New Labyrinth Microfluidic Device Detects Circulating Tumor Cells Expressing Cancer Stem Cell Marker and Circulating Tumor Microemboli in Hepatocellular Carcinoma

Shanshan Wan,Christopher Leu,Neehar D Parikh,Eric Lin,Ryan Delaney,Kaylee J Smith,Nataliya Razimulava,Thomas Plegue,Hui Guo,John Steffes,Diane M Simeone,Theodore H Welling,Ning Kuo,Tae Hyun Kim,G-Su Park,Sunitha Nagrath

doi:10.1038/s41598-019-54960-y

Abstract

Hepatocellular Carcinoma (HCC) is one of the most lethal cancers with a high mortality and recurrence rate. Circulating tumor cell (CTC) detection offers various opportunities to advance early detection and monitoring of HCC tumors which is crucial for improving patient outcome. We developed and optimized a novel Labyrinth microfluidic device to efficiently isolate CTCs from peripheral blood of HCC patients. CTCs were identified in 88.1% of the HCC patients over different tumor stages. The CTC positivity rate was significantly higher in patients with more advanced HCC stages. In addition, 71.4% of the HCC patients demonstrated CTCs positive for cancer stem cell marker, CD44, suggesting that the major population of CTCs could possess stemness properties to facilitate tumor cell survival and dissemination. Furthermore, 55% of the patients had the presence of circulating tumor microemboli (CTM) which also correlated with advanced HCC stage, indicating the association of CTM with tumor progression. Our results show effective CTC capture from HCC patients, presenting a new method for future noninvasive screening and surveillance strategies. Importantly, the detection of CTCs with stemness markers and CTM provides unique insights into the biology of CTCs and their mechanisms influencing metastasis, recurrence and therapeutic resistance.

Highlights

The incidence of hepatocellular carcinoma (HCC) has doubled in the last few decades, having the fastest rising incidence among other solid malignancies in the US1–3
Circulating tumor microemboli (CTM), which could possess higher metastatic potential and resistance to apoptosis than single Circulating tumor cell (CTC) were quantified and correlated to Hepatocellular Carcinoma (HCC) stages. These results show the capability to use our technology to further biological analysis of these rare cells in order to better understand the roles of CTCs and circulating tumor microemboli (CTM) in metastasis, therapeutic resistance, and relapse
Both HCC cell lines were larger in diameter measuring 16.62 ± 2.84 μm and 14.49 ± 2.12 μm for Hep 3B and Hep G2, respectively, compared to white blood cells (WBCs) (Supplementary Fig. 1A, n = 1000 per cell type)

Summary

Introduction

The incidence of hepatocellular carcinoma (HCC) has doubled in the last few decades, having the fastest rising incidence among other solid malignancies in the US1–3. A subset of patients with early HCC stage are eligible for potential curative strategies such as resection, ablation, or liver transplant. Reliable detection and characterization of rare CTCs in HCC patients may facilitate early detection, provide additional prognostic information, and identify mechanisms of tumor progression and metastasis. CellSearch isolates cells based on their expression of epithelial cell adhesion molecule (EpCAM) on the cell surface[13] While this technique can detect some CTCs, it fails to isolate CTCs that do not express EpCAM14, including cells that have undergone an epithelial to mesenchymal transition (EMT) which downregulates EpCAM and promotes cell mobility[15]. EpCAM-based methods would be limited in sensitivity to detect HCC CTCs17. Alternative enrichment methods and quantification markers are needed for reliable detection of HCC CTCs

Methods

Results

Conclusion