A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.
Abstract
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.
Type
Journal articleSubject
AdenomaCalcium
Humans
Microscopy, Confocal
Neoplasms
Parathyroid Glands
Parathyroid Hormone
Parathyroid Neoplasms
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https://hdl.handle.net/10161/15385Published Version (Please cite this version)
10.1371/journal.pone.0161134Publication Info
Koh, James; Hogue, Joyce A; & Sosa, Julie A (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 https://hdl.handle.net/10161/15385.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
James Koh
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
Julie Ann Sosa
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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