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

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

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

Live-cell flux response to extracellular calcium in an intact human parathyroid tumor section.

<p>Images are confocal microscopy z-stack projections from a single field taken at sequential timepoints. (A) Pre-stimulation Fluo-4-AM fluorescence (“Baseline”) and the same field one minute after calcium stimulation (“Stimulated”). (B) Detail view of two individual cells demarcated by numbered ovals captured at three sequential time points: prior to stimulation (“Baseline”), 3 minutes after calcium addition (“Stimulated”), or 2 minutes after ionomycin addition (“Ionomycin”). The accompanying graph plots Fluo4-AM fluorescence mean fluorescence intensity (MFI) over time for the two cells. Green = activated Fluo4-AM. Blue = Hoechst 33342.</p

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

Live-cell vibratome sections of human parathyroid tumor tissue.

<p>(A) Parathyroid adenoma slice culture viability. Section is stained with the propidium iodide (red) and Hoechst 33342 (blue). White dashed line indicates the plateau boundary of the vibratome cut surface fracture plane. (B) Hematoxylin/eosin stained sections of a primary parathyroid adenoma and (C) a slice culture specimen derived from the same tumor after 7 days in culture (right panel). Magnification is 200X.</p

Quantitation of parathyroid adenoma responsiveness to calcium challenge.

<p>Fluo-4-AM fluorescence in a tumor section stimulated with 2 mM calcium (upper left panel) was captured at 2 minutes post-treatment and converted to an 8-bit monochrome image for quantitation (upper right panel). Maximal responders, defined as cells exhibiting an immediate onset and sustained plateau of intracellular calcium release greater than 3X over pre-stimulation baseline, are identified as the numbered objects in the lower right panel. The total number of cells in the planar field of view is derived by counting the number of nuclei in the corresponding Hoechst emission channel (lower left panel).</p

Confocal microscopy image of CASR localization in a primary parathyroid adenoma.

<p>(A) region with primarily membrane localized CASR; (B) region with CASR confined to intracellular vesicles in a field of non-expressing cells. Images are 80 micron optical sections. Green = CASR; blue = DAPI; red = WGA, a plasma membrane marker.</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