Primary Study Of Pathological Characteristics And Immunogenicity Of Circulating Tumor Cells In Peripheral Blood Of Patients With Renal Cell Carcinoma

Objective The circulating tumor cells(CTCs)were reported to play important roles in tumor metastasis and recurrence and has been considered as a valuable biomarker for early cancer detection,diagnosis,prognosis,and prediction.CTCs were reported to have some characteristics with tumor stem cells particularly in high risk tumors,and have a "self-injection" capability that it can return to primary tumor and make it more aggressive.However,the clinical utility of CTCs in cancer immunotherapy to prevent tumor metastasis and recurrence is limited.Therefore,the aim of this study is to explore a simple way to separate and culture CTCs of patients with renal cell carcinoma(RCC),and further explore the potential application of CTCs in immunotherapy.Content The study consists of four parts: First,Estimation of progression-free survival(PFS)of 60 RCC patients who had positive CTCs in peripheral blood mononuclear cells(PBMCs).Second,optimization of culture conditions of CTCs from RCC patients.Third,observation of the morphological and pathological characteristics of CTCs.Fourth,primarily investigation of the immunogenicity of CTCs.Methods 1.The frequencies of CTCs from 60 patients with RCC were detected and the progression-free survival(PFS)of the patients was estimated using Kaplan-Meier survival curves.2.CTCs separated from mononuclear cells of 30 ml peripheral blood derived from RCC patients were cultured in vitro.Titrated rh EGF of 0,5,10,20,50 μg/ml and FBS at 10,15,20% were added to optimize the culture conditions.3.CTCs in culture were observed sequentially and their morphological and pathological characteristics were recorded.4.The dendritic cells(DCs),T lymphocytes and cytokine-induced cells(CIKs)were cultured from autologous PBMC.Cell lysate of CTCs were prepared and the antigenicity was investigated using DC-T or DC-CIK system by T cell proliferation assay and cytotoxicity activity assay.Results 1.The progression-free survival(PFS)using Kaplan-Meier survival curves revealed 23 patients(38%)with CTCs had a significant shorter median PFS compared with 37 patients without CTCs(P=0.027).No significant difference was observed in PFS between patients with CTCs≥3 and CTCs<3.2.Adherent CTCs could be observed in day 1-2 cultures.Cell started division around day 5-7 cultures and typical asymmetric division were detectable.9 cases of CTCs were successfully cultured in vitro.20μg/ml of rh EGF and 20 % fetal bovine serum could facilitate the cell growth.3.CTCs showed malignant cell characteristics of dyskaryosis and larger caryoplasm ratio.Acridine orange staining showed the cellular nucleus of CTCs were giant and presented yellow or green while the cytoplasm presented flame-like reddish yellow or staining-unconspicuous.Some CTCs differentiated into kidney tubule-like structures under electron microscopy.4.Both T cells and CIK cells exposed to CTC lysate-derived tumor antigens presented by DCs strongly proliferated and showed lytic activity in vitro to CTCs,also to primary tumor cells(PTCs).Conclusion 1.CTCs could be a valuable prognostic and predictive biomarker for patients with RCC.2.CTCs could be successfully in vitro cultured in RPMI-1640 medium supplemented with 20% rh EGF and 20μg/ml FBS.3.CTCs showed characteristics with tumor stem cells.4.CTCs could be a Specific immunogen for CSC of RCC immunotherapy.