| Background and objectivesTumor associated macrophages(TAMs)play an important role in tumor progression.Many previous studies have shown that M1 macrophages can inhibit tumor growth,but they also promote tumor progression.The protumor effects of M1 macrophages on oral squamous cell carcinoma(OSCC)should be further explored.Here,the effects of conditioned medium from M1 macrophages(M1-CM)on OSCC cell(SCC25 and CAL27)proliferation,migration,invasion,xenograft development and the role of growth differentiation factor 15(GDF15)and its downstream signal pathway in this process were evaluated.Materials and Methods1.Effects of M1 macrophages on the biological behavior of OSCC cells and the tumorigenesis abilityIn this study,the quanTIseq algorithm was used to evaluate the immune cell components of OSCC by screening OSCC transcriptome sequencing data in the cancer genome atlas(TCGA)database.The correlation analysis of infiltration and clinical feature information were also performed.Lipopolysaccharide(LPS)was used to induce THP-1 cells and the phenotype of macrophage was identified,Basic medium(BM)(RPMI 1640 containing 10%FBS)served as control group.The Ml-CM(containing 50%M1 macrophages culture supernatant+50%BM)were applied to cells.The effects of M1-CM on the proliferation of OSCC cells were observed by CCK-8,colony formation and EdU in vitro.The effects of M1-CM on the migration and invasion of OSCC cells were observed by Transwell assays.In vivo experiments,xenograft tumor assays were performed to detect the effects of M1-CM and M1 macrophages on tumorigenesis of OSCC cells.2.Mechanism of protumor effect by M1 macrophagesTCGA database was used to analyze the potential significance of GDF15 in the pathogenesis and clinical prognosis of OSCC and the correlation between GDF15 and M1 macrophages infiltration score.The M1-CM stimulated,with or without ErbB2(erb-b2 receptor tyrosine kinase 2)inhibitor,expressions of ErbB2/PI3K/AKT and ErbB2/MAPK/ErK signal pathways in SCC25 and CAL27 cell lines were detected by western blotting to analyze the role of ErbB2 in M1-CM protumor and mediating the activation of AKT and ErK signaling.The Ml-CM and TNF-α stimulated,expression of GDF15 in SCC25 and CAL27 cell lines was identified by quantitative real-time polymerase chain reaction(qRT-PCR).western blotting and enzyme linked immunosorbent assay(ELISA).The M1-CM stimulated,with or without GDF15 knockout,expression of p-ErbB2 was verified by western blot.Results1.Analysis of M1 and M2 macrophages infiltration features and the relationship with clinicopathological characteristics in OSCC.The transcriptome sequencing data of OSCC was from TCGA database.Tumor-infiltrating immune cells were estimated by quanTIseq.The results showed the number of M1 and M2 macrophages ranked first and second among the 10 kinds of immune cells.The infiltration degree of M1 and M2 macrophages in cancer was significantly higher than that in adjacent normal tissues.M1 macrophage infiltration degree was positively correlated with the clinical histological grade and lymph node stage of OSCC.These results indicated that Ml macrophages were related to poor clinical prognosis.In addition,the score ratio of M1/M2 macrophage infiltration degree in OSCC cancer tissues was significantly higher than that in adjacent tissues,and gradually increased with the clinical stage.2.Effects of M1 macrophages on the biological behavior of OSCC cells and the tumorigenesis abilityM1-CM was prepared after polarization and identification of M1 macrophages.The results of CCK-8,colony formation.EdU labeling experiments indicated that M1-CM significantly enhanced SCC25 cells proliferation vs BM.Additionally,the flow cytometry analysis results indicated no significant effect of Ml-CM on SCC25 and CAL27 cell apoptosis.Transwell assays showed that,compared with BM,M1-CM significantly increased the migration and invasion abilities of SCC25 and CAL27 cellsXenograft assay showed that Ml-CM induced SCC25 cells formed much larger tumors than SCC25 cells.Furthermore,immunohistochemical staining results revealed that the Ki67-positive cells in SCC25 cells induced by Ml-CM were markedly higher.The mixed implantation of M1 macrophages and SCC25 cells at a low proportion also significantly promoted the subcutaneous tumorigenesis of SCC25 cells.3.The regulatory mechanism of protumor effect by M1 macrophagesPan cancer analysis based on the sequencing data of 33 major cancer transcriptome in TCGA database showed that GDF15 was overexpressed in 11 types of tumor tissues,including HNSCC.Survival analysis showed that the expression of GDF15 was negatively correlated with the survival of patients with 6 types of cancer.To explore the expression of GDF15 in HNSCC/OSCC and its clinical significance,we found that the abnormal expression of GDF15 was correlated with the histopathological grade,TNM stage and lymph node stage of HNSCC/OSCC patients.In addition,OSCC immunoinfiltration analysis found that GDF15 was related to the infiltration degree of M1 macrophages.The results showed that M1-CM significantly activated ErbB2/AKT/ErK signal pathways in OSCC cells.The ErbB2 inhibitor decreased M1-CM-induced p-AKT and p-ErK expression,and inhibited Ml-CM induced SCC25 cell proliferation,and decreased the M1-CM-promoted SCC25 and CAL27 cell migration and invasionand obviously.These results indicate that the activation of PI3K/AKT and MAPK/ErK signal pathways by M1-CM in OSCC cells and the protumor effects are mediated by ErbB2.GDF15 gene and protein expression and secretion were significantly promoted by M1-CM vs BM control.TNF-α could also promote the expression of GDF15 gene.After the GDF15 gene was knocked out,p-ErbB2 expression was significantly decreased compared with cells transfected with negative vector no matter with or without M1-CM stimulation,suggesting that GDF15 is partly involved in the Ml-CMstimulated activation of the ErbB2.ConclusionsM1 macrophages promote the growth and invasion of OSCC cells by inducing GDF15-mediated ErbB2 phosphorylation and the downstream AKT and ErK signal pathways. |
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