45.08 Stem Cells and PTH Assuage Radiation-Induced Cellular Damage in a Model of Distraction Osteogenesis

Posted on January 18, 2016

A. Zheutlin1, S. Deshpande1, N. Nelson1, J. Rodriguez1, A. Donneys1, E. Carey1, S. R. Buchman1 1University Of Michigan,Craniofacial Research Laboratory, Plastic Surgery Section, Department Of Surgery,Ann Arbor, MI, USA

Introduction: Radiation-induced injury to endogenous tissue in head and neck cancer patients can impede the use of Distraction Osteogenesis (DO) as a viable option for reconstruction of the mandible. Therapeutic options which restore the viability of irradiated bone for reconstruction hold the promise to allow DO to become an effective alternative for reconstruction in patients who have undergone radiation therapy (XRT). Bone marrow stem cells possess the capability to differentiate into osteoblasts and stimulate endogenous growth factors. Additionally, intermittent parathyroid hormone is known to stimulate bone deposition. We posit that the effects of these therapeutic agents will act synergistically to improve cellularity and function, and curtail the destructive effects of XRT in a murine model of mandibular DO.

Methods: Lewis rats were divided into 5 groups: non-radiated distraction (DO), radiated distraction (xDO), radiated distraction with intermittent parathyroid hormone (PTH), radiated distraction with intraoperatively placed bone marrow stem cells (BMSC), and radiated distraction with both therapies (Combined). Groups receiving radiation were exposed to a human-equivalent dose of 35 Gy over a 5-day period. All groups underwent distraction to a distance of 5.1 mm. Coronal sections from the distraction site were stained using Hematoxylin & Eosin, Safranin O, and Gomori’s Trichrome. Statistical analysis was performed with ANOVA and subsequent Tukey or Games-Howell post-hoc tests.

Results: Rats in the combined therapy group demonstrated improved metrics of histology and histomorphometry when compared to the xDO group. Osteocyte count was significantly improved by combination therapy over xDO (p=0.00). Additionally, empty lacunae were decreased in the combined therapy group as compared to the xDO group (p=0.00) and both singular therapies. Combination therapy also improved the histomorphometric quality of the regenerate bone demonstrating less immature osteoid volume (p=0.01) and much more mature woven bone (p=0.00). Furthermore, the composition of relative mature bone volume in the combined group (0.56±0.09) approached that of the bone regenerated by non-irradiated DO (0.52±0.11), indicating that the combination therapy was able to overcome the XRT-induced devastation to the endogenous tissue.

Conclusion: In a murine model of DO, BMSCs in combination with intermittent PTH reverses XRT-induced cellular depletion, resulting in significant improvement in osteocyte presence with decreased empty lacunae. Regenerated bone treated with the combined therapy resembled bone that underwent DO without XRT, and resulted in better complete histomorphometric outcomes than bone treated with either therapy alone. Our combined therapy demonstrates the capability to assuage the damage incurred ensuing XRT and offers promise in the treatment of patients requiring mandibular reconstruction following XRT.

45.09 Transplantation of human epithelial enteroids into mice using poly (lactide-co-glycolide) scaffolds

Posted on January 18, 2016

P. H. Dedhia1, F. K. Noto3, L. D. Shea3, J. R. Spence2 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of Internal Medicine,Ann Arbor, MI, USA 3University Of Michigan,Department Of Biomedical Engineering,Ann Arbor, MI, USA

Introduction: Short bowel syndrome secondary to bowel resection for congenital anomalies, infection or inflammation, or intestinal ischemia can have devastating metabolic consequences. Supportive treatment such as parenteral nutrition is costly and has associated morbidity. Surgical intervention including bowel lengthening procedures and small bowel transplantation has met with varied success. Accordingly, alternative approaches to create tissue-engineered intestine offer novel, potentially curative therapeutic strategies to treat short bowel syndrome. Human enteroids, which are primary 3-dimensional tissue that can be generated from an endoscopic biopsy, recapitulate the complexities of the intestinal epithelium histologically and can be expanded exponentially in vitro. Consequently, enteroids can provide a limitless source of autologous intestinal epithelium. To date, successful transplantation of murine enteroids has required induction of colitis. In contrast, human enteroids have not been successfully transplanted into immunocompromised mice, likely due to inadequate supportive structures. Poly (lactide-co-glycolide) (PLG) scaffolds have been shown to provide structural support for human cells. We thus hypothesized that placement of human enteroids on PLG scaffolds embedded with growth factors would permit long-term survival of enteroids post-transplant.

Methods: We generated enteroids from human small intestinal tissue by isolating intestinal epithelium from associated structures using dispase. After expanding these enteroids in vitro, we embedded enteroid fragments and the growth factors, EGF, Noggin, and R-spondin, onto PLG scaffolds. Enteroid-laden scaffolds were then grown in vitro for 1 week and assessed for survival by calcein and ethidium homodimer staining. Enteroid-laden scaffolds were also transplanted into the epididymal fat pads of immunocompromised NSG mice and harvested at 1 and 4 weeks.

Results: Our data demonstrated that enteroids grown on PLG scaffolds were able to survive for 1 week in vitro as indicated by positive calcein staining. When enteroid-containing scaffolds were transplanted into murine epididymal fat pads, all explants demonstrated regions of cells that stained positive for human mitochondrial antigen, Cdx2, and E-cadherin after both, 1 and 4 weeks post-transplant (n = 5, and n= 4, respectively). Together, these data indicate that human enteroids persist and retain their intestinal identity when set in scaffolds and transplanted into mice.

Conclusion: This is the first report demonstrating survival of human epithelial enteroids in a transplant model. As such, these studies have important implications for short bowel syndrome, because enteroid-laden scaffolds are easily generated from autologous tissue. Furthermore, scaffolds can be designed to resemble gut tubes such that future studies may examine the ability of scaffolds to be incorporated into the small intestine.

45.03 Enhanced Serotonin Signaling Increases Enterocyte Proliferation and Axonal Growth in Small Intestinal Villi

Posted on January 18, 2016

C. J. Greig1, N. Gandotra1, J. J. Tackett1, M. C. Bamdad1, R. A. Cowles1 1Yale University School Of Medicine,Department Of Surgery, Section Of Pediatric Surgery,New Haven, CT, USA

Introduction:

The intestinal mucosa recovers from injury by accelerating enterocyte proliferation resulting in villus growth. A similar phenomenon is seen in the adapting small intestine after massive bowel resection. Serotonin (5-HT), a ubiquitous neurotransmitter in the bowel, has been implicated as an important regulator of intestinal mucosal homeostasis by promoting growth in the epithelium. The impact of 5-HT on other components of growing villi is not known. We hypothesized that 5-HT-stimulated growth in the intestinal epithelium would be associated with growth in other components of the villus such as enteric neural axonal processes.

Methods:

Enteric serotonergic signaling is inactivated by the SERT molecule located on enterocytes and enteric nerves. Knock out of SERT (SERTKO) or treatment with serotonin reuptake inhibitors (SSRIs) effectively enhances serotonergic signaling by blocking its inactivation. Therefore, 8 week-old wild type (WT), SERTKO, and SSRI-treated wild type mice were used for experiments. For SSRI treatment, a miniosmotic pump containing citalopram was inserted subcutaneously and left in place for 7 days before sacrifice. Paraffin-embedded sections were created from predefined segments of ileum harvested from each experimental group. Villus height and crypt depth were assessed in H&E-stained sections. Crypt proliferation index was calculated using Ki67 staining. Immunofluorescence microscopy with antibodies targeting Gap43, a marker of axonal growth, was performed and tiled images were obtained to visualize multiple villi in cross section. Images were analyzed with ImageJ software using integrated optical density and region of interest (ROI) tool. Adequate villi with a visualized central lacteal were identified and a ratio of Gap43 to DAPI signal was obtained to normalize differences in villus size. Data for Gap43/DAPI ratio were analyzed using one way ANOVA and two-sample Student’s t-test to ascertain statistical differences. The study protocols were approved by the Institutional Animal Care and Use Committee.

