56P - Single circulating tumor cells RNA profiling by label-free enrichment and active single cell selection on an integrated fluidic system

Date 17 December 2016
Event ESMO Asia 2016 Congress
Session Poster lunch
Topics Translational Research
Presenter Yi Fang Lee
Citation Annals of Oncology (2016) 27 (suppl_9): ix9-ix18. 10.1093/annonc/mdw574
Authors Y.F. Lee1, N. Ramalingam2, L. Szpankowski2, A. Leyrat2, N.D. Angeles2, A. Wu1, J. West2, A.A. Bhagat1
  • 1Research, Clearbridge BioMedics, 118257 - Singapore/SG
  • 2Research, Fluidigm Corporation, 94080 - San Francisco/US

Abstract

Background

Circulating tumor cells (CTCs) are disseminated tumor cells from the primary tumor into the circulatory system and are potentially “seeds of metastasis” that may initiate a secondary tumor. In spite of its biological relevance, the role of CTCs in promoting metastasis and tumor evolution remains elusive. In order to gain insights in the mechanism of circulating tumor cells survival and metastasis, we study single CTC pre-enriched using a size-based enrichment method and further singly isolated into reaction chambers by a microfluidic integrated chip for RNA sequencing (RNA-Seq).

Methods

CTCs from 7.5 ml of peripheral blood sample from breast cancer patients were enriched using ClearCell FX ®, a label- free spiral microfluidics- based system that enriches for larger cells. To differentiate larger blood cells from putative CTCs, we stained the enriched cells with Alexa 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. Singly selection of CTCs was done using PolarisTM (Fluidigm®) system which integrates fluorescence imaging, image based- cell selection and compartmentalization of cells for downstream mRNA chemistry. Full-length cDNA were generated from Polaris and the quality was checked using Bioanalyzer. Sequencing libraries were synthesized using Nextera kit and sequenced with Illumina MiSeq/NextSeq.

Results

Good quality cDNA were generated from single CTCs. Sequenced data showed high quality sequencing, with read depth of up to 2.5 million reads, with low percentage of mapped reads to ribosomal RNA and mitochondrial RNA. Unsupervised hierarchal clustering of gene expression data showed clustering by patient, but considerable heterogeneity was also observed among the CTCs from the same patient.

Conclusions

Here, we present the feasibility of integrating two microfluidics platforms to capture single CTC for transcriptome study. Preliminary analysis of our data suggests that the heterogeneity of tumor sample can be elucidated from single cell RNA-seq of CTCs.

Clinical trial indentification

Legal entity responsible for the study

Clearbridge BioMedics; Fluidigm Corporation

Funding

Clearbridge BioMedics, Fluidigm Corporation

Disclosure

Y.F. Lee, A. Wu, A.A. Bhagat: Author is an employee of Clearbridge BioMedics. N. Ramalingam, L. Szpankowski, A. Leyrat, N.D. Angeles, J. West: Author is an employee of Fluidigm Corporation.