| Background: Tumor evolution refers to the application of species evolution,ecology,and population genetics to the study of the macro and micro changes that occur when selective pressures act on tumor cells.The way of tumor evolution and the driver genes mutated during the process of tumor evolution are of great significance for the evaluation of tumor prognosis and the selection of therapeutic targets.To date,there are few studies focus on the evolutionary relationship between primary and metastatic tumors of oral squamous cell carcinoma(OSCC).The tumor samples were taken only at the late stage of tumor evolution,so it was difficult to observe the overall landscape of time dependent tumor evolution.On the other hand,there is still an uncertainty on the time point of the spreading and the charactaristics of disseminated tumor cells(DTCs)and high-frequency genes mutated during the process of spreading.Based on the mouse model of high lymphatic metastasis of OSCC,induced by 4-nitroquinoline-1-oxide(4-NQO),we found the early spreading of DTC in submandibular lymph nodes and bone marrow in the epithelial hyperplasia stage of oral primary tumor by histopathological method.However,the evolutionary relationship between primary and DTCs and high-frequency genes mutated in the spreading process are poorly understood.Objective: In this study,high throughput sequencing was used to analyze the whole genome of primary tumors,lymph nodes and bone marrow disseminated tumor cells in a mouse model of OSCC with high submandbular lymph node metastasis induced by 4-NQO.This paper attempts to reveal the characteristics of the evolution of oral squamous cell carcinoma from different temporal and spatial point of view,to infer the high-frequency mutated genes of oral squamous cell carcinoma,and to preliminarily observe the correlation between the high-frequency mutated genes acquired from whole-genome sequencing and the clinical pathological parameters of human oral squamous cell carcinoma.Methods: 1.4-NQO water drinking method was used to construct the mouse model of OSCC with high submandibular lymph nodes metastasis,and primary tumors,lymph nodes and bone marrow tissues were collected in epithelial dysplasia stage,non-lymphatic unmetastatic stage and lymphatic metastasis stage.2.Hematein and Eosin(HE)staining of mouse tongue tissue was carried out to determine the tumor pathological stage of the experimental mice.3.Lymphatic metastasis was identified by Cytokeratin(CK)immunohistochemistry(IHC)staining of submandibular lymph node.4.After single-cell suspension of lymph nodes and bone marrow were prepared,the immune cells in lymph nodes and bone marrow tissues were dislodge by CD45 Magnetic Activated Cell Sorting(MACS),and tumor cells in lymph nodes and bone marrow tissues were enriched by CD326 magnetic beads.5.CD326 flow cytometry was used to identify the separation and enrichment efficiency of MACS.6.DNA extraction,genomic amplification,purification and quality control were performed on DTCs.7.DNA extraction and purification were performed on tumor and normal tissues.8.The DNA library of each sample was built and sent to the company for sequencing,basic information such as SNV,INDEL,CNV and SV was obtained after filtering the raw sequencing data.High-frequency SNV and CNV genes were counted,tumor heterogeneity was visualized through Venn diagram based on SNV mutations,and cell purity and ploidies of each tumor sample were estimated.SNV data was screened for tumor subclonal analysis and tumor evolutionary trees constructed.9.PCR Sanger sequencing was used to validate the mutant sites of Cdh11 and Gata3 genes in the whole genome sequencing results,and the effect of mutant sites on protein function were speculated with PROVEAN.10.Samples from 20 OSCC patients were collected and RT-q PCR was performed to compare the expression levels of CDH11 and GATA3 m RNA in cancer tissues and adjacent tissues.CDH11 and GATA3 proteins were compared by Western blotting among three patients without lymphatic metastasis,three with lymphatic metastasis and three with normal mucosa.11.The expression and survival analysis data of CDH11 and GATA3 genes in TCGA database in head and neck cancer were inquired through bioinformatics website.12.Data of 83 oral squamous cell carcinoma patients was collected and the correlation among age,gender,TNM stage,Perineural Invasion(PNI),Ki67,and the expression of CDH11 and GATA3 was invstigated by IHC.Results: 1.CD326 flow cytometry results showed that CD45/CD326 magnetic beads significantly increased the proportion of tumor cells in the mixed cells in lymph nodes and bone marrow.These results were consistent with the results of the