A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.
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The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.
Published Version (Please cite this version)10.1371/journal.pone.0161134
Publication InfoKoh, James; Hogue, Joyce; & Sosa, Julie Ann (2016). A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors. PLoS One, 11(8). pp. e0161134. 10.1371/journal.pone.0161134. Retrieved from http://hdl.handle.net/10161/15385.
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Assistant Professor of Surgery
The major effort in the lab is directed towards investigating how tumor-specific dysregulation of the pRB signaling pathway affects downstream gene expression and the cellular response to DNA damage. Four projects are currently underway. First, we are utilizing a modified chromatin immunoprecipitation approach to capture and identify genomic DNA target sequences conditionally associated with pRB-containing complexes recovered from intact chromatin in untransformed primary human cells. Se
Professor of Surgery
Julie Ann Sosa, MD MA FACS is Chief of Endocrine Surgery at Duke University and leader of the endocrine neoplasia diseases group in the Duke Cancer Institute and the Duke Clinical Research Institute. She is Professor of Surgery and Medicine. Her clinical interest is in endocrine surgery, with a focus in thyroid cancer. She is widely published in outcomes analysis, as well as cost-effectiveness analysis, meta-analysis, and survey-based research, and she is director of health services research for
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