46.03 Redox Changes Following Tri-Laser Synergy with Mesenchymal Stem Cells (MSC) in Renal Fibrosis

Posted on January 18, 2016

J. Jaramillo1, R. V. Patil1, J. Yu1, A. Bartholomew1 1University Of Illinois At Chicago,Chicago, IL, USA

Introduction:
The critical organ shortage dictates a need for innovative strategies in addressing organ replacement therapies. Strategies to enhance the regenerative potential of dysfunctional kidneys, may delay or reverse organ fibrosis. While mesenchymal stem cell therapy can improve acute kidney injury, it has little effect on fibrosis. Reversal of detrimental redox states in fibrotic tissues may be necessary to fully harness the regenerative potential of these cells and to inhibit myofibroblasts which receive differentiative cues based on cell redox state. We have previously observed low level light laser therapy of 635, 532, and 405nm wavelengths to synergize with MSC in enhancing angiogenesis and reducing apopotosis. We hypothesized the pro-regenerative effects observed would correlate with an improved redox state and tested representatives of various redox pathways.

Methods:

Unilateral ureteral obstruction was used to induce fibrosis; C57BL6 fibrotic kidneys were treated in vivo with either vehicle control, or thrice weekly Low Level Light tri- Laser Therapy (635nm, 532nm, and 405 nm) with or without weekly intravenous MSC treatment for 4 weeks. For analyses, kidneys were explanted, histologically examined for quantity of fibrosis and apoptosis and expression of the following pathways implicated in redox signaling were quantitated using Q-PCR: superoxide generating oxidases, ie, NOX 1 and NOX 4, Cystathionine-β-synthase(CBS) and cystathionine-γ-lyase(CSE), which produce reactive sulfur species critical to normal mitochondrial function, NRF 1 and NRF 2 which encode anti-oxidative enzyme genes through the anti-oxidant response element (ARE), peroxiredoxins, (PRDX) which catalyze the reduction of peroxides, and SIRTUINS and HIF, which are implicated in cell signaling and survival but dependent on NAD+ as key players in the fibrosis sequence.

Results:
Compared to UUO alone, gene expressions of superoxide generators NOX1 and NOX4, respectively, were decreased by 0.72 and 0.8 times in the MSC alone group,and 0.2 and 0.5 times in the tri-laser plus MSC treatment group. Mitigation was observed by elevated PRDX5 expression, 2.5 and 3.5 times in MSC and laser+MSC treatment group, respectively, whereas PRDX1 and 2 remained unaffected. CBS and CSE expression were increased 1.5 and 2 times in MSC and laser+MSC treatment group, respectively. Sirtuins 1 and 5 were 1.5 times increased in MSC alone group. NRF1 and NRF2 decreased 0.5 times with Laser + MSC treatment. MSC or laser+MSC did not alter expression of HIF.

Conclusions:
Beneficial effects of MSC and tri-laser therapy appear to reduce superoxide generating oxidases, NOX1 & 4 and favor maintenance of cellular mitochondrial function via the hydrogen sulfide pathway as evidenced by increased CBS and CSE expression. These data will guide future strategies in optimizing the ideal redox conditions to facilitate in situ kidney regeneration.

46.04 The Effects of Clonidine on Prolonged Bone Marrow Dysfunction after Severe Trauma and Chronic Stress

Posted on January 18, 2016

I. G. Alamo1, K. B. Kannan1, T. J. Loftus1, H. Ramos1, P. A. Efron1, A. Mohr1 1University Of Florida,Trauma,Gainesville, FL, USA

Introduction:
Following severe trauma, critically injured patients develop a persistent injury-associated anemia associated with a prolonged hypercatecholamine state. Propranolol has been previously been shown to improve bone marrow (BM) progenitor cell growth, reduce hematopoietic progenitor cell (HPC) mobilization, and improve anemia. Clonidine, an alpha 2-adrenergic agonist, is sympatholytic and inhibits norepinephrine release. This study sought to investigate the effect of daily clonidine administration on BM dysfunction in a combined lung contusion (LC), hemorrhagic shock (HS), and chronic stress (CS) rodent model.

