Abstract 5362: Analysis of circulating tumor cells from prostate cancer patients
Authors: Giret Teresa , Tao Wensi , Suter Robert , Ansari Saba , Williams Sion , Ayad Nagi , Stoyanova Radka , Marples Brian , Pollack Alan 
Published: September 2020

Abstract
Background: Intermediate and high-risk prostate cancer patients are candidates for radiotherapy (RT). Evidence has shown that the presence of circulating tumor cells (CTCs) correlates with poor overall survival for patients with metastatic prostate cancer. CTCs have been reported as a better earlier predictor of treatment response than evaluation of prostate-specific antigen (PAS). The aim of our study was to identify and quantify circulating tumor cells from the peripheral blood of patients enrolled in two randomized Phase II radiotherapy clinical trials (BLaStM, NCT02307058 and CoMBINe, NCT02997709). Moreover, we investigated the genomic profiling of single CTCs using 10X Genomics to explore prognostic factors.
Design/Methods: CTCs were successfully harvested and enumerated from more than 215 patients and 500 blood samples using a microfilter system, the Circulogix FaCTChecker, and the cells of interest enriched onto CyteCatch slides. Circulating tumor cells were visualized by staining, identified and quantified for epithelial and prostate specific markers. The number of single CTCs, CTC clusters and their sum were recorded. Unfixed CTCs were enriched from whole blood from 13 prostate cancer patients at high risk of metastasis (median age of 63.5 years and median CTC value of 12 (range 0-42)) using The Parsortix™ Cell Separation System (ANGLE Inc.), a microfluidic-based platform able to capture and harvest CTCs, and single-cell RNA sequencing was then performed with Chromium (10X Genomics) and Illumina NGS sequencing technologies. Initial bioinformatic and data analysis was performed by the Biostatistics and Bioinformatics Shared Resource (BBSR) at SCCC. Subsequent principal component analysis (PCA) and gene set enrichment analysis (GSEA) were performed. Meanwhile, patient demographic and disease characteristic variables (PSA, Gleason Score (GS), T stage, % tumor in biopsies, age, race/ethnicity) were also collected and compared.
Results: Single CTCs or CTC clusters were evident in over 70% of patients analyzed using Immunofluorescence staining, with a median number of 30 (range 0-346). CTC transcriptomes at the single cell level was performed using the 10X platform, and principal component analysis discovered 16 distinct cell clusters of differential gene signatures within these cells in a t-SNE plot. Gene ontology analysis found some of these cell clusters contain differential expression pathways such as mTOR signaling (p=5.82E-05), EIF2 signaling (p=7.90E-05) and regulation of eIF4 and p70S6K signaling (p= 1.04E-04) which have been associated with prostate cancer. Gene Set Enrichment Analysis also identified distinct differential expression pathways in prostate CTCs comparing with non-CTCs.
Conclusion: We have developed a novel pipeline to harvest CTCs and analyze transcriptomes at the single-cell level. Our results suggest that gene expression profiling of CTCs can be a promising way to explore prognostic markers for predicting radiation sensitivity and metastatic risk.
Citation Format: Teresa Giret, Wensi Tao, Robert Suter, Saba Ansari, Sion Williams, Nagi Ayad, Radka Stoyanova, Brian Marples, Alan Pollack. Analysis of circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5362.