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Deformability-based separation and single-cell sequencing of circulating tumor cells in prostate cancer

University of British Columbia

Circulating tumor cells (CTCs) are malignant cells shed from a primary tumor into the bloodstream, where they have the potential to seed metastases responsible for >90% of all cancer related deaths. CTCs are particularly interesting for prostate cancer because metastases occur predominantly bone tissue, which makes biopsies difficult and low yielding. Since CTCs are accessible from peripheral blood, these cells represent a potential source of highly relevant tumor materials, which could be used to reveal new biomarkers for monitoring disease progression and evaluating drug efficacy. A key challenge in CTC isolation and characterization has been their extreme rarity in blood and their cell-to-cell heterogeneity. Both of these issues suggest the need to develop robust methods to isolate and analyze individual CTCs. This dissertation presents a new workflow to isolate, extract, and sequence single CTCs from patients with metastatic prostate cancer. Initially, we investigated the morphology of CTCs from patients with prostate cancer, and observed that CTCs and leukocytes were similar in size, but distinct in nucleus-to-cytoplasm ratio, which suggests the potential to separate CTCs based on deformability. Based on this result, a microfluidic device that separates CTCs based on cell deformability, as well as an accompanying analytical pipeline to identify CTCs using immunofluorescence, were developed, optimized, and tested. This workflow was used to successfully enumerate CTCs from 20 patients with metastatic castrate resistant prostate cancer, as well as 25 patients with localized prostate cancer. For the former cohort, we compared our process against existing technology and demonstrated 25× greater yield. We then developed a process to isolate single CTCs using laser capture microdissection for genome sequencing. Using this process, we enriched and isolated 30 single CTCs from 3 patients with metastatic prostate cancer, and sequenced 5 of these single CTCs from a patient with matched cell-free DNA. The sequencing data confirmed the presence of major driver mutations, including PTEN and TP53, as well as heterogeneous characteristics of individual CTCs. These results demonstrate the potential of our single cell sequencing workflow to discover clinically relevant mutations from single CTCs that may aid in monitoring disease progression and guiding treatment.

Text

2019-01-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/64279

Biomedical Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/