Profiling Heterogeneous Circulating Tumor Cells (CTC) Populations in Pancreatic Cancer Using a Serial Microfluidic CTC Carpet Chip

Abstract

Although isolation of circulating tumor cells (CTCs) from pancreatic adenocarcinoma patients is feasible, investigating their clinical utility has proven less successful than other cancers due to the limitations of epithelial cellular adhesion molecule (EpCAM)‐only based CTC assays. An integrated technology‐ and biology‐based approach using a microfluidic “Carpet Chip” is presented to study the biological relevance of heterogeneous CTC populations. Both epithelial CTCs (EpCs) and epithelial‐to‐mesenchymal transition (EMT)‐like CTCs (EMTCs) are isolated simultaneously from the whole blood of pancreatic cancer (PaCa) patients (n = 35) by separately targeting two surface markers: EpCAM and CD133. Recovery of cancer cell lines spiked into whole blood is ≥97% with >76% purity. Thirty‐four patients had ≥5 EpCs mL−1 and 35 patients had ≥15 EMTCs mL−1. Overall, significantly higher numbers of EMTCs than EpCs are recovered, reflecting the aggressive nature of PaCa. Furthermore, higher numbers of EMTCs are observed in patients with lymph node involvement compared to patients without. Gene expression profiling of CTCs from 17 patients reveals that CXCR1 is significantly upregulated in EpCs, while known stem cell markers POU5F1/Oct‐4 and MYC are upregulated in EMTCs. In conclusion, successful isolation and genomic profiling of heterogeneous CTC populations are demonstrated, revealing genetic signatures relevant to patient outcomes.

Detection of AXL expression in circulating tumor cells of lung cancer patients using an automated microcavity array system

“…In order to evaluate the significance of AXL‐expressing CTCs that may have undergone EMT, detection of such CTCs needs to be optimized. Methods for isolating mesenchymal‐like CTCs have been developed including immunobinding methods . CTCs are, however, highly heterogeneous, thus CTCs that can be isolated by immunobinding methods utilizing EpCAM targeting in particular may be limited to the subgroup expressing the marker of interest at a considerable level.…”

“…Among various EMT markers, VM is thought to be one of the general and well‐studied mesenchymal markers and is used in many studies . Other potential markers such as CD133, one of the cancer stem cell markers have been employed for mesenchymal‐like CTCs isolation methods . There are many reports indicating that CTCs expressing high levels of EMT markers in several cancer types such as breast, prostate, and colorectal, are often accompanied by the expression of cancer stem cell markers, suggesting the utility of incorporating stem cell markers such as CD133 .…”

“…Methods for isolating mesenchymal-like CTCs have been developed including immunobinding methods. [38][39][40] CTCs are, however, highly heterogeneous, thus CTCs that can be isolated by immunobinding methods utilizing EpCAM targeting in particular may be limited to the subgroup expressing the marker of interest at a considerable level. On the other hand, filter-based methods including our MCA system are not surface marker dependent and used for isolation of mesenchymal-like CTCs as well.…”

“…45,46 Other potential markers such as CD133, one of the cancer stem cell markers have been employed for mesenchymal-like CTCs isolation methods. 40 There are many reports indicating that CTCs expressing high levels of EMT markers in several cancer types such as breast, prostate, and colorectal, are often accompanied by the expression of cancer stem cell markers, suggesting the utility of incorporating stem cell markers such as CD133. [47][48][49] However, the relationship between EMT markers and CD133 on CTCs in lung cancer is not well-understood.…”

Microfluidic Chip-Based Cancer Diagnosis and Prediction of Relapse by Detecting Circulating Tumor Cells and Circulating Cancer Stem Cells

“…This strategy revealed interpatient heterogeneity of oncogenic signatures, such as epithelial-to-mesenchymal transition (EMT) and apoptotic-resistant mechanisms. Zeinali et al described a system of two-connected CTC carpet chips, composed of micro-sized posts, with antibody functionalization [ 43 ]. This dual capture strategy using anti-EpCAM and anti-CD133 facilitated the isolation of distinct, heterogeneous CTC populations (epithelial CTCs and EMT cells) simultaneously from pancreatic cancer patient samples with over 97% recovery and 76% purity.…”