Results:

SERTKO and SSRI-treated mice displayed increased epithelial growth with taller villi, deeper crypts and increased enterocyte proliferation rates when compared to WT mice. Eight adequate villi per animal group were analyzed for Gap43 expression. Gap43/DAPI ratios within villi were calculated and average Gap43/DAPI signal ratios (%) were 4.0(SEM 0.39, 95% CI 3.24-4.76) for WT, 5.3(SEM 0.47, 95% CI 4.39-6.21) for SERTKO and 11.0(SEM 1.53, 95% CI 8.0-14.02) for SSRI-treated animals. The differences in Gap43 expression between all animal groups were statistically significant with p<0.05 by ANOVA and t-test.

Conclusion:

Enhanced 5-HT signaling results in intestinal mucosal growth in both the epithelial cell compartment as well as the enteric nervous system. 5-HT appears be an important regulator of intestinal mucosal growth and enteric neuronal plasticity.

45.04 Synthetic HDL Nanoconjugate Targets Neuroblastoma Stem Cells, Blocking Migration and Self-Renewal

Posted on January 18, 2016

C. Subramanian1, P. T. White1, R. Kuai2, J. Moon2,5, V. P. Opipari6, B. N. Timmermann4, A. Schwendeman2, M. S. Cohen1,3 5University Of Michigan,Department Of Biomedical Engineering,Ann Arbor, MI, USA 6University Of Michigan,Department Of Pediatrics,Ann Arbor, MI, USA 1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,College Of Pharmacy,Ann Arbor, MI, USA 3University Of Michigan,Department Of Pharmacology,Ann Arbor, MI, USA 4University Of Kansas,Department Of Medicinal Chemistry,Lawrence, KS, USA

Introduction: Only 40% of patients with high-risk neuroblastoma (NB) survive beyond 5 yrs despite current treatments. We have demonstrated that 4,19,27-triacetyl withalongolide A (WGA-TA) is a novel inhibitor of the Hsp90 molecular chaperone, targeting the PI3K/Akt/NFΚ B pathways in NB. These pathways are critical for NB cancer stem cell (CSC) function and these CSCs have been ascribed the capacity to recapitulate a tumor from a few cells, enhance tumor drug resistance or its metastatic potential. While withanolides are potent against NB, they do not directly target NB cells and are difficult to solubilize in vivo. Synthetic HDL mimetic (sHDL) targets the SR-B1 receptor on the cell surface and solubilizes lipophilic drugs like WGA-TA. Since NB cells and their CSCs highly overexpress SR-B1, we hypothesize that nanoparticle conjugation of a novel mimetic sHDL nanoparticle with WGA-TA will improve targeting and inhibition of NB CSC function in vitro and in vivo to decrease tumor growth, self-renewal and invasion.

Methods: Validated human NB cells (IMR32,SH-EP,SH-SY5Y,SK-N-BE(2),and SK-N-AS) were evaluated for the expression of SR-B1 by qPCR and western blot (WB). Drug uptake in vitro was measured by fluorescence microscopy, and in vivo uptake in NB tumors (IMR32) was measured on an IVIS system using fluorescent labeled drug. Self renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Percentage viability was analyzed by Cell-Titer Glow(CTG) assay.

Results: qPCR and WB analysis revealed a 4-6 fold higher-level expression of SR-B1 in N-myc amplified NB cells compared to N-myc non-amplified or Jurkat cells(p<0.01). In vitro uptake of sHDL indicated significantly higher levels of uptake in high SR-B1 expressing (IMR32, SK-N-BE(2)) cells compared to low expressers(SH-EP, SH-SY5Y), which was almost completely blocked by excess sHDL(p<0.001). IVIS imaging of IMR32 tumors indicated highest uptake of sHDL in the tumor and liver (where it is metabolized) whereas uptake in other organs was negligible. After treatment of both IMR32 and SK-N-BE(2) cells, there was a dose dependent decrease in sphere formation starting from 8 nM [with complete blockage of self renewal at 62.5 nM (p<0.001 vs control)], and invasion and migration starting at 250 nM vs. untreated control (p<0.001). There was no change in cell viability for sHDL up to 20 µ M, whereas the sHDL-WGA-TA and WGA-TA had an IC50 value of 15-100nM which was 15-51 fold higher than the IC50 of control MRC5 cells(p<0.001).

Conclusion: Mimetic sHDL-WGA-TA is a novel treatment strategy for NB to improve drug delivery to NBs and their CSCs through SR-B1 targeting in vitro and in vivo. sHDL conjugation of withanolides enhances their ability to prevent NB tumor self-renewal and migration/invasion in vitro. Further in vivo efficacy studies are warranted to evaluate the translation effect of NB CSC inhibition on tumor growth, invasion, and metastatic spread.

45.05 Epithelial-specific Mutation of Fgfr 2IIIb is Sufficient to Cause Atresia in Mouse Embryos

Posted on January 18, 2016

A. P. Rogers1, A. Kowalkowski1, A. Reeder1, P. F. Nichol1 1University Of Wisconsin,Pediatric Surgery,Madison, WI, USA

Introduction:
Intestinal atresia can lead to chronic health issues including intestinal dysmotility, short gut syndrome, and, in extreme cases, intestinal failure. Understanding the mechanisms that lead to atresia is critical to developing preventative strategies for this defect and its sequelae. Previous work hypothesized that intestinal atresia results from persistence of a developmental epithelial plug, vascular accident, or mechanical obstruction. Fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) is a transmembrane receptor expressed only in the intestinal epithelium during early intestinal organogenesis. Homozygous mutation of Fgfr2IIIb throughout the developing embryo results in intestinal atresia of the distal colon in 100% of all embryos. However, atresias arising from epithelial-specific mutation of Fgfr2IIIb during intestinal development have never been shown. We hypothesized that conditional mutation of Fgfr2IIIb within the developing intestinal epithelium is sufficient to induce intestinal atresia. We modified the existing mouse model to directly test this hypothesis.

Methods:
The epithelial specific Cre driver Sonic Hedgehog Cre (ShhCre) was used to selectively mutate Fgfr2IIIb in the developing intestinal epithelium by mating Fgfr2IIIbLoxP/+; ShhEGFPCre/+ males to Fgfr2IIIbLoxP/LoxP females. For lineage analysis ShhEGFPCre/+ breedings were performed on a RosaLacZ background with or without Fgfr2IIIbLoxP alleles. TUNEL and BrdU staining was performed at embryonic age (E)10.5, in situ hybridization for Fgfr2IIIb was performed at E11.5. Lineage analysis was performed at E13.5. Incidence of colonic atresia was determined.

