Abstract 1997: Label-free concentration of viable breast cancer CTCs for single cell Western blotting
Authors: Sinkala Elly , Sollier Elodie , Renier Corinne , Che James , Jeffrey Stefanie S. , Herr Amy E. 
Published: August 2015

Abstract
The isolation of circulating tumor cells (CTCs) in blood can aid in cancer prognosis, characterize genetic mutations for targeted therapies, and elucidate the biological mechanisms of metastasis. While CTCs provide clinical utility, there are still several technical difficulties with CTC isolation and analysis. CTCs are very rare (1-10 cells/mL of blood) and current isolation methods rely on immunocapture, require long sample preparation, and fail to deliver viable cells. Further, recent advances in single cell analysis of CTCs have been limited to genomic studies, in which DNA/mRNA does not provide a complete picture of CTCs at the functional level, as mRNA expression may only account for a portion of protein expression. The ability to study and measure the protein expression of CTCs could provide insight into the mechanisms driving cancer progression and signaling dysregulation. Unfortunately, conventional proteomic tools (e.g. immunocytochemistry and Western blot) either do not provide sufficient specificity or sensitivity toward single cell levels. Thus, there is a lack of tools that can provide quantitative protein analysis for rare CTCs.
To address this gap, we enable rare cell Western blotting by combining isolation and proteomic technologies in a unique workflow:
(i) Label-free Vortex enrichment of rare larger cells from blood samples. Larger cancer cells are trapped within an array of microscale fluid vortices that form in simple rectangular reservoirs, while smaller blood cells pass through. The cancer cells are stably maintained in the vortices, allowing for solution exchange, followed by release upon lowering the flow rate. With this device, we were able to release a small volume (∼500 μL) of concentrated, viable cancer cells (MCF7) from healthy blood, at high throughput, capture efficiency (35%), purity (&gt;80%), and ∼10,000 fold enrichment. CTCs were also successfully extracted and enumerated from the blood of patients with breast cancer (N = 18, from 0.75 to 23.25 CTCs/mL).
(ii) High-specificity and multiplexed protein analysis of rare cancer cells with single cell Western blotting (scWB). Enriched CTCs are placed into microwells patterned into a polyacrylamide gel for the scWB assay (in-situ chemical lysis in the microwells, electrophoresis into bulk gel layer, photo-immobilization, and subsequent antibody-probing of immobilized proteins). Here, we demonstrate scWB compatibility with rare cells by spiking MCF7s into normal blood and performing protein separations. The scWB is multiplex and can assay several target proteins simultaneously. Cells stained for surface markers (anti-EpCAM) are also compatible, and the scWB can resolve complete immunocomplexes without disrupting the location of the intracellular targets (GAPDH, ERK).
With the integration of these technologies, we push the limits of quantitative protein analysis of CTCs, an essential step to better understand these rare populations.
Citation Format: Elly Sinkala, Elodie Sollier, Corinne Renier, James Che, Stefanie S. Jeffrey, Amy E. Herr. Label-free concentration of viable breast cancer CTCs for single cell Western blotting. [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 1997. doi:10.1158/1538-7445.AM2015-1997