purity of DTCs infered by genomic sequencing.2.The SNP base replacement types of primary oral squamous carcinoma cells and metastatic disseminated cells in mice were mainly C>T,followed by T>C,which was consistent with the characteristics of base mutation in human oral squamous cell carcinoma.Single base variation of Fat1 and Notch1 genes and copy number variation of Hras,Notch1 and Ccnd1 genes occurred in mouse oral squamous cell carcinoma.3.The results of SNV,CNV and SV all showed that there was significant heterogeneity among primary tumor cells,DTCs in lymph nodes,and DTCs in bone marrow.4.We obtained a total of 238 tumor-related SNV,and 120 high-frequency mutated genes with mutation frequency ≥5%.The top 10 high frequency mutated genes were: Sirpb1 a,Cdh11,Smarca4,Fat1,Gata3,Notch1,Usp32,Cdk12,Cic,Creb3l2.5.The regions with significant chromosome amplification included Muc16,Pik3,bcl2,etc.,while the regions with significant deletion included Hras,Notch1,Apc2,Ccnd1,Batf2,Dapk3,Smarca4,Traf2,Traf3,Cdk3,cdh4,etc.6.It was found that there were subclones from primary sites in the submandibular lymph nodes and bone marrow samples of mice with oral squamous cell carcinoma at the stage of dysplasia and lymph node non-metastasis,suggesting early spread of oral squamous cell carcinoma.7.There were multiple subclones disseminated to lymph nodes and bone marrow from primary tumors,suggesting that oral squamous cell carcinoma was polyclonal disseminated.8.Late evolutionary subclones were found in the DTCs of lymph node and bone marrow during tumour metastatic stage,suggesting the characteristics of persistent spread of the primary oral cancer.9.The results of tumor phylogenetic trees showed that there were private subclones of tumor cells existed in lymph nodes and bone marrow in the same mouse,suggesting that different microenvironments may change the evolution trajectory of tumor cells.10.Compared with adjacent tissues,m RNA expression levels of CDH11 and GATA3 genes in OSCC tissues were significantly increased(P < 0.05),and CDH11 and GATA3 proteins were highly expressed in oral squamous cell carcinoma tissues(P<0.05).The expression levels of CDH11 proteins in patients with lymph node metastasis were higher than those without lymph node metastasis(P<0.05).11.CDH11 was negatively expressed in normal oral mucosa,and the positive expression rate in oral squamous cell carcinoma patients was51.81%.Among them,the positive expression rate of CDH11 in patients with N1 and N2 lymph node metastasis was higher than that in patients without metastasis(P < 0.05).The positive expression rate of CDH11 in patients with PNI was higher than that in patients without PNI(P < 0.05).GATA3 was negatively expressed in normal oral mucosa,and the positive expression rate in oral squamous cell carcinoma patients was 27.71%.There was no significant correlation between its positive expression rate and clinical pathological parameters of oral squamous cell carcinoma.Conclusion: 1.CD45/CD326 immunomagnetic bead separation method can effectively enrich disseminated tumor cells in lymph nodes and bone marrow.2.The 4-NQO mouse oral squamous cell carcinoma model has similar chromosomal variation with human oral squamous cell carcinoma,suggesting that this model can be used as an genomic animal model for the study of human oral squamous cell carcinoma.3.The significant differences of chromosomal variation between primary and metastatic tumor cells in the 4-NQO mouse models of oral squamous cell carcinoma,suggesting significant tumor heterogeneity between these tumor cells.4.The lymph nodes and bone marrow disseminated tumor cells druing the stage of precancerous in 4-NQO mouse model accepted tumor subclones from primary tumors,suggesting that tumor cells may have spread to lymph nodes and bone marrow in the precancerous stage,oral squamous cell carcinoma may have the characteristics of early spreading.Moreover,tumor evolution analysis suggested that oral squamous cell carcinoma has the characteristics of polyclonal and persistent spreading.5.In the 4-NQO mouse model of oral squamous cell carcinoma,private subclones were found in disseminated cells of lymph nodes and bone marrow from the same mouse,suggesting that the metastatic microenvironment may change the evolutionary trajectory of disseminated tumor cells.6.CDH11 and GATA3 m RNA as well as CDH11 and GATA3 proteins were highly expressed in human oral squamous cell carcinoma tissues.High expression of CDH11 is associated with lymph node metastasis and PNI in oral squamous cell carcinoma and may be a marker of metastasis or prognosis of oral squamous cell carcinoma. |
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