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...

Nuclear factor-κB (NF-κB) is a family of transcription factors that play a key role in cell survival and proliferation in many hematological malignancies, including multiple myeloma (MM). Bortezomib, a proteasome inhibitor used in the management of MM, can inhibit both canonical and noncanonical activation of NF-κB in MM cells. However, we previously reported that a significant fraction of freshly isolated MM cells harbor bortezomib-resistant NF-κB activity. Here, we report that hyaluronan and proteoglycan link protein 1 (HAPLN1) is produced in bone marrow stromal cells from MM patients, is detected in patients' bone marrow plasma, and can activate an atypical bortezomib-resistant NF-κB pathway in MM cells. We found that this pathway involves bortezomib-resistant degradation of the inhibitor of NF-κB (IκBα), despite efficient bortezomib-mediated inhibition of proteasome activity. Moreover, HAPLN1 can also confer bortezomib-resistant survival of MM cells. We propose that HAPLN1 is a novel pathogenic factor in MM that induces an atypical NF-κB activation and thereby promotes bortezomib resistance in MM cells.

Chemosensitivity and resistance assays (CSRAs) aim to direct therapy based upon ex vivo response of patient tumor cells to chemotherapeutic drugs. However, successful CSRAs have yet to be developed. Here, we exposed primary CD138+ multiple myeloma (MM) cells to bortezomib, a clinical proteasome inhibitor, in microfluidic-cis-coculture (MicroC3) incorporating patient’s own CD138− tumor-companion mononuclear cells to integrate some of the patients’ own tumor microenvironment components in CSRA design. Statistical clustering techniques segregated MicroC3 responses into two groups which correctly identified all seventeen patients as either clinically responsive or non-responsive to bortezomib-containing therapies. In contrast, when the same patient MM samples were analyzed in the absence of the CD138− cells (monoculture), the tumor cell responses did not segregate into clinical response clusters. Thus, MicroC3 identified bortezomib-therapy MM patient responses making it a viable CSRA candidate toward enabling personalized therapy.