3D Culture of Circulating Tumor Cells for Evaluating Early Recurrence and Metastasis in Patients with Hepatocellular Carcinoma

Purpose: Circulating tumor cells (CTCs) are considered to be a key factor involved in tumor metastasis. However, the isolation and culture of CTCs in vitro remains challenging, and their clinical application for predicting prognosis and survival is still limited. The development of accurate evaluating system for CTCs will benefit for clinical assessment of HCC. Methods: Density gradient centrifugation and magnetic separation based on CD45 antibody were used to isolate CTCs. 3D culture was used to maintain and amplify CTCs and HCC cells. Cellular immunofluorescence was used to identify CTCs and spheroids. The cutoff value of CTC spheroid was calculated using X-tile software. The relationship between clinicopathological variables and CTC spheroids in HCC patients is analyzed. In vivo models were used to evaluate tumor growth and metastasis of CTC spheroids. Results: Patient-derived CTCs/HCC cells were isolated and expanded to form spheroids using 3D culture. CTC spheroids could be used to predict short-term recurrence of CTCs compared with conventional CTC enumeration. Different cell lines exhibited different formation rates and grew to different sizes. Identification of CTC spheroids revealed that EpCAM and beta-catenin were expressed in spheroids derived from HCC cells and in the HCC/CTCs. EpCAM-positive HCC cells exhibited improved spheroid formation in 3D culture and were more tumorigenic and likely to metastasize to the lung in vivo. Abnormal activation of the Wnt/beta-catenin signaling pathway was observed in EpCAM positive cells. Conclusion: CTC spheroids could predict prognosis of HCC more precisely compared with conventional CTC enumeration. EpCAM may participate in the formation and survival of CTC spheroids which dependent on Wnt/beta-catenin signaling pathway.