Real-Time Monitoring of Rare Circulating Hepatocellular Carcinoma Cells in an Orthotopic Model byIn VivoFlow Cytometry Assesses Resection on Metastasis

Zhi-Chao Fan,Wei-Zhong Wu,Xun-Bin Wei,Jian Zhou,Guang-Da Liu,Xiao-Ying Tan,Xiao-Fu Weng,Jun Yan

doi:10.1158/0008-5472.can-11-3733

Abstract

The fate of circulating tumor cells (CTC) is an important determinant of metastasis and recurrence, which leads to most deaths in hepatocellular carcinoma (HCC). Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying, and monitoring patients with metastatic diseases. In vivo flow cytometry has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and noninvasively. Here, we combine in vivo flow cytometry technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our in vivo flow cytometry has approximately 1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We found a significant difference in CTC dynamics between orthotopic and subcutaneous tumor models. We also investigated whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our results show that the number of CTCs and early metastases decreases significantly after the resection. The resection prominently restricts hematogenous metastasis and distant metastases. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel in vivo flow cytometry technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy.

Highlights

Hepatocellular carcinoma (HCC) is one of the prevalent human cancers worldwide ranking third for mortality and fifth for estimated new cases annually [1]
circulating tumor cells (CTC) in orthotopic GFP-labeled HCC tumor mice can be readily counted flowing through the ear microcirculation by in vivo flow cytometry (Fig. 1A)
hematoxylin and eosin (H&E) analysis of GFPþ cells sorted by fluorescence-activated cell sorting (FACS) in peripheral blood confirmed that the cells we considered as CTCs were tumor cells (Fig. 1D)

Summary

Introduction

Hepatocellular carcinoma (HCC) is one of the prevalent human cancers worldwide ranking third for mortality and fifth for estimated new cases annually [1]. Tumor recurrence and metastasis are still the major obstacles for long-term survival [2, 3]. The metastasis is reported to correlate with the presence of circulating tumor cells (CTC) in the vasculature as a conse-. Authors' Affiliations: 1Department of Chemistry, 2Liver Cancer Institute, Zhongshan Hospital, 3Institutes of Biomedical Sciences, Fudan University; 4Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai; and 5Department of Surgery, Fujian Provincial Tumor Hospital, Teaching Hospital of Fujian Medical University, Fuzhou, China. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Methods

Results

Conclusion