Methods:
Male Sprague-Dawley rats (n=4-6/group) were subjected to CS, LCHS, and LCHS/CS ± daily clonidine (75ug/kg). Animals underwent two hours of daily restraint stress until the day of sacrifice (day 7). BM cellularity and growth of BM CFU-GEMM, BFU-E and CFU-E colonies were assessed. Peripheral blood was analyzed for hemoglobin (Hgb) and flow cytometry for mobilization of HPCs (CD71+CD117+). Data was presented as mean±SD; *p<0.05 vs. untreated counterpart by t-test.

Results:
Seven days after LCHS alone, BM cellularity and CFU-GEMM, BFU-E, and CFU-E growth remains 10-15% suppressed as compare to naïve (Table). With the addition of clonidine, BM cellularity improved by 19% and BFU-E and CFU-E colony growth was restored to naïve levels. In the more severe and clinically relevant model of LCHS/CS, after seven days there is a 27% suppression of BM cellularity and persistent anemia. Daily administration of clonidine following LCHS/CS resulted in a 26% improvement in BM cellularity and a significant recovery in BM CFU-GEMM, BFU-E, and CFU-E colony growth by 16, 37, and 32% (Table). Furthermore, the use of clonidine following LCHS/CS led to an 85% decrease in HPC mobilization to the peripheral blood and significantly improved Hgb (13.9±0.5* vs. 10.6±0.8 g/dL).

Conclusion:
Following lung contusion, hemorrhagic shock, and chronic stress, the use of clonidine increased BM cellularity, prevented prolonged suppression of BM HPC growth, reduced mobilization of HPC, and improved anemia. Clonidine has been shown to be protective by reducing the sympathetic effects on BM function following trauma and chronic stress. Therefore, reduction of the prolonged hypercatecholamine state following trauma is therapeutic goal that is beneficial for BM function.

45.10 Human Adipose Stromal Cells Increase Survival and Mesenteric Perfusion After Intestinal Ischemia

Posted on January 18, 2016

D. L. Doster1, A. R. Jensen1, E. B. Hunsberger1, M. C. Yoder1, T. A. Markel1 1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction: Intestinal ischemia can quickly escalate to bowel necrosis and perforation. Transplantation of stem cells presents a novel treatment modality for this problem. Recent work suggests that bone marrow-derived stromal cells can protect the intestine from ischemia. However, the low proliferative potential and difficulties in isolating these cells make them difficult to implement on a large scale. Human adipose-derived stromal cells (ASC) have shown greater proliferative potential and are more readily isolated. We hypothesized that: 1) ASCs would increase seven day survival following intestinal ischemia, and 2) improved survival with ASC therapy would be associated with improved post-ischemic mesenteric perfusion.

Methods: Cells were cultured on polystyrene flasks at 37C in 5% CO2 in air. Human ASCs were grown in EGM2 with FBS and human nTERT keratinocytes (differentiated cell control) were cultured in Epilife media with keratinocyte growth factor. Two million cells in 250ul of PBS were used for experimentation. Adult male C57Bl6J mice (8-12 weeks, 20-30g) were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root was identified, and baseline intestinal perfusion was determined using Laser Doppler Imaging. Intestinal I/R was established by temporarily occluding the superior mesenteric artery for 60 minutes with a non-crushing clamp. Following ischemia, the clamp was removed and the intestines reperfused. Immediately prior to abdominal closure, vehicle, ASCs, or keratinocytes were injected into the peritoneum. Animals were allowed to reperfuse for 12 or 24 hours (perfusion studies), or 7 days (survival studies). After these designated times, animals were reanesthetized and a final perfusion scan was obtained. Perfusion data is expressed as a percentage of baseline. Groups were compared with student’s t test and p<0.05 was significant.

Results: Animals administered ASCs following intestinal ischemia had significantly greater 7 day survival compared to vehicle or keratinocytes (ASC: 80% survival, keratinocytes: 20%, vehicle 40%, p<0.05). Additionally, ASCs facilitated better post-ischemic recovery of mesenteric perfusion compared to vehicle or keratinocytes at 24 hours (ASC: 66.0+/-11.8%; PBS: 25.6 +/- 6.1%; keratinocytes: 25.0 +/-11.4%, p<0.05) but not at 12 hours post-ischemia.

Conclusion: Adipose stromal cells attenuate post-ischemic intestinal injury and promote survival by enhancing mesenteric vascular perfusion. Further studies are needed to identify the mechanism that these cells utilize to promote improved perfusion. Understanding these mechanisms may allow for the design of stem cells that promote maximum effect during therapeutic use.

