Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism

AbstractPrimary 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

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.

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

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors

<div><p>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.</p></div

Ionomycin-induced flux response in a viable human parathyroid tumor section.

<p>Images are z-stack projections captured with a 20X immersion confocal objective. Images of the same field taken prior (A, C) or 60 seconds after ionomycin addition (B, D). Upper panels are single frame fluorescence emission images; lower panels are three dimensional histogram plots of the same image fields. Blue = Hoechst 33342. Green = activated Fluo4-AM.</p

Intracellular flux response to extracellular calcium stimulation is CASR-dependent.

<p>Slice culture specimens were challenged with extracellular calcium in the presence of the CASR-specific calcimimetic cinacalcet or the CASR-specific calcilytic agent NPS2143. (A) Single frame immersion confocal images of Fluo-4AM fluorescent intensity at time 0 (“Baseline”) or 1 minute after calcium addition (“Stimulated”), in the presence of 1 mM calcium stimulus alone or in the presence of 1 mM calcium plus 2 micromolar cinacalcet. (B) Quantitation of the image data in (A). Mean fluorescent intensity (MFI) with standard deviation error bars for the cells in each field are plotted for the four conditions shown. Intensity values were captured at the same time points shown in (A). (C) Single frame images of fluorescence intensity before (“Baseline”) and 1 minute after stimulation (“Stimulated”) with 3 mM calcium in the presence or absence of the CASR inhibitory agent NPS2143 at a concentration of 300 nM. (D) Quantitation of the image fields shown in (C).</p

Kinetic profiles of parathyroid tumor cell flux responses.

<p>Y-axis = mean fluorescence intensity. X-axis = time in seconds.</p

Calcium response setpoint curve.

<p>The proportion of cells exhibiting a maximal flux response profile is plotted as a function of log (calcium concentration). Data points are calculated from fields of at least 600 cells at each calcium concentration level.</p

Calcium-sensitive modulation of PTH abundance in live parathyroid adenoma tissue.

<p>(A) Tumor sections from three different parathyroid adenoma specimens were challenged by exposure to elevated extracellular calcium (5 mM, right column) and compared to matched sections maintained under normocalcemic conditions (1.25 mM, left column). PTH reactivity is shown in red; nuclei are shown in blue. Fold suppression is calculated as the relative change in mean fluorescence intensity of the anti-PTH signal (AlexaFluor555-conjugated secondary antibody) normalized to baseline fluorescence under normocalcemic conditions. (B) PTH decrement following parathyroidectomy. Circulating levels of intact PTH were determined intraoperatively prior to tumor resection (time 0), at 10 minutes post-resection, and at 5 minute intervals thereafter.</p

Alignment of live-cell calcium response behavior with PTH abundance in a parathyroid adenoma.

<p>(A) Prestimulation baseline flux activity at time 0; (B) same field of view at 2 minutes after stimulation with 2 mM calcium; (C) same field after fixation and immunofluorescent staining for PTH (red); (D) image overlay of (B) and (C). Flux images (A, B) are single timepoint frames from the same time-ordered image stack. Anti-PTH reactivity is visualized by an AlexaFluor555 secondary antibody. Magnification = 200X.</p