Molecular Characterization Of Individual Circulating Tumor Cells By Rna Sequencing

Background: Numbers of circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. Such association with clinical outcome suggests that CTCs play an important role in metastatic process. Molecular characterization of CTCs can provide important insights into phenotypic characteristics of cells necessary to participate in this process. Moreover, detailed molecular characterization of CTCs at various points of the disease course may be useful in understanding of evolution of tumors and aid in selection of optimal therapeutic regimens. Like most other CTC isolation instruments CellSearch is a CTC-enrichment device that efficiently captures CTCs while also retaining few thousands non-specifically captured white blood cells. To obtain deep molecular profiles of CTCs isolation of single cells is required and development of specialized next-generation sequencing protocols is necessary. Method/Results: We developed a workflow to enable isolation of single CTCs or pools of few CTCs following enrichment with the FDA approved CellSearch platform. Experiments with cancer cell lines spiked into donor blood showed 80% cellular recovery from blood using CellSearch and subsequent 40-50% capture of individual cancer cells using Fluidigm C1 Single Cell system. Following analysis of gene expression via RNAseq expression of ∼5000 genes can be detected in isolated tumor cells. We will discuss reagents and molecular approaches utilized to ensure efficient isolation and quality assessment of nucleic acids recovered from isolated tumor cells. We will discuss impact of sample preservation has on transcriptome profiling. We will share insights into prostate and lung cancers that one can gain from such analysis. Conclusion: Characterization of individual CTCs by RNA sequencing is a promising approach to characterize metastatic processes and tumor evolution. Citation Format: Jaymala Patel, Brad Foulk, Vipul Bhargava, Denis A. Smirnov. Molecular characterization of individual circulating tumor cells by RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2014-3063