Results:
ShhCre eliminated Fgfr2IIIb expression specifically from the intestinal epithelium in Fgfr2IIIbLoxP/r; ShhEGFPCre/+ mouse embryos resulting in distal colonic atresias in all homozygous conditional mutants by E13.5 (40/40) where as control littermates did not form atresias (0/155) (P<0.01). TUNEL staining was increased in the proximal colon, but largely absent from the distal colon at E10.5. BrdU labeling was diminished throughout the colon at this same time point. Lineage analysis demonstrated that atresias were fully formed by E13.5 and featured an absence of both colonic epithelium and surrounding mesenchyme.

Conclusion:
This data supports our hypothesis that epithelial-specific mutation of Fgfr2IIIb is sufficient to induce distal colonic atresias in mice. ShhCre eliminates expression of the Fgfr2IIIb conditional alleles from the intestinal epithelium; a tissue layer separate from the one that gives rise to the vasculature of the intestine. Thus, our findings also suggest that disruptions in epithelial development alone are sufficient to induce atresias and point to a potential mechanism separate from vascular accidents in the etiology of these defects.

45.06 Delivery Of Mesenchymal Stem Cells In Biomimetic Engineered Scaffolds Promotes Healing Of Diabetic Ulcers

Posted on January 18, 2016

H. HE1,2, T. Foster1, R. Assi1, K. Stamati3, H. Bai1, Y. Huang4, F. Hyder4, D. Rothman4, C. SHU5, S. Homer-Vanniasinkam3, U. Cheema3, A. Dardik1,6, A. Dardik1,6 1Yale University School Of Medicine,Vascular Biology And Therapeutics Program And The Department Of Surgery,New Haven, CT, USA 2The Third Xiangya Hospital Of Central South University,Department Of Vascular Surgery,changsha, HUNAN, China 3University College London,Division Of Surgery And Interventional Science,London, LONDON, United Kingdom 4Yale University,Departments Of Diagnostic Radiology And Biomedical Engineering,NEWHAVEN, CONNECTICUT, USA 5The Second Xiangya Hospital Of Central South University,Department Of Vascular Surgery,Changsha, HUNAN, China 6VA Connecticut Healthcare System,Department Of Surgery,West Haven, CT, USA

Introduction: Lower extremity wounds remain an important complication in patients with diabetes. Mesenchymal stem cells (MSC) are known to enhance wound healing. We hypothesize that delivery of MSC in a biomimetic collagen scaffold improves wound healing in a diabetic mouse model.

Methods: Bone marrow derived MSC were suspended in high density type I collagen scaffold sheets and rolled into a spiral configuration. Real-time oxygen measurements confirmed cells in the core having restricted oxygen exposure. These scaffolds were implanted adjacent to lower extremity wounds in diabetic C57BL/6 mice; a splinted excisional back wound model was used to test wound healing without contraction. Daily rates of wound healing were measured and immunohistochemical analysis was performed to identify changes in cell composition and growth factor expression. Nanoparticle labeling of MSC was used to monitor cell retention within the scaffold.

Results:The scaffold core was confirmed to be hypoxic with a resultant increase in VEGF release. Diabetic mice with leg wounds showed significantly increased wound healing with the scaffold compared to controls (64% closure vs. 49% with MSC alone vs. 37% with acellular scaffold; day 1; p=0.004). Diabetic mice with splinted back wounds also showed enhanced wound healing compared to controls (36% MSC scaffold vs. 31% acellular scaffold; day 4; p≤0.0001). Examination of cells surrounding the scaffolds showed increased proliferation without increased apoptosis, increased VEGF expression and capillary density, and increased numbers of macrophages, fibroblasts, and smooth muscle cells in mice treated with MSC containing scaffolds. Wound healing was further enhanced with the addition of laminin to the collagen scaffold.

Conclusion:MSC within a biomimetic collagen scaffold enhance wound healing in a translationally relevant diabetic mouse model. Tissue engineering approaches can create niche-like environments that promote stem cell survival and function, increasing their potential applications for therapeutic use.

45.01 MYCN Deregulation is a Critical Tumor Initiating Event During Human Neural Crest Stem Cell Development

Posted on January 18, 2016

D. Bashllari2, R. Van Noord2, A. Opipari2, E. Lawlor2, V. Castle2, E. Newman2 2University Of Michigan,Pediatrics, Obstetrics And Gynecology, And Pediatric Surgery,Ann Arbor, MI, USA

Introduction: Neuroblastoma (NB) is a developmental cancer that arises from neural crest stem cells (NCSC). The amplification of the transcription factor MYCN predicts poor prognosis and correlates with an undifferentiated cell phenotype. MYCN is expressed during normal NCSC development, regulating neural progenitor cell proliferation and differentiation at distinct time points. Our preliminary work has shown that deregulation of MYCN during normal human NCSC differentiation increases cell proliferation, motility, migration, and invasion in-vitro. To further determine how MYCN contributes to NB tumorigenesis, we generated a novel in-vivo human model to test our hypothesis that MYCN deregulation during early NCSC development is a critical component of tumor initiation.

Methods: Human embryonic stem cells (hESC) were plated on a mouse embryonic fibroblasts feeder layer and grown in stromal-derived inducing activity (SDIA) media to differentiate into NCSC. NCSC were collected using staining for p75 and HNK1+ neural markers by FACS. Double positive sorted cells were then transduced with a doxycycline inducible V5-tagged MYCN cDNA lentiviral construct. MYCN expression was confirmed by Western blot and immunocytochemistry (ICC). NCSC-MYCN+ cells in a 1:1 mix with matrigel were flank-injected into NOD/SCID mice, while NCSC-MYCN- cells were injected into the opposite flanks. Mice were fed doxycycline water to induce MYCN expression. Animals were monitored daily for health and tumor formation. Resultant tumors were excised, characterized, and examined for tumor markers using paraffin-embedded slides stained for H/E, MYCN, tyrosine hydroxylase (TH), Chromogranin A, and TrkB.

Results: NCSC-MYCN+ cells formed robust tumors at flank injection sites that were metastatic to the abdominal cavity, while NCSC-MYCN- flanks did not form tumors. Primary and metastatic tumors were locally invasive and morphologically consistent with poorly differentiated NB, with immature neuroecterdermal cells that palisaded around blood vessels, forming pseudorosettes and brisk mitotic rates. Resultant tumors stained positive for TH, Chromogranin A, TrkB, and MYCN. Sections of lymph nodes and abdominal spread sites were consistent with NB and stained positive for all markers as well.

Conclusion: Our results are progress towards establishing the first human model of NB using hESC and human NCSC, and further implicate MYCN in early NCSC tumor initiation. These data demonstrate that forced constitutive expression of MYCN is sufficient for NCSC tumor initiation in-vivo that is capable of metastatic spread, and pave the way for studies to determine how NB tumors develop.

45.02 Organoid Units Repopulate Neurons and Glia in Human Intestinal Organoid Tissue-Engineered Intestine

Posted on January 18, 2016

M. M. Wieck1, C. R. Schlieve1, W. N. El-Nachef1, X. Hou1, S. Huang2, J. R. Spence2, T. C. Grikscheit1 1Children’s Hospital Los Angeles,Los Angeles, CA, USA 2University Of Michigan,Ann Arbor, MI, USA

Introduction:
Human embryonic or induced pluripotent stem cells (hESC, iPSC) differentiated into human intestinal organoids (HIOs) has potential to be a scalable tissue source for generating tissue-engineered intestine. Previous studies have demonstrated epithelial architecture and some enzymatic function in HIOs that replicates native intestine, but the resultant tissue lacked elements of the enteric nervous system (ENS). In contrast, intestine derived from multicellular organoid units (OU) derived from postnatal human or mouse intestine contain neurons and glia. We therefore hypothesized that co-implantation of HIOs with murine OU would generate tissue-engineered intestine with ENS components.

