Live Single Cell Imaging of Human Parathyroid Tumor Tissue Reveals Functional Heterogeneity in Calcium Sensing

Presentation Number: LB-OR01-6
Date of Presentation: March 5th, 2015

James Koh*1, Joyce Hogue1, Yuli Wang2, Matthew DiSalvo2, Nancy Lynn Allbritton2, Yuhong Shi3, John A Olson Jr.3 and Julie Ann Sosa4
1Duke University Medical Center, Durham, NC, 2University of North Carolina at Chapel Hill, 3University of Maryland School of Medicine, Baltimore, MD, 4Duke University, Durham, NC

Abstract

We have recently shown that human parathyroid adenomas derived from patients with primary hyperparathyroidism (PHPT) are comprised of distinct cellular subpopulations with variable clonal origin and calcium responsiveness [1].  To gain a clearer understanding of the relationship between cellular identity, tumor composition, and clinical biochemistry in PHPT, we developed a novel single cell platform and computational analysis tool for quantitative evaluation of parathyroid tumor behavior. Dispersed parathyroid adenoma cells prepared from surgically resected tumor tissue are plated onto an indexed array composed of a large number of micron-scale elements (microrafts) doped with paramagnetic nanoparticles. The microrafts serve as releasable, optically transparent, individually addressed culture sites for isolated parathyroid cells.  Quantitative live-cell image analysis of calcium flux visualized by Fluo-4-AM epifluorescence was performed, and the cells were subsequently probed in situ for expression of the calcium sensing receptor (CASR), a canonical component in the extracellular calcium-signaling pathway.  Using this system, we found that the reactivity of individual parathyroid tumor cells to extracellular calcium stimulus is highly variable, with discrete patterns of kinetic response observed both between and among parathyroid tumor isolates. When challenged with 3 mM calcium, the proportion of cells exhibiting a rapid and transient response ranged from 7.1-27.7%, while sustained signaling responses were seen in 1.0–48.1%.  Non-responders at this calcium concentration ranged from 30.8-68.2%. When examined in relationship to each individual cell’s behavior, CASR abundance was not found to be a determinant of calcium responsiveness.  56.2 percent of rapid transient responder cells and 38.4 percent of sustained responder cells did not express detectable CASR protein.  Conversely, 32.3 percent of non-responsive cells were CASR-positive. Finally, we generated dose-response curves to calculate calcium EC50 values from a series of parathyroid adenomas and found that the tumors appeared to group into two distinct categories with respect to calcium responsiveness.  One group manifested a mean EC50 of 2.40 mM (95% CI 2.37–2.41), closely aligned to the published normal range [2].  The second group was clearly less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI 3.45–3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-categorization of PHPT tumors, possibly reflecting distinct etiological mechanisms.  Prospective identification of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.

 

Disclosure: NLA: Founder, Cell Microsystems. Nothing to Disclose: JK, JH, YW, MD, YS, JAO Jr., JAS