description:Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

Dataset Information

M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma.

Publications

M1-like tumor-associated macrophages activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma.

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