Abstract LB-280: Gene expression profiling of individual circulating tumor cells from non-small cell lung cancer (NSCLC) patients via integrated nanotechnologies
Authors: Park Seung-min , Wong Dawson J. , Ooi Chin Chun , Nair Viswam S. , Vermesh Ophir , Lee Sang Hun , Suh Susie , Lee Luke P. , Wang Shan X. , Gambhir Sanjiv S. 
Published: August 2015

Abstract
Background: Circulating tumor cells (CTCs), defined as epithelial cells shed from a primary tumor into the bloodstream, are valuable prognostic, and possibly diagnostic, biomarkers that contain actionable genetic information for cancer treatment. Unfortunately, the rarity of CTCs in comparison to other blood components necessitates high-throughput separation technologies for efficient enrichment and practical downstream analysis. Moreover, genetic data extraction from CTCs currently suffers from a dearth of reliable analytical methods capable of handling low cell numbers. Technological innovations are urgently required to developing platforms that can help to optimize cancer management.
Aim: To measure gene expression profiles of individual CTCs for cancer management via an integrated nanoscale platform.
Methods: We have developed a protocol to effectively enrich rare cells via a magnetic sifting technology, whose methodology is based on using magnetic nanoparticles to tag CTCs in conjunction with magnetic filtration to enable high-throughput enrichment with release capability. This magnetic sifter offers 1) high capture efficiency at fast flow rates due to extreme field gradients at the pore edges, 2) high throughput due to the density of pores (∼200 pores/mm2), 3) scalability via standard lithographic fabrication, and 4) harvesting of viable cells. For subsequent characterization, a robust nanowell-based assay was designed to circumvent experimental errors associated with ensemble measurements through detection of mRNA transcripts directly from single CTCs (using one-step RT-PCR). Using standard photolithography, 25,600 nanowells are positioned on top of polydimethyl-siloxane (PDMS) and designed for subsequent RT-PCR reaction from a single CTC. These massive single-cell arrays are able to isolate up to thousands of individual NSCLC cells to measure gene expression. Also, this device is easily interrogated by conventional fluorescence microscopy to detect a candidate panel of genes on CTCs that are relevant for cancer detection or therapy monitoring. Nanowell is innovative as a low-cost (PDMS-based), easily scalable (from currently 25k to more than 100k nanowells), adjunctive solution to existing diagnostic methods.
Results and Discussion: To date, we have assayed 23 NSCLC patients using the MagSifter & Nanowell and detected CTCs with valid biomarkers (hTERT and cMET). Each 4-mL whole blood sample was processed within one working day using the current workflow. From these samples, individual CTCs were assessed for hTERT and cMet expression. Single CTCs displaying hTERT only, cMet only, and both were evident upon fluorescent imaging. Direct comparison with CTC enumeration confirms better sensitivity by Nanowell assay, since the Nanowell utilizes PCR amplification in fluorescence as a signal generator. We believe this is the first demonstration of ex vivo visualization of gene expression from individual lung cancer CTCs.
Citation Format: Seung-min Park, Dawson J. Wong, Chin Chun Ooi, Viswam S. Nair, Ophir Vermesh, Sang Hun Lee, Susie Suh, Luke P. Lee, Shan X. Wang, Sanjiv S. Gambhir. Gene expression profiling of individual circulating tumor cells from non-small cell lung cancer (NSCLC) patients via integrated nanotechnologies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-280. doi:10.1158/1538-7445.AM2015-LB-280