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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.

SUBMITTER: Koh J

PROVIDER: S-EPMC4990350 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Koh James J Hogue Joyce A JA Sosa Julie A JA

PloS one 20160818 8

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-m ...[more]

PMID: 27537691

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Project description:BackgroundMobilization of intra and extracellular calcium is required for platelet activation, aggregation, and degranulation. However, the importance of alterations in the calcium-platelet axis after injury is unknown. We hypothesized that in injured patients, in vivo initial calcium concentrations (pretransfusion) predict ex vivo platelet activation and aggregation, viscoelastic clot strength, and transfusion of blood products. We additionally hypothesized that increasing calcium concentrations ex vivo increases the expression of platelet activation surface receptors and platelet aggregation responses to agonist stimulation in healthy donor blood.MethodsBlood samples were collected from 538 trauma patients on arrival to the emergency department. Standard assays (including calcium), platelet aggregometry (PA) and thromboelastometry (ROTEM) were performed. In PA, platelet activation (prestimulation impedance [Ω]) and aggregation responses to agonist stimulation (area under the aggregation curve [AUC]) with adenosine diphosphate (ADP), thrombin receptor-activating peptide, arachidonic acid (AA), and collagen (COL) were measured. Multivariable regression tested the associations of calcium with PA, ROTEM, and transfusions. To further examine the calcium-platelet axis, calcium was titrated in healthy blood. Platelet aggregometry and ROTEM were performed, and expression of platelet glycoprotein IIb/IIIa and P-selectin was measured by flow cytometry.ResultsThe patients were moderately injured with normal calcium and platelet counts. Higher calcium on arrival (pretransfusion) was independently associated with increased platelet activation (prestimulation, Ω; p < 0.001), aggregation (ADP-stimulated, AUC; p = 0.002; thrombin receptor-activating peptide-stimulated, AUC; p = 0.038), and clot strength (ROTEM max clot firmness; p < 0.001), and inversely associated with 24-hour transfusions of blood, plasma, and platelets (all p < 0.005). Up-titrating calcium in healthy blood increased platelet activation (prestimulation, Ω; p < 0.001), aggregation (ADP, AA, COL-stimulated AUCs; p < 0.050), and expression of P-selectin (p = 0.003).ConclusionInitial calcium concentrations (pretransfusion) are independently associated with platelet activation, aggregation, clot-strength, and transfusions after injury. These changes may be mediated by calcium driven expression of surface receptors necessary for platelet activation and aggregation. However, the therapeutic benefit of early, empiric calcium repletion in trauma patients remains undefined.Level of evidencePrognostic, level V.

Project description:Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp-derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP scaffolds are intercalated between dentin and the odontoblast region, a finding that has significant implications when considering these materials as pulp-capping agents.

Dataset Information

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

Publications

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

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