Abstract 5581: Single-cell genomic analysis of patient-derived circulating tumor cells in pancreatic cancer
Authors: Ball Harrison , Rupp Brittany , Owen Sarah , Smith Kaylee , Gunchick Valerie , Keller Evan T. , Sahai Vaibhav , Nagrath Sunitha 
Published: April 2023

Abstract
Metastatic pancreatic cancer suffers from an extremely low five-year survival rate of only 3%. While patients with localized disease show marginally better survival rates, 52% of pancreatic cancers are not diagnosed until metastases have already developed. There is therefore a critically unmet need in the early diagnoses of pancreatic cancer patients. Liquid biopsies have emerged as a promising tool for cancer diagnosis through the analysis of various blood-based biomarkers. Among these, circulating tumor cells (CTCs) stand out as a promising target of interest. Over the past decade, CTCs have been identified as a precursor to metastasis. CTCs have also been shown to harbor mutations matching those of the primary tumor, making them a potential surrogate for biopsied tissue. However, CTCs are an extremely heterogenous population of cells, making bulk analysis challenging. In this study, we demonstrate a workflow for the isolation and genomic characterization of single CTCs from pancreatic cancer patients. CTCs are first enriched using a size-based microfluidic system, the Labyrinth, developed in the Nagrath lab. Single CTCs are subsequently isolated using the DEPArray Nxt system, followed by whole genome amplification (WGA) and low-pass sequencing. The pipeline was optimized using three commercially available pancreatic cancer cells lines (Panc-1, Capan-2, and BxPC-3) and two pancreatic cancer CTC cell lines previously developed in-house (CTC-CL-1 and CTC-CL-2). Finally, the workflow was validated using a blood sample from a stage IV pancreatic ductal adenocarcinoma patient. Following WGA, the average nucleic material concentration of the cell lines was 26.24 ± 16.28 ng/μL and 14.43 ng/μL for cells isolated from the patient sample. The average fragment size was 746 ± 232.79 bp for cell line cells and 507 bp for patient sample cells. This was within the expected range of 100-2000 bp. Both CTC cell lines show gains on parts of chromosome 8q (Myc), 10q (FGFR2) and 17q. Myc is a known oncogene and FGFR2 has been associated with cell growth, migration, and invasion. There are also noticeable differences between the two CTC-CL-1 cells, with one cell showing a 3p12.3-3p26.3 gain (CTNNB1), and the other showing a loss of 6q12-6q15 and gain of 6q16.1-6q27 (Myb, ESR1). A patient sample CTC and patient-matched WBC were isolated and sequenced. The CTC has a noticeable gain of chromosome 1q and loss of chromosome 5q. A gain in chromosome 1q has been seen previously in pancreatic cancer and chromosome 5q is the location of multiple oncogenes including APC, CSF1R and FGFR4. The loss of APC is known to affect Wnt/β-catenin signaling, causing an increase in CD34 expression which is linked to increase in cell invasion and migration. These results demonstrate an efficient workflow for the single cell genomic analysis of pancreatic CTCs. Further studies can be conducted to characterize the heterogenous landscape of CTCs in pancreatic cancer.
Citation Format: Harrison Ball, Brittany Rupp, Sarah Owen, Kaylee Smith, Valerie Gunchick, Evan T. Keller, Vaibhav Sahai, Sunitha Nagrath. Single-cell genomic analysis of patient-derived circulating tumor cells in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5581.