Microfluidic immunocapture of circulating pancreatic cells using parallel EpCAM and MUC1 capture: characterization, optimization and downstream analysis

Abstract

We have developed and optimized a microfluidic device platform for the capture and analysis of circulating pancreatic cells (CPCs) and pancreatic circulating tumor cells (CTCs). Our platform uses parallel anti-EpCAM and cancer-specific mucin 1 (MUC1) immunocapture in a silicon microdevice. Using a combination of anti-EpCAM and anti-MUC1 capture in a single device, we are able to achieve efficient capture while extending immunocapture beyond single marker recognition. We also have detected a known oncogenic KRAS mutation in cells spiked in whole blood using immunocapture, RNA extraction, RT-PCR and Sanger sequencing. To allow for downstream single-cell genetic analysis, intact nuclei were released from captured cells by using targeted membrane lysis. We have developed a staining protocol for clinical samples, including standard CTC markers; DAPI, cytokeratin (CK) and CD45, and a novel marker of carcinogenesis in CPCs, mucin 4 (MUC4). We have also demonstrated a semi-automated approach to image analysis and CPC identification, suitable for clinical hypothesis generation. Initial results from immunocapture of a clinical pancreatic cancer patient sample show that parallel capture may capture more of the heterogeneity of the CPC population. With this platform, we aim to develop a diagnostic biomarker for early pancreatic carcinogenesis and patient risk stratification.

Characterization of microfluidic shear-dependent epithelial cell adhesion molecule immunocapture and enrichment of pancreatic cancer cells from blood cells with dielectrophoresis

“…4,[11][12][13][14] A majority of immunocapture techniques use the epithelial marker EpCAM (epithelial cell adhesion molecule), which has been reported to have oncogenic potential, 15 is correlated with proliferation in cancer cell lines, 16 and has been used to identify CTCs in many cancers. 11,13,[17][18][19][20][21][22][23] However, EpCAM varies in expression level between cancers and potentially fails to capture more invasive CTCs that have undergone the epithelial-to-mesenchymal transition (EMT). [24][25][26] Despite differences in cell surface antigen expression levels, a majority of cancer cells are vastly different from blood cells in cellular composition and morphology, which leads to their distinct electrical properties and dielectrophoretic response.…”

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

“…Anti-EpCAM antibody coating has been demonstrated to have an outstanding cell capture efficiency in both static and dynamic systems. [98][99][100][101] In the case of CTCs, most positive enrichment is grounded on the EpCAM antibodies. For example, He et al proposed a biocompatible and surface roughness controllable nano-film which is composed of TiO 2 nanoparticles for highly efficient CTC capture and in-situ identification by immunocytochemistry ( Fig.…”

Enhancing sensitivity and specificity in rare cell capture microdevices with dielectrophoresis

“…28 We have previously reported particle advection and cell capture simulations that predict transport, collision behavior, and capture probability in these obstacle arrays due to fluid advection and immunocapture; 11,28,29 these simulations have been adapted to study a range of target cells. 30,31 In this study, we expand those existing numerical simulations to also include the effect of DEP forcing, identifying geometries and applied AC electric fields that are optimized for DEP-immunocapture. We build on our previous characterization studies 14 to inform a Monte Carlo cell capture simulation, and use that simulation to identify an optimized geometry to enhance the capture of pancreatic cancer cells with pDEP, while rejecting most contaminating leukocytes by with nDEP.…”