Integrated Workflow for the Label-Free Isolation and Genomic Analysis of Single Circulating Tumor Cells in Pancreatic Cancer

Abstract

As pancreatic cancer is the third deadliest cancer in the U.S., the ability to study genetic alterations is necessary to provide further insight into potentially targetable regions for cancer treatment. Circulating tumor cells (CTCs) represent an especially aggressive subset of cancer cells, capable of causing metastasis and progressing the disease. Here, we present the Labyrinth–DEPArray pipeline for the isolation and analysis of single CTCs. Established cell lines, patient-derived CTC cell lines and freshly isolated CTCs were recovered and sequenced to reveal single-cell copy number variations (CNVs). The resulting CNV profiles of established cell lines showed concordance with previously reported data and highlight several gains and losses of cancer-related genes such as FGFR3 and GNAS. The novel sequencing of patient-derived CTC cell lines showed gains in chromosome 8q, 10q and 17q across both CTC cell lines. The pipeline was used to process and isolate single cells from a metastatic pancreatic cancer patient revealing a gain of chromosome 1q and a loss of chromosome 5q. Overall, the Labyrinth-DEPArray pipeline offers a validated workflow combining the benefits of antigen-free CTC isolation with single cell genomic analysis.

Label-Free Separation of Circulating Tumor Cells and Clusters by Alternating Frequency Acoustic Field in a Microfluidic Chip

“…Although the concentration of CTCs is relatively low, making accurate separation of CTCs a great challenge, and its advantage over other liquid biopsy objects (such as ctDNA) is that CTCs are viable intact cells, which can accurately provide genomic information of cancer patients [ 32 ]. Recently, studies have isolated CTCs from pancreatic cancer patients and identified new single-cell copy number variations [ 33 ]. In addition, patient-derived CTCs cell lines can also be cultivated [ 34 ].…”

CellMag‐CARWash: A High Throughput Droplet Microfluidic Device for Live Cell Isolation and Single Cell Applications

“…Commercial single-cell microfluidic technologies are available, including the DEPArray (DiElectroPhoresis Array), [25] which manipulates single cells in a chamber using dielectrophoresis, and integrated fluidic circuits (IFC) which moves single cells into chambers using a system of pressure-controlled microfluidic valves. [26] Both technologies have been used in multiple studies to obtain single cell data, [27,28] but they suffer from limitations, such as lengthy processing time, lack of validation for use in live cells (DEPArray), and the ability to only collect a limited number of cells (IFC). [29,30] Droplet-based microfluidics can enable compartmentalization and analysis of analytes in a high throughput fashion.…”