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

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2016

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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.

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10.1371/journal.pone.0161134

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Koh, James, Joyce A Hogue and Julie A Sosa (2016). A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors. PLoS One, 11(8). p. e0161134. 10.1371/journal.pone.0161134 Retrieved from https://hdl.handle.net/10161/15385.

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Scholars@Duke

Koh

James Koh

Assistant Professor of Surgery

The major effort in the lab is directed towards investigating how tumor-specific dysregulation of the pRB signaling pathway affects downstream gene expression and the cellular response to DNA damage. Four projects are currently underway. First, we are utilizing a modified chromatin immunoprecipitation approach to capture and identify genomic DNA target sequences conditionally associated with pRB-containing complexes recovered from intact chromatin in untransformed primary human cells. Second, we are investigating functional heterogeneity amongst closely related components in the pRB pathway. Specifically, we are conducting comparative analyses of the INK4 proteins p16INK4a and p18INK4c and their preferred target kinases, the cyclin dependent kinases cdk4 and cdk6. Third, in collaboration with Dr. Jeff Marks, we are developing a mammary gland organoid approach to quantitate and analyze parity-dependent DNA damage checkpoint responses in the context of the primary human mammary tissue. Finally, we are engaged in a collaborative effort with Dr. Francis Ali-Osman to investigate the role of the glutathione-S-transferase protein P1 (GSTP1) in conferring chemotherapeutic drug resistance in human gliomas. To date, we have made significant progress in these projects. We have isolated a collection of genomic clones that are preferentially bound by pRB in senescent primary human mammary epithelial cells, and we intend to characterize these candidate regulatory elements as potential downstream targets of pRB-mediated gene regulation. Interestingly, the majority of the captured sequences do not contain canonical E2F binding sites, a finding that supports our approach of seeking pRB-bound sequences without the limitation of prior assumptions regarding the identity of the DNA-binding transcription factor bound by the pRB complex. In our comparative study of the closely related INK4 proteins p16INK4a and p18INK4c, we have found that differential substrate kinase preference may provide a molecular explanation for why p16INK4a but not p18INK4c is selectively targeted for inactivation in human tumors. In an extension of our previously published studies in T-cell acute lymphoblastic leukemias, we have determined that p18INK4c is highly expressed in a series of medulloblastoma cell lines (derived by Dr. Hai Yan), and that these same cell lines have selectively lost p16INK4a expression. Re-introduction of p16INK4a into these cells induces complete cell cycle arrest, but exogenous expression of p18INK4c has no effect on proliferation. Molecular analysis of p16INK4a and p18INK4c complexes in these cells indicates that p16INK4a associates preferentially with cdk4 whereas p18INK4c binds cdk6. Although cdk4 and cdk6 are highly similar (71% amino acid identity) and are generally assumed to be functionally redundant, we have found that these two kinases differ in their ability to bypass INK4-protein induced cell cycle arrest. In the context of medulloblastomas, we are currently testing the hypothesis that cdk4 and cdk6 execute opposing functions, with cdk4 activity driving proliferation and cdk6 activity inducing differentiation and cell cycle exit. A manuscript describing these findings is currently in preparation.

Previously, we have observed that mammary glands isolated from age-matched parous and nulliparous mice differed in their response to gamma-irradiation. Essentially, glands isolated from nulliparous animals failed to undergo a DNA damage checkpoint arrest, whereas glands from parous animals ceased cellular proliferation following exposure to mutagenic insult. In collaboration with Dr. Jeffrey Marks, we will analyze luminal and basal epithelial primary cells isolated from human reduction mammoplasty tissue with the goal of identifying parity- and compartment-dependent differences in checkpoint functional response.

In collaboration with Dr. Francis Ali-Osman, we are investigating the role of GSTP1-containing protein complexes in mediating drug resistance in gliomas. Our approach exploits Dr. Ali-Osman’s extensive background in chemotherapeutic drug resistance and GSTP1 activity and my laboratory’s expertise in identifying and characterizing protein:protein interactions and functional determinants of checkpoint response and apoptosis. Through biochemical enrichment and utilization of the new Duke Proteomics facility, we have begun systematically identifying GSTP1-associated proteins from extracts of cultured human glioma cells. We will then determine how the interacting proteins contribute to GSTP1-mediated chemotherapeutic drug resistance and other functional readouts of GSTP1 activity. Using this approach, we have found that the tissue transglutaminase TGM2 forms a dynamic, non-covalent complex with GSTP1 in actively dividing gliomas, and that this complex confers resistance to clinically important DNA-damaging drugs such as cisplatin (manuscript in preparation). In the coming year, we plan to extend these results to in vivo systems and investigate whether interfering peptides that disrupt the complex could serve as sensitizing agents to improve chemotherapeutic response to cisplatin.

Sosa

Julie Ann Sosa

Professor of Surgery

Julie Ann Sosa, MD MA FACS is Chief of Endocrine Surgery at Duke University and leader of the endocrine neoplasia diseases group in the Duke Cancer Institute and the Duke Clinical Research Institute. She is Professor of Surgery and Medicine. Her clinical interest is in endocrine surgery, with a focus in thyroid cancer. She is widely published in outcomes analysis, as well as cost-effectiveness analysis, meta-analysis, and survey-based research, and she is director of health services research for Duke Surgery. Her research group is multi-disciplinary, and she has collaborators in health services research and outcomes, biostatistics, geriatrics, endocrinology, oncology, vascular surgery, breast surgery, pharmacology and cancer biology, and stem cell research. She is a member of the Board of Directors of the International Thyroid Oncology Group, and has been a PI or investigator in a number of clinical trials for advanced thyroid cancer. She is Deputy Editor of JAMA-Surgery, and is on the editorial boards of the Annals of Surgery, Annals of Surgical Oncology, Endocrine, Hormones and Cancer, Journal of Thyroid Research, and the International Journal of Endocrine Oncology. She is Associate Editor of the World Journal of Surgery and the Journal of Surgical Research, and is editor of the endocrine tumors section of Current Opinion in Oncology. She has served on ATA guidelines committees for hyperthyroidism and thyroid nodules and differentiated thyroid cancer, and on the NCCN guidelines committee for neuroendocrine tumors. Dr Sosa is the recipient of grants from the NIH/NCI, Paget Foundation, the Association for Academic Surgery, the Donaghue Foundation, the American Geriatrics Association/Hartford Foundation, and the Connecticut Stem Cell Research Fund. She was born in Montreal and raised in upstate New York. She received her AB at Princeton, her MA at Oxford, and her MD at Johns Hopkins, where she also completed the Halsted residency program and a fellowship.


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