Partial thermal imidization of polyelectrolyte multilayer cell tethering surfaces (TetherChip) enables efficient cell capture and microtentacle fixation for circulating tumor cell analysis.

Julia A Ju,Keyata N Thompson,Rachel M Lee,Michele I Vitolo,Christopher M Jewell,Trevor J Mathias,Stephen J P Pratt,Stuart S Martin,Eleanor C Ory,Cornell J Lee

doi:10.1039/d0lc00207k

Abstract

The technical challenges of imaging non-adherent tumor cells pose a critical barrier to understanding tumor cell responses to the non-adherent microenvironments of metastasis, like the bloodstream or lymphatics. In this study, we optimized a microfluidic device (TetherChip) engineered to prevent cell adhesion with an optically-clear, thermal-crosslinked polyelectrolyte multilayer nanosurface and a terminal lipid layer that simultaneously tethers the cell membrane for improved spatial immobilization. Thermal imidization of the TetherChip nanosurface on commercially-available microfluidic slides allows up to 98% of tumor cell capture by the lipid tethers. Importantly, time-lapse microscopy demonstrates that unique microtentacles on non-adherent tumor cells are rapidly destroyed during chemical fixation, but tethering microtentacles to the TetherChip surface efficiently preserves microtentacle structure post-fixation and post-blood isolation. TetherChips remain stable for more than 6 months, enabling shipment to distant sites. The broad retention capability of TetherChips allows comparison of multiple tumor cell types, revealing for the first time that carcinomas beyond breast cancer form microtentacles in suspension. Direct integration of TetherChips into the Vortex VTX-1 CTC isolation instrument shows that live CTCs from blood samples are efficiently captured on TetherChips for rapid fixation and same-day immunofluorescence analysis. Highly efficient and unbiased label-free capture of CTCs on a surface that allows rapid chemical fixation also establishes a streamlined clinical workflow to stabilize patient tumor cell samples and minimize analytical variables. While current studies focus primarily on CTC enumeration, this microfluidic device provides a novel platform for functional phenotype testing in CTCs with the ultimate goal of identifying anti-metastatic, patient-specific therapies.

Highlights

Tumor metastasis is the leading cause of cancer-associated mortality, yet it remains the most poorly understood component of cancer progression.[1]
By replacing our lipid formaldehyde crosslinking step with a partial thermal imidization crosslinking reaction on the nanometer-thin polyelectrolyte multilayer (PEM) deposited onto Ibidi microfluidic slides, we extend the applications of this technology with the proven capabilities of cell capture, crosslinked stabilization, wash tolerance, and archival tumor cell fixation
Live tumor cells can be tethered to surfaces engineered with PEM+DOTAP, but are not well-retained during routine washing procedures that are required for many laboratory procedures.[5]

Summary

Introduction

Tumor metastasis is the leading cause of cancer-associated mortality, yet it remains the most poorly understood component of cancer progression.[1] During metastasis, circulating tumor cells (CTCs) are shed from the primary tumor into the vasculature where they get transported to distant organs to seed and regrow. Studies have shown that CTCs serve as an early indicator of disease spread and survival in metastatic cancer patients.[2,3] CTCs are extremely rare cells (1 CTC per 1 billion blood cells in patients with advanced cancer) and are difficult to isolate and characterize, our understanding of their biological properties is limited.[4] Technical challenges of imaging nonadherent cells have led to the reliance on data generated solely from adherent cells, which is not necessarily predictive of treatment outcome To overcome these obstacles, our lab 2872 | Lab Chip, 2020, 20, 2872–2888

Methods

Results

Conclusion