Methods:
HIOs were generated as previously described with sequential application of Activin A followed by FGF4 and CHIR99021 to promote patterning of intestinal spheroids. Spheroids were placed in Matrigel with growth medium that contained EGF, R-spondin 2 (RSPO2) and Noggin (NOG) to further promote HIO growth as previously described (Spence 2011, Watson 2014). Intestine from actinGFP mice were resected and then mechanically and enzymatically digested to form GFP OU. In the first group, 20 HIOs were pipetted onto the surface of a 3 mm polyglycolic/poly-L lactic acid tube. In the second group, GFP OU were co-implanted with HIOs. Both constructs were implanted into the omentum of adult irradiated NOD/SCID gamma mice. Harvest was at 12 weeks. Immunofluorescent staining with neuronal markers β III tubulin (TUJ1) and neuronal nuclei (NeuN), and glial marker glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100b) identified ENS elements. Human and murine elements were differentiated by staining for human specific human mitochondria (hMito) and GFP

Results:
Implantation of HIOs with and without OU formed small intestinal epithelium with villus-crypt architecture and all differentiated epithelial cell types surrounded by smooth muscle. All the epithelium was either hMito+, confirming HIO origin, or GFP+, indicating OU origin. Intestine generated from HIOs alone did not contain any neurons or glia. However, co-implantation of HIOs with OU resulted in hMito+ epithelium and adjacent subepithelial and myenteric GFP+ neurons and glia. GFAP and S100b localized to different subpopulations of glia. Tuj1 and GFAP co-stained a subset of GFP+ structures.

Conclusion:
Co-implantation of OU with HIO generates tissue-engineered intestine with human epithelium and OU-derived neurons and glia in the subepithelial and myenteric layers, thus replicating the structure of normal human intestine including ENS components. This demonstrates that implantation of supplemental and separate cells with HIOs can successfully generate tissue-engineered intestine with ENS elements.

44.08 Minnelide Potentiates the Antitumor Effects of Current Chemotherapy in the Treatment of Pancreatic Cancer

Posted on January 18, 2016

S. Modi1, B. Giri1, K. Majumder1, S. Banerjee1, V. Dudeja1, A. K. Saluja1 1University Of Minnesota,Surgery,Minneapolis, MN, USA

Introduction: Gemcitabine with nab-paclitaxel is the current benchmark in the treatment of metastatic pancreatic ductal adenocarcinoma (PDA); however, the survival benefit of this combination is around 6 weeks more than gemcitabine alone. Drug combinations that help in reducing the doses of these drugs while providing better treatment response are required. Minnelide (triptolide prodrug, currently in phase I trials) has been shown to be effective against pancreatic cancer in preclinical models. In this study, we evaluated the efficacy of Minnelide in combination with reduced doses of standard therapy in PDA.

Methods: In vitro, pancreatic cancer cells (S2-VP10, S2-013 and PANC-1) were treated with low doses of paclitaxel (0-25nM), low dose of triptolide (25nM), and a combination of both for 24-72h. Cell viability was measured by the WST-8 assay. Cancer cell death by apoptosis was evaluated by measuring cleaved caspase-3 levels using flow cytometry. Cell cycle analysis was done using propidium iodide staining.

In vivo, combination therapy was tested in various models of PDA. Xenograft, subcutaneous, and orthotopic models were established using S2-VP10 and S2-013 pancreatic cancer cells. The immunocompetent subcutaneous model was established using KPC derived cells in C57BL/6J wild type mice. Due to the development of fatal immune reaction in mice treated with nab-paclitaxel (due to human albumin), equivalent doses of paclitaxel (dissolved in DMSO) were used in immunocompetent mice while immunodeficient mice were treated with nab-paclitaxel. Tumors were harvested, and tissues were used for various experiments.

Results: In vitro, proliferation of pancreatic cancer cells was markedly inhibited by the combination of triptolide and paclitaxel. Low doses of triptolide and paclitaxel led to significantly increased levels of cleaved caspase-3 and cleaved PARP suggesting that apoptotic cell death was induced in these cells. The combination therapy also led to an increased number of these cells trapped in the G2/M phase of the cell cycle.

In vivo: In the s/c model, combination of low doses of Minnelide and paclitaxel inhibited tumor progression. The tumor volumes in various groups were: Minnelide: 75.4 ±25 %, paclitaxel: 50.0 ±3%, Minnelide + paclitaxel: 11.0 ±1%. In the metastasis orthotopic cancer model, median survival (in days) of animals in different treatments groups was vehicle (13.0), Minnelide (20.5), paclitaxel (21.5) and combination (all alive). The combination of Minnelide and low doses of gemcitabine + nab-paclitaxel was effective in decreasing the tumor burden in orthotopic models and increasing survival of tumor-bearing mice. Besides decreasing tumor burden, combination treatment significantly reduced cancer-related morbidity by decreasing ascites and metastasis.

Conclusion: Minnelide synergizes with standard of care chemotherapy, reduces the doses of these toxic drugs and achieves better efficacy in treatment of PDA.

44.09 FoxO-dependent Gene Networks Associate with Cachexia in Pancreatic Cancer.

Posted on January 18, 2016

D. Delitto1, S. M. Judge1, R. N. Nosacka1, T. J. George1, K. E. Behrns1, S. J. Hughes1, S. M. Wallet1, A. R. Judge1, J. G. Trevino1 1University Of Florida,Gainesville, FL, USA

Introduction: Pancreatic cancer (PC) is associated with a high rate of cachexia, which contributes significantly to patient morbidity and mortality. However, mechanisms underlying muscle wasting in the human disease remain incompletely described, in part due to limited translational models. We hypothesize that the development of translational models of pancreatic cancer cachexia will allow for the delineation of potential therapeutic targets in cachexia. To test our hypothesis, we propose to 1) establish the first described PDX model of cancer cachexia and 2) examine skeletal muscle in patients and corresponding patient-derived xenograft (PDX) models to further support a central role for transcription factors associated in PC-associated cachexia.

Methods: Rectus abdominis muscle was biopsied from surgically resected PC patients and non-cancer pancreatitis controls. Pancreatic cancer PDX models were derived in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and skeletal muscle was harvested for histologic investigations on ultrastructural disorganization and qRT-PCR for atrophy-related transcription factors differentially regulated in PC patients.

Results: Rectus biopsies from patients with PC displayed marked muscle fiber atrophy, increased extracellular space, greater variation in fiber size and shape and more centralized nuclei compared to controls. These architectural abnormalities were also present in mice bearing xenografts representative patients with muscle wasting. Further, skeletal muscle from patients with PC and mice bearing PDX demonstrated high expression of the same Forkhead box O (FoxO)-dependent, atrophy-associated transcription factors.

Conclusions: Preoperative muscle wasting in PC is associated with characteristic architectural abnormalities and elevated FoxO-dependent transcriptional activity. Mice bearing patient-derived xenografts demonstrate comparable muscle pathology and transcriptional activity. These results thereby provide a valid translational model of cachexia which supports a central role for FoxO1 and FoxO3a in PC-associated muscle wasting.

