Abstract 2923: Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients
Authors: Ramalingam Naveen , Lee Yi Fang , Szpankowski Lukasz , Leyrat Anne , Fowler Brian , Tan Jovina , Lu Chong Tracy , Angeles Ninez Delos , Sanada Chad , Greene Cassandra , Hukari Kyle , Wu Andrew , Yap Yoon-Sim , West Jay , Bhagat Ali Asgar 
Published: July 2017

Abstract
Background Label-free methods for isolating circulating tumor cells (CTCs) are attractive because they provide an opportunity to analyze a larger set of CTCs that may otherwise be missed due to variable or no expression of protein (label) markers. Understanding genetic and functional heterogeneity in CTCs allows us to gain insight into the mechanisms underscoring metastasis, drug resistance, and tumor aggressiveness. Currently, a simple workflow for isolation and molecular characterization of single CTCs by mRNA sequencing is lacking. In order to address this challenge, we developed a label-free workflow to isolate CTCs from breast cancer patients for full-length mRNA sequencing analysis by integrating the ClearCell® FX System with the Polaris™ system. The ClearCell FX system processes blood samples from cancer patients and enriches for CTCs in a label-free antibody-independent manner. The low level of nonspecifically isolated white blood cells from ClearCell FX is further depleted on the Polaris system by negative enrichment of viable CTCs. This unique integration of systems will enable researchers to perturb single CTCs in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis.
Method and Results CTCs from 7.5 mL of peripheral blood sample from breast cancer patients were enriched using ClearCell FX. To differentiate larger blood cells from putative CTCs, we stained the enriched cells with Alexa Fluor® 647-conjugated CD45 and CD31 to identify leukocytes and endothelial cells, respectively. Calcein AM (live cell marker) and CellTracker™ Orange (universal cell marker) were added to identify live cells. Single CTCs were selected on Polaris (Fluidigm) system, lysed and reverse-transcribed, and cDNA were preamplified on the Polaris integrated fluidic circuit (IFC). Sequencing libraries were generated using the Nextera® kit and sequenced on Illumina® MiSeq™ and NextSeq™ systems. We successfully processed blood samples from four patients. Sequenced data showed high-quality metrics, with read depth of up to 2.5 million reads (MiSeq) or 60 million reads (NextSeq), with a low percentage of mapped reads to ribosomal RNA and mitochondrial RNA. Unsupervised hierarchical clustering of gene expression data showed clustering by patient, but considerable heterogeneity was also observed among the CTCs from the same patient. We will provide insights into full-length mRNA transcriptome of single CTCs from triple negative breast cancer patient.
Conclusion We present the feasibility of integrating two microfluidics platforms to capture single CTCs for transcriptome and functional study. Our data suggests that the heterogeneity of tumor sample and characterization of metastatic processes can be elucidated from single-cell mRNA sequencing of CTCs.
Citation Format: Naveen Ramalingam, Yi Fang Lee, Lukasz Szpankowski, Anne Leyrat, Brian Fowler, Jovina Tan, Chong Tracy Lu, Ninez Delos Angeles, Chad Sanada, Cassandra Greene, Kyle Hukari, Andrew Wu, Yoon-Sim Yap, Jay West, Ali Asgar Bhagat. Label-free enrichment and integrated full-length mRNA transcriptome analysis of single live circulating tumor cells from breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2017-2923