Abstract 4272: An integrated platform for quantifying gene expression in co-cultured cells

• Published in: Cancer research (Chicago, Ill.) Vol. 72; no. 8_Supplement; p. 4272
• Main Authors: Berry, Scott, Singh, Chandresh, Kueck, Jessica, Alarid, Elaine, Beebe, David
• Format: Journal Article
• Language: English
• Published: 15.04.2012
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2012-4272

Abstract The quantification of mRNA is a ubiquitous and critical tool for understanding cellular mechanisms in cancer. While RT-PCR is often the endpoint, the success of the analysis depends not only on the PCR reaction, but also on an entire process flow linking living cells to the PCR endpoint. For cultured cells, this process flow includes the culture itself, cell lysis, mRNA extraction and purification, and RT-PCR. While much research has been targeted to streamlining and increasing throughput of the PCR process, the remainder of the process flow has largely been neglected. In this study, we link a new mechanism for purifying mRNA, Immiscible Filtration Assisted by Surface Tension (IFAST), with cell culture on a single platform. IFAST uses an immiscible liquid barrier (e.g., oil) to separate the cell culture / lysis region from an elution buffer. Using paramagnetic particles (PMPs) that selectively bind mRNA, we extracted mRNA from the lysate by using a magnet to draw the PMP-captured mRNA through the immiscible phase. This process, which takes only seconds, replaces multiple washing steps required by current mRNA isolation protocols, which typically take 15-60 minutes to complete. The simplicity of IFAST enables facile integration of culture and mRNA extraction on an easy-to-use chip that requires only a micropipette and magnet to operate. Once proof-of-concept was demonstrated with a single cell type, the platform was expanded to incorporate co-culture of two cell types, with breast cancer cells and stromal cells in separate compartments connected via diffusion ports to allow cytokine exchange. Each compartment has an IFAST device, such that mRNA can be collected independently from each side. RT-PCR expression levels from the integrated culture / IFAST platform had less variance than similar experiments with independent culture and mRNA purification components, possibly due to the elimination of error during transfer. mRNA purified with the integrated device had a yield and purity similar to “gold standard” kits. Breast cancer cells co-cultured with bone marrow stromal cells showed increased proliferation and morphological changes relative to breast cancer cells cultured alone. Additionally, mRNA extracted from these cells using the integrated device showed transcriptional changes consistent with estrogen response, even in hormone-independent conditions. We have developed a new platform linking compartmentalized cell co-culture with NA isolation. This technology simplifies and accelerates two processes ubiquitous in cancer biology, enabling the collection of additional endpoints with finite resources while reducing error associated with manipulation. Furthermore, co-cultures of breast cancer with stroma from a metastatic site induced proliferation and transcriptional signaling associated with pro-growth conditions, illustrating the utility of this platform for studying the metastatic microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4272. doi:1538-7445.AM2012-4272