44.10 Pharmacologic Ascorbate is a Potential Novel Therapy for Treatment of Gastric Adenocarcinoma

Posted on January 18, 2016

F. K. Houwen1, B. R. O’Leary1, C. L. Johnson1, B. G. Allen1, G. Buettner1, J. J. Cullen1, D. R. Spitz1, J. J. Mezhir1 1University Of Iowa,Surgery,Iowa City, IA, USA

Introduction: Gastric adenocarcinoma (GAC) is a lethal disease due to resistance to current available therapies and its propensity for metastasis. Pharmacological Ascorbate has been shown to increase the cytotoxicity of chemotherapy and radiation in solid tumors secondary to generation of toxic hydrogen peroxide (H2O2). We therefore hypothesize that Ascorbate, when combined with current multimodality therapies, will increase cytotoxicity in tumor growth and metastasis cell models of GAC.

Methods: Human GAC cell lines MKN-45 and AGS, and normal human stomach/intestinal cell line, Hs738st/Int, were evaluated for clonogenic survival after treatment with clinically relevant doses of Ascorbate (25pmol/cell) and pertinent multimodality therapies including 5-flurouracil (5FU, 5uM), and Ionizing Radiation (IR, 1.8Gy). Cytotoxicity of AGS cells was evaluated with Ascorbate both with and without cisplatin (0.5uM) and irinotecan (2.5uM). GAC cell lines were treated with bovine catalase and deferoxamine (DFO) plus diethylene triamine pentaacetic acid (DTPA) to confirm the mechanisms of H2O2-mediated cell death following Ascorbate exposure in the model systems. Tumor cell invasive capacity was measured using matrigel migration and invasion assays.

Results: Ascorbate reduced GAC clonogenic survival by 50% (p <0.0001) compared to control. Ascorbate when combined with 5-FU or IR resulted in significant increases in cell death compared to 5-FU or IR alone. Triple therapy with Ascorbate, 5FU, and IR showed enhancement in cytotoxicity that was significantly increased over the standard multimodality therapy regimen (Figure, p <0.01). Ascorbate increased cell death when combined with irinotecan and cisplatin compared to either chemotherapy drug alone. Ascorbate combined with catalase or DFO+DTPA yielded a full rescue of cytotoxicity from the toxic effects of Ascorbate treatment (p<0.0001) thereby confirming the dependence on H2O2 levels and catalytic metal ions for cellular death to occur. Ascorbate significantly reduced tumor cell invasion by 57% (p < 0.003) and tumor cell migration by 61% (p < 0.002) compared to control. Normal gastric cell clonogenic survival was not altered by treatment with Ascorbate, suggesting treatment selectivity for malignant cells and safety in normal cells.

Conclusion: Pharmacologic Ascorbate is highly toxic to GAC cells at pharmacologic concentrations while having no effect on normal cells; Ascorbate also reduces tumor cell invasive capacity. Our data confirm that Ascorbate exerts its cytotoxicity via generation of H2O2 and is dependent upon the presence of catalytic metal ions. These highly translational data suggest that Pharmacological Ascorbate is a potential novel therapy for the treatment of GAC.

44.04 Anti-tumor activity of BRAF inhibitor and IFNα combination in BRAF mutant melanoma

Posted on January 18, 2016

C. Ferrone1,2, F. Sabbatino1,2, Y. Wang1,2, G. Scognamiglio3, E. Favoino1,2, S. A. Feldman4, V. Villani1,2, K. T. Flaherty5, S. Nota6, D. Giannarelli7, E. Simeone3, A. M. Anniciello3, G. Palmieri8, S. Pepe9, G. Botti3, P. A. Ascierto3, C. Ferrone1,2 1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA 2Harvard School Of Medicine,Department Of Surgery,Brookline, MA, USA 3Istituto Nazionale Tumori Fondazione,Naples, CAMPANIA, Italy 4National Cancer Institute,Surgery Branch,Bethesda, MD, USA 5Massachusetts General Hospital,Department Of Medical Oncology,Boston, MA, USA 6Massachusetts General Hospital,Department Of Orthopaedic Surgery,Boston, MA, USA 7Regina Elena National Cancer Institute,Rome, LAZIO, Italy 8Institute Of Biomolecular Chemistry,Sassari, SARDINIA, Italy 9University Of Salerno,Baronissi, SALERNO, Italy

Introduction: BRAFV600E mediated-MAPK pathway activation is associated in melanoma cells with IFNAR1 down-regulation. IFNAR1 regulates melanoma cell sensitivity to IFNα, a cytokine currently used for the adjuvant treatment of melanoma patients. These findings in conjunction with limited therapeutic efficacy of BRAF-I prompted us to examine whether the efficacy of IFNα therapy of melanoma harboring BRAFV600E can be increased by its combination with BRAF-I.

Methods: BRAF/NRAS genotype, ERK activation, IFNAR1 and HLA class I antigen expression were tested in 60 primary melanoma tumors of treatment naïve patients. The effect of BRAF-I on IFNAR1 expression was assessed both in melanoma cell lines and in tumor biopsies of BRAFV600E metastatic melanomas. The anti-proliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination was tested both in vitro and in vivo utilizing melanoma cell lines, HLA class I antigen-MA derived peptide complex-specific T cells and immunodeficient mice.

Results:IFNAR1 level was significantly (P<0.0001) lower in BRAFV600E primary melanoma tumors than in BRAF wild type tumors. IFNAR1 down-regulation was reversed by BRAF-I treatment both in melanoma cell lines and in tumor biopsies from metastatic melanoma patients. IFNAR1 level in the melanoma tumors analyzed was increased as early as 10-14 days following the beginning of the treatment. These changes were associated with an increased susceptibility of melanoma cells to the anti-proliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination both in vitro and in vivo.

Conclusion:The results of this study provide a strong rationale for the novel clinical trials implemented in BRAFV600E melanoma patients with BRAF-I and IFNα combination.

44.05 Novel HDAC Inhibitor AB3 Regulates NF-kB and Notch Signaling in Medullary Thyroid Cancer Cell Lines

Posted on January 18, 2016

S. K. Golden1, H. Jin1,3, V. Jain1, A. Ma1, A. Marin1, C. Matsumura1, R. Jaskula-Sztul1,3, S. Miyamoto2, H. Chen1,3 1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Oncology,Madison, WI, USA 3University Of Alabama,Surgery,Birmingham, Alabama, USA

Introduction: Medullary thyroid cancer (MTC) is a neuroendocrine malignancy arising from the calcitonin-producing C-cells of the thyroid gland. To date, no effective systemic therapies for MTC have been established. AB3 is a novel histone deacetylase inhibitor (HDACi) that has been shown to diminish MTC cell line proliferation by inducing apoptosis. Preliminary studies have suggested that phenotypic regulation by AB3 may be mediated by Notch3 signaling; however, AB3's broader effects on gene expression and its mode of Notch3 activation are not well understood.

Methods: The effects of AB3 treatment on transcription were investigated using a DNA microarray. Human MTC cell lines (MZ-CRC-1 and TT) were treated with 2 uM AB3 or DMSO vehicle control. The microarray results were filtered to remove non-coding genes, unnamed genes, and genes with an absolute fold change <1.5 or ANOVA p-value >0.05 in either cell line. Selected DNA microarray results were then validated by qRT-PCR. Lastly, a luciferase reporter construct harboring deletion fragments of the Notch3 upstream region was used to map AB3 responsive elements within the Notch3 promoter. Luciferase activity of the constructs and a promoter-less backbone were measured following treatment with AB3 (a Notch3 inducer) or vehicle control.

