High Throughput Microfluidic Labyrinth for the Label Free Isolation of CTCs for Single Cell Gene Expression Profiling

Abstract

Circulating tumor cells (CTCs) present in the blood are the seeds of metastasis and are of high biological and clinical relevance. Single-cell technologies are playing an increasing role in profiling CTCs in the peripheral blood for detection and real time monitoring of cancer metastasis. CTCs also help in identifying distinct drivers of metastasis. However, current approaches are limited to subjective selection of CTCs based on biomarkers, which hinders the unbiased comprehensive study of CTCs on a single cell level. We present a unique label-free microfluidic “Labyrinth” device to isolate CTCs at a high throughput of 2.5mL mL of blood per minute, offering the first biomarker independent single cell isolation and genomic characterization platform to study heterogeneous CTC subpopulations in cancer patients. The Labyrinth takes advantage of inertial forces on the microscale in curved geometries to differentially focus cells based on the size difference between CTCs (15-25 µm) and blood cells (2-12 µm). This novel strategy of multi-course path traversing across inner loops to outer loops yielding highest hydrodynamic path length enabling focusing of both CTCs and white blood cells (WBCs) differentially, leading to high recoveries (> 90%) and efficient separation of WBCs from CTCs, resulting in high purity of CTCs in the final product (~ 600 contaminating WBCs per mL remained), even in whole blood samples without any pre-processing. CTCs were successfully isolated from 56 breast cancer (9.1 CTCs/mL average, range 2-31/mL) and 20 pancreatic cancer (51.6 CTCs/mL average, range 11-115/mL) patients. We detected not only CTCs typically defined by epithelial markers, but also significant numbers of CTCs (> 50%) lacking epithelial markers but expressing mesenchymal and cancer stem cell (CSC) markers. Patient samples were then analyzed using single cell multiplex gene expression. Seventy single cells were successfully recovered and used to identify different subpopulations of CTCs based on their genetic signature, unlike other methods where a positive or negative selection based on protein expression is used. Interestingly, both inter- and intra-patient molecular heterogeneity at the single cell level in CTCs were observed with cells expressing genes uniquely related to epithelial, mesenchymal-epithelial transition (MET), and epithelial- mesenchymal transition (EMT) phenotypes. The Labyrinth platform allows a thorough molecular understanding of the heterogeneity among CTCs. This platform also shows CTCs potential as a biomarker to non-invasively evaluate tumor progression and response to treatment in cancer patients. As a truly biomarker free isolation platform, Labyrinth is also adopted for the isolation of CTCs from various types of cancers, including but not limited to adenoid cystic carcinoma (ACC), hepatocellular carcinoma (HCC), and lung cancer. Besides enumeration, the isolated CTCs were used in a wide range of studies, such as ex vivo culture of live CTCs from pancreatic patients and generating xenograft tumor model in mice. Beyond CTC isolation, Labyrinth was applied in the study of skeletal muscle satellite cells in collaboration with Dr. Brian C. Syverud and Prof. Lisa M. Larkin. All in all, Labyrinth device offers a microfluidic technology to address the need for efficient isolation of rare cells and enables downstream studies on the target cells.