46.01 Cells from In Vivo Models of Heterotopic Ossification Exhibit Increased Osteogenic Properties

Posted on January 18, 2016

J. Drake1, S. Agarwal1, K. Shigemori1, S. Loder1, C. Hwang1, S. Li1, Y. Mishina1, S. Wang1, B. Levi1 1University Of Michigan,Ann Arbor, MI, USA

Introduction: Large burns and high-energy trauma can lead to heterotopic ossification (HO), a process by which pathologic, ectopic bone forms within soft tissue. Management of HO is limited by efficacy of available treatments, difficulty identifying at-risk patients, and high recurrence rates following surgical excision. The cellular and molecular basis of HO is unknown. Here we investigate whether the cells themselves or their environment drive HO formation. We demonstrate that human and mouse cells isolated from sites of HO retain increased osteogenic capacity when cultured outside of an inflammatory environment.

Methods: Human cells were cultured from HO and surrounding normal bone. Mouse cells were obtained from two models including trauma-induced and genetic HO. Cells from the trauma induced model were isolated from the tendon transection site of mice which had undergone a dorsal burn with tendon transection (burn/tenotomy) at 1, 2, and 3 weeks after injury, a model that reliably produces HO. In the genetic HO model (Nfatc1-cre/caACVR1fl/wt) normal and HO-derived osteoblasts were isolated from 1, 2, and 3 week old mice. Osteogenic differentiation was assessed for by alkaline phosphatase production, alizarin red stain for mineral deposition, RNA expression, and protein expression. Cell proliferation was also assessed.

Results: Human HO cells showed increased osteogenic signaling compared to human osteoblasts from non-HO bone (Fig. 1). Cells isolated from the burn/tenotomy mice 2 and 3 weeks after injury demonstrated significantly increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with controls. Similarly, HO-derived cells from our genetic HO model in 2 or 3 week old mice exhibited increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with non-HO osteoblasts from the same mice (Fig. 2). Finally, targeting these cells with inhibitors of smad5 phosphorylation (LDN-193189) decreased osteogenic capacity by alkaline phosphatase and alizarin red quantification (p<0.05), consistent with its effect on HO formation in our trauma model.

Conclusion: In vitro analysis demonstrates significant differences in cellular behavior with regard to proliferation and osteogenic differentiation in HO models when cells are cultured separate from their in vivo environment. This data suggest that changes in cell behavior drive the process of HO as cell characteristics are preserved after they are removed from their environment. Furthermore, the striking differences in these HO-derived cells from normal cells suggests that they may be used for in vitro assays to study potential therapies targeting HO development.

46.02 Combined Effects of Insulin and Exercise on Muscle Function in Severe Burn

Posted on January 18, 2016

M. R. Saeman1, K. DeSpain1, L. A. Baer2, J. Song1, C. E. Wade2, S. E. Wolf1 1University Of Texas Southwestern Medical Center At Dallas,Surgery,Dallas, TEXAS, USA 2University Of Texas Health Science Center At Houston,Surgery,Houston, TEXAS, USA

Introduction: Muscle loss is a consequence of severe burn and critical illness increasing sepsis risk and recovery time. Previously, a rat model of hindlimb unloading after burn found bedrest contributes significantly to atrophy. In this model, we found that exercise increased the function and changed the fiber composition of slow-twitch muscle. We hypothesize that insulin with exercise will further diminish the loss of muscle function in burn with bedrest.

Methods: Twenty-four male Sprague-Dawley rats received a full thickness 40% total body surface area (TBSA) burn and were randomly assigned (n=6) to vehicle without exercise (V/No), insulin (pro zinc 40U daily) without exercise (I/No), vehicle with exercise (V/Ex), or insulin with exercise (I/Ex). All animals were placed in a tail traction system for hindlimb unloading to mimic bed rest immediately following burn. The exercise group was trained to perform twice daily weighted resistance climbing of 1 meter with 5 repetitions. On day 14 in situ isometric forces of the left soleus and plantaris muscles were measured. Fatigue measurement was performed in only the soleus. Statistical analysis was performed with Sigma Plot using Student’s t-test or ANOVA where appropriate.

