Chorom Pak

Microscale functional cytomics for studying hematologic cancers.

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-κB and STAT3 in multiple myeloma cells exposed to different conditions, including cocultured bone marrow stromal cells. We further assessed its utility by analyzing NF-κB 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.

A New Alpha in Line Between KRAS and NF-κB Activation?

Bang and colleagues report a novel role for GSK-3α, rather than the well-studied GSK-3β, as the link between oncogenic KRAS and the canonical and noncanonical activation pathways of NF-κB in pancreatic cancer. Although the mechanism through which it promotes noncanonical activation remains unclear, the authors show that GSK-3α binds and stabilizes TAK1-TAB complexes to constitutively activate canonical NF-κB signaling. Consequently, the inhibition of GSK-3α retards pancreatic cancer growth in vitro and in vivo, thereby revealing this relatively less-studied kinase as a potential therapeutic target for treatment of KRAS-positive pancreatic cancer.

Abstract 3159: Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay

BACKGROUND: Renal cell carcinoma (RCC) is one of the top ten causes of cancer death in the United States. The last ten years have shown a dramatic increase in the number of available treatment options, however metastatic RCC remains largely incurable. The classes of drugs that have been developed can be divided into agents that target the Vascular Endothelial Growth Factor (VEGF) pathway (Sunitinib, Sorafenib, Pazopanib, Axitinib, Bevacizumab), those that target the mammalian Target of Rapamycin (mTOR) pathway (Everolimus, Temsirolimus) and immune based therapies (IL-2 and PD-1 inhibition). There are currently limited biomarkers to guide clinical decisions, mainly due to the lack of tumor cells for longitudinal molecular analysis. Circulating tumor cells (CTCs) are potential a source of tumor cells that can be identified from a blood draw for serial analysis. CTCs have not been reliably detected in RCC due to the significant heterogeneity and high rate of false positive events in this disease when EpCAM has been used. We sought to identify RCC CTCs with alternative markers, such as carbonic anhydrase IX (CAIX) which is found in greater than 90% of clear cell RCC tumors. METHODS AND RESULTS: To evaluate for the presence and subtypes of CTCs from patients with RCC, we utilized a multi-parametric flow cytometry assay. We evaluated heterogeneity across subpopulations of putative CTCs with Epithelial Cell Adhesion Molecule (EpCAM), Carbonic Anhydrase IX (CAIX), Carbonic Anhydrase XII (CAXII), PAX8, and Cytokeratin (CK). Negative controls for immune and endothelial events were performed with markers for CD45, CD14, CD34, CD11b and CD61. We tested twenty blood samples from patients with RCC at the Dana Farber Cancer Institute and University of Wisconsin Carbone Cancer Center. CTC frequency in RCC ranges from 0-5410 CAIX+/CK+ events with a median of 24.5 putative CTCs/7.5mL of blood. A subset of patients with radiographic progression had a higher number of CTCs with a median of 295.2 CTCs/7.5 mL. A range of 1-231 EpCAM+/CK+ events were identified with a median of 5.5 CTCs/7.5mL. There was low frequency of events being CAIX+/EpCAM+/CK+, with a median of 1 CTC/7.5mL of blood. Assay specificity was dramatically improved through the combination of multiple positive markers with stains for immune and endothelial cells given frequent non-specific staining for cytokeratin in RCC blood samples. CONCLUSIONS: CTCs can be identified in patients with RCC using non-traditional markers. CAIX is a more sensitive marker than EpCAM to identify putative CTCs from patients with RCC. Specificity in the assay is critical given the high frequency of false positive events identified if only CD45 is used as a marker for immune cells. Our investigation is ongoing for further molecular characterization of orthogonal endpoints in identified CTCs. Future directions include longitudinal monitoring of CTCs during treatment with VEGF inhibitors. Citation Format: Joshua A. Desotelle, Chorom Pak, Erika Heninger, Jennifer L. Schehr, Rana R. McKay, Benjamin K. Gibbs, Craig Norton, Toni K. Choueiri, Joshua M. Lang. Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3159.

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

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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