Results: 67,528 genes were assessed by DNA microarray after AB3 treatment. Of these, 2,243 showed differential expression after filtering. Genes affected by AB3 treatment included members of the nuclear factor-κB (NF-κB) pathway and its targets: NFKB1, ATM, PARP1, XIAP, IκBα, cyclin D1, and BCL2 (down-regulated), as well as SENP2 (up-regulated). These results were consistent with NF-κB pathway down-regulation and were validated by qRT-PCR. Interestingly, qRT-PCR showed substantial increases in Notch 1-3 transcription, including a 33 fold increase in Notch3. Deletion mapping identified the potential AB3-responsive elements within the human Notch3 promoter as a 31-bp fragment located 109 to 140 nucleotides upstream of the Notch3 start codon.

Conclusion: Down-regulation of the NF-κB pathway by AB3 was demonstrated by DNA microarray and validated by qRT-PCR. NF-κB is a major stress-responsive transcription factor whose activation is associated with intrinsic cancer cell resistance to chemo- and radiation therapy. Understanding the mechanism by which AB3 down-regulates NF-κB could provide insight into improving HDACi-based therapies. Furthermore, we describe for the first time the potential HDACi binding site within the Notch3 promoter, which could determine the mechanism(s) of HDACi regulation of Notch signaling.

44.07 Radiotherapy Enhances Natural Killer Cell Homing and Function in Canine Bone and Soft Tissue Sarcoma

Posted on January 18, 2016

J. S. Park1, S. Grossenbacher2, J. Modiano3, J. Miller4, E. Ames2, S. Mac2, A. Monjazeb5, M. Kent6, W. Culp6, M. Chen7, W. J. Murphy2, R. Canter1 1University Of California – Davis,Surgical Oncology/Surgery,Sacramento, CA, USA 2University Of California – Davis,Dermatology,Sacramento, CA, USA 3University Of Minnesota,Veterinary Medicine,Minneapolis, MN, USA 4University Of Minnesota,Internal Medicine/Oncology,Minneapolis, MN, USA 5University Of California – Davis,Radiation Oncology,Sacramento, CA, USA 6University Of California – Davis,Veterinary Medicine,Sacramento, CA, USA 7University Of California – Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA

Introduction: We have previously shown that combination radiotherapy (RT) and Natural Killer (NK) immunotherapy is effective in diverse pre-clinical models of human solid malignancies. Since bone and soft tissue sarcomas commonly afflict dogs, and canine clinical trials are a tremendous resource, particularly for novel immunotherapy protocols, we hypothesized that dog PBMC-derived NK cells could be similarly expanded and activated ex vivo as a precursor for a canine combination radioimmunotherapy clinical trial.

Methods: Dog NK cells were isolated from 10-15 mL fresh PBMCs using Ficoll separation and CD5 depletion. Isolated NK cells (CD3+, CD5dim, TCR-) were expanded via co-culture with irradiated (100Gy) K562-C9-mIL21 for 2-3 weeks in 100IU/mL recombinant human IL-2. Canine osteosarcoma (OSCA) tumor lines and fresh canine primary sarcomas were evaluated for susceptibility to NK killing before/after RT in vitro and in xenograft experiments with NSG mice. NK cytotoxicity was assessed in 4-16 hour killing assays by Flow cytometry using a BD Fortessa cell sorter (BD Biosciences) with 7-Aminoactinomycin as cell viability marker. Parametric and non-parametric statistical tests were performed as appropriate.

Results: NK expansion was successful in 14/20 donors (including 9 tumor-bearing dogs) from baseline 4.5(±1.9) x106 cells to 103.5(±29.1) x106, mean increase 23.2X (±2.3). Canine NK cells were also responsive to human cytokines (IL-2, IL-12, and IL-18), but expansions were lower (1.6-3.5 fold expansion over 14 days). NK cytotoxicity to OSCA78, OSCA32, and NK-sensitive CTAC cells in vitro increased in a dose-dependent fashion reaching 74 – 88% cytolysis at effector:target ratios of 10:1 – 20:1 (P<0.001). RT augmented NK cytotoxicity with greatest synergy at 2.5-5 Gy RT in 4-hour killing assays (1.3-3.4X increased killing, P<0.01). At doses of 10 Gy and/or 16-hour killing assays, only minor differences in overall killing were observed. Similar results were observed with RT sensitization to NK killing in primary canine sarcomas. In a dog sarcoma PDX model using focal RT, intravenous NK transfer, and hydrodynamic human IL-15 for in vivo NK support, focal RT increased NK homing to tumors by 3.8X±0.3 (P<0.001).

Conclusion: NK cell homing and effector functions are increased following RT in canine models of sarcoma. Dog sarcoma appears to be a valuable model to facilitate clinical translation of NK immunotherapy.

44.02 Cancer Associated Fibroblast Induction and Maintenance Are Differentially Regulated

Posted on January 18, 2016

A. N. Kothari1,2, C. E. Weber1,2, V. Chang2,4, M. L. Arffa2,4, M. A. Zapf4, W. K. Syn3, P. C. Kuo1,2, Z. Mi1,2 1Loyola University Medical Center,Department Of Surgery,Maywood, IL, USA 2Loyola University Chicago,Cardinal Bernardin Cancer Center,Maywood, ILLINOIS, USA 3The Institute Of Hepatology,Barts Health NSH Trust,London, UK, United Kingdom 4Loyola University Chicago Stritch School Of Medicine,Maywood, IL, USA

Introduction:
Tumor growth and metastasis rely on interactions between cancer cells and the surrounding tumor microenvironment (TMEN). Major regulatory components of the TMEN are cancer-associated fibroblasts (CAF), of which a significant proportion originate from mesenchymal stem cells (MSC). Osteopontin (OPN) is secreted by tumor cells and can induce the transformation of MSCs to CAFs. Our objective was to determine how tumor-derived OPN regulates time-dependent plasticity of the CAF phenotype in a hepatocellulcar carcinoma (HCC) model.

Methods:
Experiments were conducted using HepG2 (high OPN expressing) and Hep3B (low OPN expressing) HCC cell lines. All co-culture experiments were performed in a permeable support system with MSCs seeded in the lower compartment and HepG2/Hep3B cells in removable inserts (pores =0.4 µm). Measurement of active TGF- β was done using ELISA. Relative mRNA expression of MSC/CAF signature genes (α-SMA, tenacin-C, vimentin) were quantified by using RT-PCR. Changes in MSC cell surface markers were measured using flow cytometry. Mean values reported ± SD.

Results:
HepG2 cells co-cultured with MSCs induced the mRNA expression of CAF genes (α-SMA ddCT=11.7±0.1) at 72h while Hep3B cells did not in similar culture conditions (α-SMA ddCT=1.6±0.1, p<0.001). Functional activation of CAFs was measured using active TGF-β protein expression at baseline and after 72h co-culture (477.5±61.4 pg/mL vs. 1,527.2±21.2 pg/mL, p<0.001). Blockage of OPN with aptamer (APT) at onset of co-culture inhibited the transformation of MSCs to CAFs indicating an essential role for OPN in the initiation of the CAF phenotype. To measure the impact of tumor-derived OPN on CAF phenotype maintenance, activated CAFs were treated with OPN APT while remaining in co-culture. CAF signature gene mRNA expression remained elevated. In contrast, when HepG2 cells were completely removed from co-culture, CAF gene mRNA expression returned to baseline levels (Figure). Expression of MSC cell surface markers CD105, CD146, and CD90 were compared at each time point, with CD105 being down-regulated at 72h of co-culture. After withdrawal of HepG2 cells, CD105 remained down-regulated suggesting the CAF did not revert back to its original MSC phenotype.

