OncoTargets and Therapy (Apr 2021)
3D Culture of Circulating Tumor Cells for Evaluating Early Recurrence and Metastasis in Patients with Hepatocellular Carcinoma
Abstract
Cong-Li Hu,1,2 Yan-Jun Zhang,3 Xiao-Feng Zhang,2 Xiang Fei,4 Hai Zhang,5 Chun-Guang Li,4 Bin Sun2 1Translational Medicine Center, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 201620, People’s Republic of China; 2Department of Molecular Oncology, Eastern Hepatobiliary Surgical Hospital & National Center for Liver Cancer, Second Military Medical University, Shanghai, 200438, People’s Republic of China; 3School of Health and Social Care, Shanghai Urban Construction Vocational College, Shanghai, 201415, People’s Republic of China; 4Department of Thoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, 200438, People’s Republic of China; 5Department of Pharmacy, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, 201204, People’s Republic of ChinaCorrespondence: Bin SunDepartment of Molecular Oncology, Eastern Hepatobiliary Surgical Hospital & National Center for Liver Cancer, Second Military Medical University, Shanghai, 200438, People’s Republic of ChinaEmail [email protected] LiDepartment of Thoracic Surgery, Changhai Hospital, Second Military Medical University, 225 Changhai Road Shanghai, 200438, People’s Republic of ChinaEmail [email protected]: 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 β-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/β-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/β-catenin signaling pathway.Keywords: 3D culture, circulating tumor cells, early recurrence, metastasis, hepatocellular carcinoma