Results: There was no significant change in animal body mass between treatments. The physiological cross sectional area (PCSA) of the plantaris increased with combined insulin and exercise. The tetanic (Po) and twitch (Pt) muscle functions were significantly elevated in the plantaris of I/Ex. However, there was no change in the tetanic force when normalized to PCSA (Po/CSA). The soleus had significant elevation of Po, Pt, Po/CSA, fatigue maximum, and fatigue minimum in I/Ex. Please refer to the table for specific values and significance.

Conclusion: Insulin and resistance exercise have a positive combined effect on the hindlimb muscle function in this model of critical illness. The plantaris muscle demonstrated increased physiological cross sectional area with increased force suggesting a net increase in muscle fibers. Previously, we found no change in plantaris with exercise only. The soleus demonstrated a change in the specific force of the muscle and fatigue functions indicating a change in the composition of muscle fiber types.

45.07 Targeting the ECM: An Innovative Strategy to Improve Pulmonary Hypertension in CDH.

Posted on January 18, 2016

S. Balaji1, M. Shah3, M. Phillips3, X. Wang1, C. Moles1, P. L. Bollyky2, S. E. Mclean3, S. G. Keswani1 1Baylor College Of Medicine & Texas Children’s Hospital,Pediatric Surgery,Houston, TX, USA 2Stanford Univeristy School Of Medicine,Division Of Infectious Diseases,Palo Alto, CA, USA 3University Of North Carolina,Division Of Pediatric Surgery,Chapel Hill, NC, USA

Introduction:
The management of pulmonary arterial hypertension (PAH) is challenging in patients with congenital diaphragmatic hernia (CDH) and is a significant source of morbidity and mortality in these patients. In CDH, the pulmonary arteries have thick walls due to smooth muscle cell hyperplasia, increased collagen deposition, and marked inflammation, leading to fibrotic remodeling of the perivasculature. Adult models of PAH and fibrosis demonstrate a dysregulation of hyaluronan (HA) metabolism in the pulmonary vasculature, which may contribute to increased inflammatory response. The role of HA has not been examined in neonatal PAH and CDH.

Methods:
The main challenge in studying CDH is the lack of a viable congenital murine model that develops PAH. We are characterizing the phenotype of the Slit3-/- mouse which shows a CDH at birth with a resultant increase in right ventricular pressures (surrogate for pulmonary arterial pressures) and evidence of vascular wall remodeling in the pulmonary arteries that mimics the human phenotype. To determine the dysregulation of HA metabolism in CDH, lungs from Slit3-/- or WT-control mice at 8-12 weeks (n=3-4/group) were harvested. RNA was isolated from frozen lung samples, and hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-2) were measured (RT PCR). HA expression was determined in paraffin-embedded sections (HA-binding protein, staining). Data represented as mean+/-SD; p<0.5 denotes significance; t-test.

Results:
Compared to WT mice, the Slit3-/- mice progressively develop right ventricular remodeling, thickening of the pulmonary arteries, increased collagen, and TGFβ1 expression. Slit3-/- murine lungs expressed a significant (5-fold) increase in HAS1 gene expression (11.11+/-3.34 vs. 2.3+/-1.93, p<0.05) compared to WT-controls. There was no significant difference in the other 2 hyaluronan synthases, HAS2 (0.84+/-0.41 vs. 0.84+/-0.34) or HAS3 (0.69+/-0.24 vs. 1.15+/-0.8). Interestingly, the gene expression of hyaluronidases was also significantly higher in Slit3-/- murine lungs compared to WT-controls, including Hyal1 (3.24+/-0.68 vs. 1.02+/-0.11, p<0.01) and Hyal2 (2.58+/-0.57 vs. 0.84+/-0.15, p<0.01). HA staining demonstrated significantly more and dysregulated HA in the perivasculature in the Slit3-/- murine lungs.

Conclusion:
Using a Slit3-/- murine model, we demonstrated altered HA metabolism in CDH lungs. We also showed an increase in HA expression in the perivasculature in the lungs of Slit3-/- mice. These data provide evidence for a possible role of HA metabolism in the development of PAH in CDH, and HA may represent a novel therapeutic target. Targeting the ECM of the pulmonary vasculature would represent a paradigm shift in treatment of PAH in CDH patients.

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.

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