Conclusion:
We demonstrate OPN is critical for inducing the MSC to CAF transformation in an in vitro HCC model. After CAF activation, tumor-derived OPN is not required for continued maintenance of CAF indicating a secondary signal pathway responsible for stabilizing CAFs. These findings are the first to demonstrate CAF phenotypic plasticity relies on separate initiating and maintenance signaling pathways.

44.03 Circulating Microvesicles, Exosomes, are Enriched in Melanoma and Correlate with Tumor Burden.

Posted on January 18, 2016

D. DePeralta1, W. Michaud1, M. Hammond1, G. Boland1 1Massachusetts General Hospital,Surgical Oncology/Surgery,Boston, MA, USA

Introduction: Circulating microvesicles, exosomes, have been described since 1981 [1], but their specific role in cancer biology remains unclear. They are small membrane vesicles (40-100nm) that contain microRNAs (miRNAs), messenger RNAs (mRNAs), DNA, and proteins. Given the variety of nucleic acids contained by exosomes, there is interest in their use as biomarkers of cancer. Studies show that circulating serum exosomes contain RNA signatures representative of the parental tumor [2, 3]. Applications of circulating exosomes to tumor diagnosis have been described for various cancers [4-6]. The work herein is focused on utilizing exosomes as markers of response to therapy, as well as to identify potential mediators active in this process. Our pilot set of serum samples have been collected at both pre-treatment and on-treatment timepoints. Our hypothesis is that there may be exosomal signatures that correspond with treatment response or resistance to current therapies.

Methods: We have collected serial tumor and blood samples from patients with metastatic melanoma enrolled on clinical trials with various therapies since these trials opened. Tissue and blood are collected using protocols approved by the institutional review board (IRB). Exosome isolation has been performed by both serial centrifugation and exoRNeasy serum/plasma kits (Qiagen Inc.). Assesment of exosome size and concentration is done using Nanosight analyses. Western blots have been performed for CD63/flotillin, markers associated with exosomes. We have also isolated exosomes from normal healthy donors to serve as a control.

Results: We can demonstrate a purified exosomal population (Fig. 1A) and an increased exosomal concentration in patients with metastatic melanoma as compared to normal controls (Fig. 1B). In all samples assessed (n=94) exosome concentration correlates with tumor burden, decreasing after successful treatment (surgical or systemic) and increasing at the time of clinical progression (representative patient shown in Fig. 1C). We can now model these findings in an in vitro system, showing that non-cancer cells have a much lower concentration of exosomal proteins in cell culture as compared to those derived from melanoma cell lines (data not shown).

Conclusion: Circulating microvesicles are enriched in melanoma patients as compared to normal healthy controls, a finding replicable in an in vitro system. In patient samples, the concentration correlates with tumor burden, suggesting potential utility as a circulating biomarker of disease. Efforts at profiling the mRNA/miRNA content of these exosomes is ongoing and may shed light on the mechanisms of response or resistance to current therapies.

43.09 mTOR Signaling is Required for Adaptation and Epithelial Cell Proliferation after Bowel Resection

Posted on January 18, 2016

R. C. Sun1, J. Guo1, B. W. Warner1 1Washington University,Pediatric Surgery/General Surgery,St. Louis, MO, USA

Introduction: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is important for intestinal stem cell renewal and maintaining mucosa structure. It is also essential for Wnt-driven intestinal tumor initiation and growth. Rapamycin can bind the mTORC1 complex resulting in inhibition of downstream signals and intestinal regeneration. Intestinal adaptation is a phenomenon that occurs after small bowel resection (SBR) with increased crypt depth and villus height which is believed to be driven by enhanced enterocyte proliferation. Our objective was to investigate whether mTOR signaling is necessary for small bowel adaptation after SBR.

Methods: Male C57/Bl6 mice,aged 8 weeks underwent a 50% proximal SBR. Postoperatively,mice were randomized to receive daily injections of rapamycin (n=8) or vehicle (n=7). On postoperative day 7,mice were harvested and intestine was collected for histology to measure structural adaptation by counting crypt depth and villus height. Rates of enterocyte proliferation were measured by counting the number of p-histone 3 stained cells per 20 crypts. Western blot confirmed rapamycin inhibition of mTOR signaling by probing for pS6. To see whether rapamycin affects the integrity of intestinal structure at baseline, rapamycin (n=4) and vehicle (n=5) were injected daily in two separate groups of non-operative mice. After 7 days,mice were harvested and histology and proliferation were compared. Student’s t-test was used to statistically compare groups with p<0.05 defined as significant.

Results:The degree of adaptation was diminished in the rapamycin treated group compared to controls after proximal 50% SBR (Figure). Similarly,rates of proliferation significantly dropped in rapamycin treated group compared to vehicle treated group (2.1±0.1 vs 4.5±0.3, p-value<0.01). Western blot showed a decrease in pS6 signaling which confirms mTOR signaling inhibition by rapamycin within the enterocytes. Daily injections of rapamycin in non-operated C57/Bl6 mice did not affect crypt depth (74.9±3.5µm vs 72.5±3.5µm,p-value=0.7) or villus height (219.8±9.9µm vs 215.9±8.0µm, p-value=0.8) when compared to vehicle injections.

Conclusion:mTorC1 pathway may play a critical role in intestinal adaptation post SBR. Rapamycin diminishes the degree of adaptation and enterocyte proliferation after SBR. However,rapamycin was given systemically in this study and tissue specific contribution of mTOR signaling in adaptation could not be determined. This is the first time mTor pathway has been studied in the context of resection-associated adaptation. Further studies will focus on the role of enterocyte specific mTOR signaling involved in intestinal adaptation.

43.10 Increased Cystathionine-β-Synthase Drives Adenoma to Adenocarcinoma Progression

Posted on January 18, 2016

J. R. Zatarain1, C. Phillips1, P. Johnson1, S. Widen1, T. G. Wood1, N. Druzhyna1, B. Szczesny1, C. Szabo1, C. Chao1, M. R. Hellmich1 1University Of Texas Medical Branch,Galveston, TX, USA

Introduction: Our laboratories have recently reported, cystathionine-β-synthase (CBS) is selectively upregulated in colorectal cancer (CRC), compared to patient-matched normal colonic mucosa. CBS produces the gaseous transmitter hydrogen sulfide (H2S), which we showed promotes CRC growth and metastasis by stimulating tumor cell bioenergetics (i.e., product of ATP for cell proliferation), and creating a localized H2S-rich microenvironment that stimulates tumor angiogenesis and local vasodilatation to feed and disseminate the growing cancer cells. However, the role of CBS/H2S in the progression of disease from adenoma to adenocarcinoma is unknown. The purpose of this study was to determine whether forced overexpression of CBS in a non-malignant colonic cell line (NCM356) would induce a malignant phenotype.

