The Establishment And Accessment Of Genetically Characterized OSCC Patient-derived Xenograft

Purpose:Oral squamous cell carcinoma(OSCC)is the most common malignant tumor in the oral and maxillofacial region with 5-year survival rate is approximately50%-60%.The patients with advanced OSCC have poorer prognosis.The genetic landscapes of oral squamous cell carcinoma(OSCC)have been revealed and preclinical model that can represent the inter-patient and intra-tumoral heterogeneity is essential to understand the molecular diversity of OSCC and achieve the goal of personalized therapies.The aim of the study is to investigate the establishment,high-throughout technique characterization of patient-derived xenograft(PDX)mouse model as well as determine if YAP could serve as a novel therapeutic target in OSCC.Methods:1.OSCC tumor specimens were collected and subcutaneously implanted into the immune-compromised NOD/SCID mice.Once the size of the xenograft derived from the patient(named P0)reached 1000-1500 mm~3 in mouse,xenograft was surgically excised and small tumor fragments were re-transplanted to the nude mice(named P1,P2,P3…)for tissue expansion.We conducted stability assessment comparing the histological and genetic characteristics between the xenograft and the parental tumor.We then annotated a panel of PDX models using clinical information and high throughout techniques including whole exome sequencing and gene microarrays.2.68 primary OSCC patients were enrolled in this study.YAP expression in tissue specimens were investigated by using immunohistochemistry staining.YAP putback plasmid with specific mutation sites after YAP-shRNA interference was constructed by site-directed mutagenesis.Cell scratch and cell invasion assay were performed after YAP-siRNA interference or YAP putback again in TB2T4 and SCC23 cells.Results:1.A panel of OSCC PDX models were successfully established and systematically annotated with clinical information and genetic features including:anatomical site,TNM stage,primary or recurrent tumor,mutational status,copy number variation status and global gene expression pattern.The histological and genetic characteristics of xenografts were highly consistent with the corresponding patients’tumor.The mutational status of previously reported significantly mutated genes including TP53,NOTCH1 and CDKN2A were detected in our PDX models.Meanwhile,copy number variation of EGFR,CCND1 and CDKN2A were also identified.The PDX models were then classified based on the clinical or genetic annotations.2.Nuclear and cytoplasmic expression of YAP was increased in primary OSCC tissue samples(P<0.05).Nuclear expression of YAP was associated with lymph node metastasis(P<0.001)and 5-years overall survival rate(P=0.008).YAP knockdown greatly suppressed tumor cell migration and cell invasion both in TB2T4 and SCC23cells(Scramble vs YAP-shRNA,P<0.01),but these abilities were rescued subtotally when YAP was put back to the YAP-shRNA stable cells(Control vs YAP putback,P<0.01).Conclusion:1.A panel of OSCC PDX models could be established with clinical specimens using standardized process and the tumor take rate could reach about 50%in NOD/SCID immuno-deficient mice.2.The established PDX models we developed could resemble the complex histopathological features and genetic diversity of OSCC.PDX derive from different anatomical sites showed both genetic similarity and specificity.3.YAP expression in OSCC was associated with lymph nodal metastasis and YAP might play an essential role on the invasion ability of OSCC cells.As an important proto-oncogene,YAP protein can be used as an important therapeutic target.Further translational study is warranted in genetically characterized PDX model.