Microscale functional cytomics for studying hematologic cancers

Abstract

An important problem in translational cancer research is our limited ability to functionally characterize behaviors of primary patient cancer cells and associated stromal cell types, and relate mechanistic understanding to therapy selection. Functional analyses of primary samples face at least 3 major challenges: limited availability of primary samples for testing, paucity of functional information extracted from samples, and lack of functional methods accessible to many researchers. We developed a microscale cell culture platform that overcomes these limitations, especially for hematologic cancers. A key feature of the platform is the ability to compartmentalize small populations of adherent and nonadherent cells in controlled microenvironments that can better reflect physiological conditions and enable cell-cell interaction studies. Custom image analysis was developed to measure cell viability and protein subcellular localizations in single cells to provide insights into heterogeneity of cellular responses. We validated our platform by assessing viability and nuclear translocations of NF-B and STAT3 in multiple myeloma cells exposed to different conditions, including cocultured bone marrow stromal cells. We further assessed its utility by analyzing NF-B activation in a primary chronic lymphocytic leukemia patient sample. Our platform can be applied to myriad biological questions, enabling high-content functional cytomics of primary hematologic malignancies. (Blood. 2012;119(10):e76-e85) IntroductionOne challenging area of translational cancer research is the difficulty of performing functional analyses of primary patient samples to increase our understanding of human cancer biology. Highly sensitive genomic and proteomic methods, approaches that primarily evaluate the state of cancer samples, have significantly added to, and continue to increase, our understanding of the biology and the stratification of human malignancies. [1][2][3][4] In contrast, functional analyses, assessing biological responses to various experimental conditions, with primary patient samples are challenging for two reasons: (1) standard in vitro models and cell culture platforms (eg, Petri dishes, well plates, Transwell inserts) do not accurately reflect the complex spatiotemporal dynamics of the physiological microenvironment 5,6 ; and (2) current laboratory techniques often require more biological starting material than can be adequately obtained from patients. For example, electrophoretic mobility shift assays (EMSAs) to detect transcription factor-DNA interactions typically require a minimum of 10 5 to 10 6 cultured cells per condition (ie, per lane). 7,8 This quantity, in some cases, may not be obtainable from patient samples with particularly low cell counts. EMSAs also belong to a broad class of populationaverage cellular assays that provide only a single readout for the entire cultured cell population. Such population-average approaches not only restrict experimentation to samples with abundant populations and limit the number of...

MicroC3: an ex vivo microfluidic cis-coculture assay to test chemosensitivity and resistance of patient multiple myeloma cells

“…Moreover, extramedullary myeloma tissue sampling often yields lower amounts of tumor cells than standard bone marrow biopsy-derived material. Previously we reported the development of a microfluidic culture platform that enables culturing and functional analysis of low numbers of suspension cells, such as blood cancer tumor cells, in coculture with other cell types placed in different compartments permitting communication through soluble factors via diffusion ports 31 .…”

Characterizing asthma from a drop of blood using neutrophil chemotaxis

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IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation

“…The relative nucleus and cytoplasmic signal intensities were analyzed as described previously (54). Briefly, binary masks were created from both DAPI and NEMO fluorescence images by standard thresholding algorithms in ImageJ.…”

“…2A). We have recently developed a method to semiquantitatively analyze relative nuclear and cytoplasmic signal intensities of protein factors in individual cells (54). By measuring the changes in nuclear-cytoplasmic intensity ratios before and after VP16 treatment in NEMO-WT and NEMO-DN clones (ϳ1000 -3000 individual cells at each time point for each clone), we detected increases in nuclear NEMO intensities in NEMO-WT clones in response to VP16 treatment, but such increases were significantly reduced in NEMO-DN clones (Fig.…”