Methods: NCM356 (parental) cells express a very low level of endogenous CBS, similar to normal colonic mucosa, and are non-tumorigenic when xenografted into immune compromised mice. CBS overexpressing (NCMCBS) and control (NCMvec) cells where produced by transducing parental cells with a lentiviral vector containing a CBS cDNA or the empty vector, respectively. Western blotting was used to assess CBS protein levels. Levels of H2S were visualized in intact cells using the H2S-specific fluorescent probe, AzMC (7-azido-4-methylcoumarin). The amount of H2S was quantified by measuring the conversion of CBS substrates L-cysteine and L-homocysteine in protein extracts using AzMC. Cell growth rates where determined with a Coulter Counter. aminooxyacetic acid (AOAA) was used to inhibit CBS activity. Cell migration and invasion assay were performed in Boyden chambers with NIH3T3 conditioned media as a chemo-attractant. Anchorage-independent growth was assessed by soft-agar assay. Statistical significance (p≤0.05) was calculated using ANOVA or non-parametric Student t-test.

Results:NCMCBS cells showed a marked increase in H2S levels by microscopy and a 4-fold increased in NCM-CBS protein extracts, compared to either NCMvec or the parental cell line. CBS overexpression enhanced proliferation rate (p<0.03 NCMCBS vs. NCMvec or parental), cell migration, invasion through matrigel (p<0.01), and colony formation in soft agar (p<0.01). The stimulatory effects of CBS overexpression on NCM cell migration and invasion were inhibited by AOAA (p<0.01). Exosome-wide sequence analyses of the parental cells identified inactivating mutations in the APC and TP53 tumor suppressor genes, and an activating mutation in KRAS.

Conclusion:Colorectal carcinogenesis requires the accumulation of multiple mutations and the derangement of normal cellular metabolism. Our data demonstrate that forced overexpression of CBS can induce a malignant (i.e. invasive) phenotype in a cell line that is normally non-invasive, suggesting that CBS may play a role in adenoma to adenocarcinoma progression during colorectal carcinogenesis.

44.01 Activation of Protein Phosphatase 2A Inhibits Neuroblastoma Tumor Growth

Posted on January 18, 2016

E. F. Garner1,2, A. M. Waters1, L. L. Stafman1, J. E. Stewart1, E. Mroczek-Musulman2, E. A. Beierle1 1University Of Alabama,Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama,Pathology,Birmingham, Alabama, USA

Introduction:
Neuroblastoma is the most common extra-cranial solid tumor in children. Despite advances in therapy, the outcomes for children with advanced-staged disease remain dismal. The role of protein phosphatase 2A (PP2A) as a tumor suppressor has been characterized in other cancers. We hypothesized that increasing tumor suppressor PP2A activity would inhibit tumorigenesis in neuroblastoma cells.

Methods:
Institution approval was obtained from IRB (X111123007) and IACUC (09064). Immunohistochemical staining for the endogenous PP2A inhibitor, I2PP2A, was completed on 25 human neuroblastoma specimens. Slides were scored by a blinded pathologist and a stain score calculated based upon the formula of percent of cells staining positive (0-100%) × intensity of the stain (0-3) resulting in a score from 0-300. Human neuroblastoma cell lines SH-EP and WAC2 were utilized. Activation of PP2A was accomplished with multiple approaches: transient inhibition of the endogenous inhibitor (I2PP2A) with siRNA and stable knockdown with shRNA; non-specific activation with forskolin; direct activation with small molecule FTY720. Migration and invasion assays were performed with Transwell plates. Proliferation was measured using CellTiter96 assay. Stable I2PP2A knockdown clones in the SH-EP and WAC2 cell lines were used for in vivo studies. Empty vector (shEV) control and shI2PP2A cells (2.5 × 106 cells) were injected into the right flank of nude mice (N=5/group) and animals monitored for tumor growth for 2 months. Data reported as median (range) or mean ± SEM and Student’s t-test was used to compare data between groups, with p ≤ 0.05 considered significant.

Results:
I2PP2A staining was present in 22 of the 25 human specimens examined and the median stain score was 140 (0-180), indicating presence of the endogenous PP2A inhibitor. Protein for I2PP2A and PP2A was detected in both neuroblastoma cell lines. Transient inhibition of I2PP2A with siRNA significantly decreased migration and invasion in both cell lines. Further, small molecule activation of PP2A with forskolin or FTY720 significantly decreased proliferation in both cell lines. Finally, stable clones with knock-down of I2PP2A, the endogenous PP2A inhibitor, allowing activation of PP2A, showed significantly decreased tumor growth in both the SH-EP (1211 ± 587 mm3 vs. 347 ± 149 mm3, shEV vs. shI2PP2A, p=0.04) and WAC2 (1714 ± 210 mm3 vs. 1031 ± 139 mm3, shEV vs. shI2PP2A, p=0.01) cell lines.

Conclusion:
The activation of PP2A in neuroblastoma cell lines by inhibition of the endogenous inhibitor (I2PP2A) and direct activation with forskolin or FTY720 significantly inhibited cellular proliferation, migration and invasion. Also, knockdown of the PP2A inhibitor, I2PP2A, led to inhibition of neuroblastoma tumor growth in vivo. These results suggest a role for activation of PP2A tumor suppressor as a novel therapeutic strategy for neuroblastoma.

43.06 TNF-α Stimulates Colonic Myofibroblast Migration via COX-2 and Hsp27

Posted on January 18, 2016

S. Saini1, T. Liu1, L. Chen1, J. Yoo1 1Tufts Medical Center,Colon And Rectal Surgery,Boston, MA, USA

Introduction: Crohn’s disease (CD) is a chronic inflammatory enteropathy characterized by the presence of fibrotic strictures. Myofibroblasts (MFB) are stromal cells of the GI tract that are found in increased numbers in the lamina propria of patients with CD and represent the key effector cells involved in pathologic fibrosis during chronic inflammation. MFB are a known target of TNF-α, a key pro-inflammatory cytokine strongly implicated in the pathophysiology of CD. However, the precise mechanisms through which TNF-α contributes to fibrosis remain incompletely understood. Here, we demonstrate for the first time that TNF-α increases MFB-induced collagen production and MFB migration through the COX-2 and Hsp27 pathways.

Methods: The human colonic MFB cell line 18Co was grown to confluence on 35x10mm cell culture dishes and was used from passages 8-14. An in vitro wound-healing assay was used to assess the effect of TNF-α (12 ng/ml) on MFB migration over a period of 24 hours in the presence or absence of several inhibitors (NS398 (10 μM) and SB203580(10 μM)). Hsp27 siRNA was used to block the expression of Hsp27 in the 18Co cells, and to evaluate the influence of Hsp27 on colonic MFB migration. Antibodies to collagen (COL1A2), pHsp27, and COX-2 were used to evaluate their expression levels using western blotting.

Results:Exposure of 18Co cells to TNF-α increased collagen type I expression in a time-dependent fashion and increased MFB migration over 24 hrs. Incubation of 18Co cells with TNF-α for 4 hours also led to the increased expression of COX-2 and stimulated rapid phosphorylation of Hsp27 at Ser82. TNF-α-mediated expression of COX-2 and phosphorylation of Hsp27 were both inhibited by the P38 MAPK inhibitor SB203580. TNF- α-induced MFB migration was significantly inhibited by SB203580 (p<0.05), as well as NS398 (p<0.05), a direct inhibitor of COX-2. Hsp27 siRNA was effective in blocking the expression of Hsp27, and also inhibited MFB migration.

Conclusion:TNF-α increases collagen production and stimulates cell migration in human colonic myofibroblasts through P38 MAPK-mediated activation of COX-2 and Hsp27. Both COX-2 and Hsp27 appear to regulate myofibroblast cell migration, which may play an important role in mucosal healing in the setting of inflammation. Further elucidating these inflammatory signaling pathways may lead to novel therapeutic targets for the treatment of Crohn’s related fibrosis and strictures.

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