| Background: Oral squamous cell carcinoma(OSCC)is the most common pathological type of malignant tumors in the head and neck.Despite advances in OSCC diagnosis,surgery,and tumor treatment,the 5-year survival rate for patients in the past 30 years is still about 50%.Since chronic inflammation is usually associated with a poor prognosis,it is important to understand how the tumor microenvironment is involved in the progression of OSCC.The internal structure of tumors is much more complicated than normal tissues.Its specific tumor microenvironment(TME)is composed of various stromal cells and immune cells and runs through all stages of the disease,from early tumorigenesis to tumor progression and metastasis.Studies have reported that tumor associated macrophages(tumor associated macrophages,TAMs)account for 5-50% of the volume of malignant solid tumors in the immune cells associated with TME,and they are involved in regulating cancer-related inflammation,immune escape,matrix remodeling and metastasis.Cytokines and pro-inflammatory factors from TAMs play a key role in different stages of malignant transformation.TAMs can be divided into two types,one is classically activated M1 macrophages,which inhibit tumor development,and the other is selectively activated M2 macrophages,which can promote tumor development,but the specific mechanism is still unclear.Dual-specificity phosphatase-1(Dusp1),also known as mitogen-activated protein kinase phosphatase(Mkp-1),is the earliest discovered member of Mkps.It can inhibit the expression of p38,Erk and Jnk by dephosphorylating the threonine-glutamate-tyrosine motif on mitogen-activated protein kinases(MAPKs).Dusp1 has been shown to be involved in different biological processes,such as metabolic signaling,skeletal muscle function,inflammation,and cancer development.Dusp1 expression is reduced in the primary tumors of bladder cancer,breast cancer,prostate cancer and colon cancer,and also appears low in nearly 80% of metastatic tissues.Another study of ovarian cancer has shown that Dusp1 may play a key role in inhibiting the formation of silk feet and thus in cell movement.In breast cancer,activation of p38 and Erk signaling pathways can induce M2 type macrophages to produce IL-8,which promotes tumor migration and invasion.At present,most studies on Dusp1 expression in tumor tissues are limited to in situ hybridization and immunohistochemistry to detect mRNA and protein levels.It is not clear whether Dusp1 expression directly affects inflammatory immune cells in TME.Our research group tested the expression of Dusp1 in human OSCC tissues by immunohistochemistry,Western Blot and Realtime PCR,and found that Dusp1 was low in OSCC tissues and was closely related to tumor progression.Based on the results of previous studies and literature,this study intends to determine the role of Dusp1 deletion in the tumor microenvironment on the occurrence and development of oral squamous cell carcinoma,and to initially reveal the cellular mechanisms that Dusp1 guides TAMs recruitment,polarity,and affects OSCC metastasis,so as to provide a theorerical basis for the in-depth study of OSCC targeted drugs.Materials and Methods:1.Experimental materials:Dusp1 gene systemic knockout mice(C57BL/6),Dusp1 gene bone marrow conditional knockout mice(C57BL/6),mouse homologous oral cancer cell line MOC2.2.Experimental methods:Tumor cell culture,TCGA database analysis,mouse subcutaneous tumor formation,immune cell sorting,macrophage culture,scratch experiment,flow cytometry technology,H&E staining,immunohistochemical staining,mRNA extraction and Nanostring,4NQO drug induction Tumors in mice.Result:1.DUSP1 expression in OSCC tissue based on TCGA databaseComparing the expression of DUSP1 gene in oral squamous cell carcinoma and normal oral mucosa tissues in the TCGA database,we found that the expression of DUSP1 in OSCC tissues was significantly lower than that in normal oral mucosa tissues(P<0.05);At the same time,we compared the expression of DUSP1 in normal mucosal tissues with that in cancer tissues from patients with cervical lymph node metastasis at different N0,N1,N2,and N3 stages.As a result,the expression of DUSP1 from oral cancer tissues of patients cervical lymph node metastasis N0,N1,and N2 stages was also significantly lower than that from normal