Abstract 3197: Functional microscale cell-based systems for studying hematologic cancers in complex microenvironments

Abstract

Abstract Our ability to improve treatments for blood-related cancers such as multiple myeloma (MM) is currently hindered by limitations in lab procedures for studying cell signaling mechanisms in drug resistance. Conventional assays for studying signal transduction (gel shift assays) require large quantities of cell sample (> 100,000 cells per condition) that often cannot be acquired safely and reliably from primary bone marrow aspirations of different patients. Furthermore, in vitro assays often do not adequately represent key aspects of the complex bone marrow microenvironment, including spatial organization of stromal cell types. The objective of this study was to develop a new functional assay using microfluidics technology to examine signal transduction events, specifically in improved in vitro microenvironments, for non-adherent cells such as those associated with hematologic cancers, and for low cell number applications (∼1000 cells per condition) including patient samples. Successful development of this assay would provide an alternative to gel shift assays and streamline experiments. We developed microscale cell culture chambers that used a surface tension-based passive pumping method for fluidic delivery, obviating the need for external pumps and tubing commonly used in microfluidic systems, thus reducing dead volume and cell loss. To validate the platform, non-adherent RPMI8226 cells (human MM cell line) were cultured, treated with cytokines/drugs, fixed, and immunostained to determine cytoplasmic versus nuclear localization of NF-κB subunit RelA as a measure of canonical NF-κB activation. Fluorescent images of RelA were analyzed using custom image analysis for calculating ratio of nuclear to cytoplasmic signals (intensity ratio, IR) at single cell resolution. IR values of cells in a population were calculated to determine overall nuclear translocation and NF-κB activation. Results showed fluorescence immunostaining coupled with custom image analysis was able to detect dose-dependent TNF-α activation of NF-κB, and dose-dependent inhibition of NF-κB activity by bortezomib treatment. Ongoing work involves applying the platform to study effects of various drug treatments on NF-κB signaling in primary patient MM cells in monoculture and in coculture with bone marrow stromal cells. Our results demonstrate the platform is a functional, enabling tool for studying single-cell NF-κB signal transduction with low numbers of non-adherent cells. The platform is extensible to other hematologic cancers, applicable for other transcription factors (e.g., STAT3), amenable to coculture studies, and has potential to enable novel experiments with limited primary samples that may have significant impact in translational research. 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 3197. doi:1538-7445.AM2012-3197