Abstract
Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4-AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was 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-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.
Highlights
Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder, with an overall incidence of 15–30 per 100,000 individuals in the USA. [1,2,3]
We have recently shown that parathyroid adenomas are comprised of functionally distinct cellular subtypes that differ in their relative sensitivity to calcium stimulation despite equivalent levels of calcium sensing receptor (CASR) expression in each population [21]
Utilizing the system’s capacity to align post-assay immunofluorescence image analysis with the individual record of each cell’s functional response, we show that the presence of detectable CASR protein is not the sole determinant of calcium sensitivity in parathyroid adenoma cells
Summary
Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder, with an overall incidence of 15–30 per 100,000 individuals in the USA. [1,2,3]. We have recently shown that parathyroid adenomas are comprised of functionally distinct cellular subtypes that differ in their relative sensitivity to calcium stimulation despite equivalent levels of CASR expression in each population [21]. This evidence of intratumoural heterogeneity in the composition and biochemical behaviour of parathyroid adenomas calls into question the assumption of CASR down-regulation as an obligate mechanism in PHPT and highlights the need for a means to interrogate intrinsic calcium responsiveness of individual parathyroid tumour cells as a more direct functional readout of the underlying calcium sensing deficit
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