tissues,(P<0.05).2.Result for subcutaneous tumor formation in Dusp1 global knockout miceThe C57/BL6 mouse homologous cell line MOC2 was used to conduct subcutaneous tumor formation experiments in Dusp1 systemic knockout mice.The results showed that Dusp1 knockout mice had significantly faster subcutaneous tumor formation than wildtype mice(P<0.05).3.Result for lymph node metastasis of the transplanted cancer in the floor of mouth in Dusp1 global knockout miceThe C57/BL6 homologous mouse cell line MOC2 was used for in situ tumor formation experiments on the floor of the mouth of Dusp1 global knockout mice(due to back tumor formation,metastatic lymph nodes cannot be obtained under limited conditions),and H&E staining was used to detect the rate of lymph node metastasis in mice.The results showed that the submental lymph node metastasis rate of Dusp1 systemic knockout mice was 62.5%(10/16),and that of wild type was 31.25%(5/16).4.Results for cancer formation in Dusp1 bone marrow conditional-knockout mice induced by 4NQOBased on the above experimental results,in order to further confirm the effect of Dusp1 on cancer formation,we set up cancer model in Dusp1 bone marrow conditional knockout mice induced by 4-nitroquinoline oxide(4-nitroquinoline oxide,4NQO).As a result,Dusp1 bone marrow conditional knockout mice produced OSCC.Dusp1 bone marrow conditional knockout mice showed significantly faster tumor progression,a relatively larger tumor area,and a less differentiated pathological type(P<0.05).5.Detection for CD45+ inflammatory cells in cancer tissues of Dusp1 global knockout mice.Histochemical staining method was used to detect CD45+ inflammatory cells in the tumors of Dusp1 knockout mice.The results showed that CD45 positive cells significantly increased in the center of the tumor of Dusp1 knockout mice compared to those in wildtype(P <0.05).However,the number of CD45 positive cells showed no significant difference at the edge of tumor between the Dusp1 knockout mice and wild-type(P >0.05).Subsequently,we sorted single cells labeled with CD45+ magnetic microbeads from cancer tisuues of Dusp1 knockout mice.As a result,we obtained CD45+ inflammatory cells from cancer tissues of Dusp1 knockout mice for further use.6.Inflammatory cell components in TME of the Dusp1 global knockout miceThe prepared CD45+ cell suspension was tested by flow cytometry,and various inflammatory cell components from TME in Dusp1 global knockout mice were compared with those in wild-type mice.The results showed that compared with wild-type mice,the number of M2 macrophages of the TME significantly increased(P<0.05),while B cells significantly decreased in Dusp1 knockout mice(P<0.05).There is no difference in the number of CD4+ T cells,CD8+ T cells,Treg,Gr-1+ cells,M-MDSC,PMN-MDSC,M1 macrophages,NK cells or dendritic cells between Dusp1 knockout mice and wild-type mice(P>0.05).7.Effect of TAMs obtained from Dusp1 knockout mice on the migration of MOC2cellsThe sorted TAMs in Dusp1 knockout and wild-type mice were cultured separately,and the serum-free culture medium supernatant was then used for MOC2 cell scratch experiment.As a result,the scratch healing speed of MOC2 cells was significantly accelerated in Dusp1 knockout mice compared to that in wild-type mice(P<0.05).8.Screening and functional analysis of differentially expressed genes in TAMs of Dusp1 knockout miceTAMs sorted by magnetic bead labeling were used for Nanostring analysis.As a result,63 differential genes were obtained,of which 30 genes were upregulated and 33 genes downregulated.According to GO function analysis,we found that several differently expressed genes were shown to participated in signal pathways for MAPK,Notch,bone marrow-derived related genes and cytokine/trend factors,angiogenesis-related differential genes,and metabolic emergency-related genes.Conclusion: 1.Dusp1 knockout promotes the occurrence and development of oral squamous cell carcinoma;2.Dusp1 knockout promotes the transition of tumor-related macrophages to M2 type,which may be one of reasosn for the migration and metastasis of OSCC;3.Dusp1 knockout may participate in the occurrence and development of OSCC via some signaling pathways involving bone marrow-derived genes,cytokines,chemokines. |
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