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

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.

Inertial focusing of circulating tumor cells in whole blood at high flow rates using the microfluidic CTCKey™ device for CTC enrichment.

Circulating tumor cells (CTCs) are extremely rare cells shed from tumors into the blood stream. These cells can provide valuable information about their tumor of origin and direct treatment decisions to improve patient outcomes. Current technologies isolate CTCs from a limited blood volume and often require pre-processing that leads to CTC loss, making it difficult to isolate enough CTCs to perform in-depth tumor analysis. Many inertial microfluidic devices have been developed to isolate CTCs at high flow rates, but they typically require either blood dilution, pre-processing to remove red blood cells, or a sheath buffer rather than being able to isolate cells directly from whole blood. To decrease the need for pre-processing while increasing CTC yield, we developed an inertial device, the CTCKey™, to focus CTCs in whole blood at high throughput yielding a concentrated product stream enriched for CTCs. The CTCKey™ consists of two sections to create CTC enriched blood that can be further processed using any CTC isolation device to selectively isolate the CTCs. A thorough analysis was performed using the MCF7 breast cancer cell line spiked into bovine serum albumin (BSA) solutions of varying concentrations, as well as whole blood to characterize the focusing patterns of the CTCKey™. At the optimal flow rate of 2.4 mL min-1, the CTCKey™ reduces the CTC containing blood volume by 78%; the CTCs from 1 mL of blood are now in 0.22 mL of blood. The CTCKey's™ ability to concentrate CTCs from a large original blood volume to a smaller, highly concentrated volume enables a much greater blood volume to be interrogated by downstream isolation and characterization methods despite their low volume input limitations.