| Objective: Oral squamous cell carcinoma(OSCC)is the most common malignancy of the oral and maxillofacial head and neck,accounting for almost 90%.In recent years,with the improvement of traditional treatment methods,such as surgery,radiotherapy and chemotherapy,the survival rate of patients has been improved to a certain extent,but the5-year survival rate is still about 50%.The main cause of death in patients with OSCC is the metastasis to distant organs,especially lung metastasis.Therefore,to explore the mechanism of lung metastasis of OSCC is of great significance for developing new therapeutic drugs,improving the prognosis and prolonging the survival rate of patients.The process of metastasis is very complex,which involves tumor cells growing in the primary site,entering the blood vessels,surviving in the peripheral blood circulation,penetrating the blood vessels,arriving at the site of metastasis,colonizing,growing and multiplying.Myeloid Cells,which are derived from bone marrow,mainly include tumor associated macrophages(TAMs)and myeloid derived suppressor cells(MDSCs).TAMs and MDSCs are widely abnormal in tumor tissues,which are closely related to the growth,invasion and metastasis of cancer.Studies have shown that myeloid cells can directly affect the proliferation,apoptosis and migration of tumor cells.On the other hand,it can promote tumor progression by affecting other tumor stroma,such as promoting tumor angiogenesis and providing nutrition for tumor growth.Inhibiting the immune response of T cells and helps the immune escape of tumor cells;and remodeling the extracellular matrix promotes invasion and metastasis of cancer cells.However,the role of myeloid cells in OSCC,especially its effect on metastasis,is not well understood.The aim of this study was to investigate the effects of myeloid cells on the growth and metastasis of OSCC,so as to provide a theoretical basis for the treatment of oral carcinoma in the future.It has been found that induced nitric oxide synthase(i NOS)is highly expressed in various tumor tissues and mainly produced by myeloid cells.It can degrade arginine to produce NO,peroxynitrate and other metabolites,and promote the growth of tumor cells.Meanwhile,i NOS is also an important factor regulating the functions of TAMS and MDSCs.Overexpression of i NOS in oral cancer is often associated with poor prognosis.L-NIL is a selective inhibitor of i NOS,which can inhibit tumor growth through a variety of pathways,but its effect on the growth and metastasis of OSCC is still unclear.This study was designed to investigate the effect of L-NIL on the growth and metastasis of OSCC in vivo and its mechanism.Methods: 1.Immunohistochemical staining was used to detect the distribution and types of macrophages and MDSCs in OSCC.2.PMA and IL-4 were used to induce THP-1cells to polarize into M2-type TAMs in vitro.q PCR and immunofluorescence were used to identify the phenotype of M2-type TAMs.3.Tumor cells were treated with conditioned media of M2 type TAMs,q PCR and immunofluorescence were used to detect the role of M2 TAMs on EMT of tumor cells.4.q PCR and immunofluorescence were used to detect the effects of M2-type TAMs on the stemness of tumor cells.5.Scratch and Transwell invasion assay were used to observe the effect of M2-type TAMs on the migration and invasion of tumor cells.6.MDSCs cells were depleted by anti-GR1 antibody in vivo,and its effects on the metastasis of OSCC were detected.7.Flow cytometry and immunohistochemistry were used to verify the depletion of MDSCs in tumors and spleen tissues.8.The distribution of i NOS in cancer tissues was detected by immunohistochemistry 9.The effect of L-NIL on the growth of OSCC in mice was observed in vivo.10.Lung metastasis of OSCC was determined with in vivo imaging system and HE sraining with or without L-NIL treatment.11.Kaplan-Meier method was used to analyze the survival curve of mice.12.Nitrate/nitrite kit was used to detect changes in NO metabolites.13.The effect of L-NIL on the proliferation and apoptosis of tumor was detected by immunohistochemistry.14.The effect of L-NIL on immune microenvironment was detected by mass flow cytometry.15.The effect of L-NIL on TAMs and MDSCs in tumor tissues and spleen was further verified by immunohistochemistry and flow cytometry.16.Cytokine array was used to detect the changes of cytokines in plasma and tumor tissues.17.The effect of CXCR2 inhibitor on lungmetastasis was observed in vivo.18.Flow cytometry and immunohistochemical staining were used to identify the effect of CXCR2 inhibitor on MDSCs infiltration.19.Statistical analysis: all in vitro experiments were repeated at least 3 times,and in vivo animal experiments were repeated at least 2 times,and the experimental results were expressed according to the mean ± standard deviation.Student's t test was used for statistical analysis of the two groups of data conforming to normal distribution.Non-parametric Mann-Whitney U test was used to analyze the data that did not conform to the normal distribution.P<0.05 indicated a statistically significant difference.Results: 1.Immunohistochemical staining showed that a large number of CD68+TAMs were distributed in oral cancer tissues,mainly manifested as CD163+ M2 phenotype,and more CD68+TAMs were found in cancer tissues than in normal epithelial tissues.At the same time,we found that the CD14+ M-MDSCs and CD15+ PMN-MDSC in cancer tissues were significantly more than those in normal epithelium.2.q PCR results showed that the expression of M2 macrophage related genes CD163,CD206 and Arg1 were increased after 48 h induction with PMA+ IL4 compared with PMA alone,and the immunofluorescence results further showed that CD163 and CD206 proteins were also increased.3.q PCR results showed that M2 macrophages promoted EMT of tumor cells.Immunofluorescence staining further showed that E-cadherin decreased,while Vimentin increased with M2 CM treatment.4.q PCR and immunofluorescence results showed that cancer cells obtained stemness after treatment with M2 TAMs CM,the stemness related genes Sox2,Ocy4 and Nanog were up-regulated,and the expressions of stemness proteins CD44 and CD105 were increased.5.The scratch and invasion experiments showed that M2 TAMs could enhance the migration and invasion ability of tumor cells.6.In vivo application of anti-Gr1 neutralizing antibody can effectively deplete MDSCs,and the removal of MDSCs can significantly inhibit lung metastasis of OSCC.7.The result of immunohistochemical staining showed that i NOS was highly expressed in cancer tissues.8.In vivo imaging and HE staining showed that the i NOS inhibitor L-NIL could inhibit the growth and metastasis of OSCC in mice compared with the control group.9.Immunohistochemical staining showed that proliferation of Ki67+ decreased and apoptosis of Cleaved caspase3+ increased in the L-NIL treatment group.10.The results of mass flow cytometry showed that the MDSCs in tumor tissues were significantly reduced with L-NIL treatment,while the change of TAMs was not particularly significant.11.Further verification by flow cytometry and immunohistochemistry showed that the Ly6G+ and Ly6C+ MDSCs in tumor tissues were decreased after L-NIL treatment,but there was no effect on MDSCs in spleen.Similarly,F4/80+ macrophages,CD163+M2macrophages,and CD86+M1 macrophages in the tumor and spleen were not significantly altered.12.Cytokine array results showed that the CXCL5 chemokine in plasma of mice treated with L-NIL was significantly reduced.13.In vivo application of CXCR2 inhibitor SB225002 can significantly inhibit the infiltration of MDSCs in tumors.Compared with the control group,lung metastasis was significantly reduced in the CXCR2 inhibitor group.Conclusion: Myeloid cells promote invasion and metastasis of OSCC.The i NOS inhibitor L-NIL inhibited the growth and metastasis of OSCC by inhibiting CXCL5 mediated MDSCs infiltration;The CXCR2 inhibitor SB225002 could inhibit lung metastasis. |
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