62.05 Bowel Dysfunction in Hirschsprung Disease is Associated with an Overabundance of Nitrergic Neurons

Posted on January 26, 2017

L. S. Cheng1,2, D. M. Schwartz2, R. Hotta2, H. K. Graham2, A. M. Goldstein2  1University Of California – San Francisco,Surgery,San Francisco, CA, USA 2Massachusetts General Hospital,Pediatric Surgery,Boston, MA, USA

Introduction: Recent studies have suggested that patients with Hirschsprung disease (HD) have abnormal neurotransmitter expression in the ganglionated proximal colon, and that these alterations may cause persistent bowel dysfunction even after pullthrough surgery. We sought to quantify the proportion of nitrergic neurons in the ganglionic colon of HD patients and to relate these findings to functional outcome.

Methods: The proximal resection margin from 17 patients with colonic HD who underwent a pullthrough procedure and colorectal tissue from 4 age-matched control patients were immunohistochemically examined to quantify the proportion of nitric oxide synthase (NOS) expressing neurons. The incidence of constipation, incontinence, and enterocolitis in HD patients was assessed retrospectively and correlated with the proportion of nitrergic neurons. Neuronal subtypes in the ganglionic colon of the Edrnb-/- mouse model of HD were also studied.

Results: Mice with HD had a significantly higher proportion of NOS+ neurons in ganglionic colon than their wild-type littermates (32.0 ± 5.6% vs. 19.8 ± 1.2%, p<0.01). Likewise, patients with HD also had significantly more NOS+ neurons in ganglionic colon than control patients (18.4 ± 4.6% vs. 13.1 ± 1.9%, p<0.01; see Figure). Patients with HD who experienced constipation or enterocolitis postoperatively tended to have a higher proportion of NOS+ neurons (21.4 ± 3.9% vs. 17.1 ± 4.1%, p=0.06). Furthermore, patients with a proportion of NOS+ neurons in the upper quartiles of all HD patients were significantly more likely to have constipation than those in the lower quartiles (75% vs. 14%, p<0.05).

Conclusion: In patients with HD, an overabundance of nitrergic neurons in the proximal resection margin may predict bowel dysfunction following pullthrough surgery.

62.04 ML327 Induces H3K9 Acetylation and Differentiation in Neuroblastoma

Posted on January 26, 2017

E. J. Rellinger2, C. Padmanabhan2, B. T. Craig2, H. An2, J. Qiao2, J. Zhu2, A. Waterson2, C. Lindsley2, D. Beauchamp2, D. H. Chung2  2Vanderbilt University Medical Center,Section Of Surgical Sciences,Nashville, TN, USA

Introduction: Neuroblastoma is the most common extracranial solid tumor in children with significant clinical heterogeneity, ranging from spontaneous regression to overwhelming metastasis. Retinoic acid is utilized as a pro-differentiating agent in the treatment of children with high-risk disease. We have previously demonstrated that ML327 blocks N-MYC expression and established neuroblastoma xenograft growth. Herein, we hypothesized that ML327 exerts its anti-tumorigenic effects in neuroblastoma by inducing differentiation.

Methods: Human neuroblastoma cell line, BE(2)-C (I-type), was used for our study. For all trials, BE(2)-C cells were pre-treated with ML327 for 7 days and subsequently reared in the absence of compound for the experimental duration. We determined sphere-forming capacity using serial limited dilution analysis as a measure of neuroblastoma stemness. Tetrazolium-based assay and adherent colony formation were our surrogates for proliferation, and soft colony agar formation was our measure of anchorage-independent growth. Murine subcutaneous xenografts were performed to evaluate for tumor-initiating capacity.  Gene expression profiling of ML327-treated cells was performed by paired-end RNA sequencing.

Results: Pre-treatment with ML327 decreased adherent colony formation eight-fold (p<0.0001) and induced a four-fold decrease in soft colony agar growth (p<0.0001). In addition, neurosphere formation was completely abrogated with ML327 pretreatment  (p<0.0001). Notably, ML327 pretreatment also blocked the ability of BE(2)-C cells (0 vs. 8/10 tumors; p<0.0001) to develop subcutaneous xenografts (Figs A,B). Prolonged (7d) treatment with ML327 induced transcriptional upregulation of 2,473 genes and repression of 338 genes (log fold change >2). These striking transcriptional changes were associated with early global enhancement of both H3K4me3 and H3K9Ac within 2h of experimental treatment.

Conclusion: ML327 induces global epigenetic changes (H3K4me3 and H3K9Ac) consistent with transcriptional activation in neuroblastoma and inhibits neuroblastoma growth, at least in part, by inducing neuroblastoma differentiation. ML327 may be an effective lead compound for the development of novel therapeutics in patients with high-risk neuroblastomas.

 

62.03 Enhanced serotonin signaling stimulates mucosal growth along the entire small intestine

Posted on January 26, 2017

C. J. Greig1, R. A. Cowles1  1Yale School Of Medicine,Department Of Surgery,New Haven, CT, USA

Introduction:

Intestinal adaptation is regulated by a variety of factors and is thought to be most robust in the ileum. In patients with short bowel syndrome (SBS), resection of the distal intestine is associated with more severe disease and worse outcomes which may be explained by the regional differences in adaptive potential. Enteric serotonin (5-HT) signaling is known to induce mucosal growth in the distal small bowel but its actions at other sites have not been characterized. We hypothesized that enhanced 5-HT signaling would stimulate mucosal growth in all areas of the small intestine, potentially allowing for the development of novel therapies for SBS patients with distal resections.

Methods:

The serotonin reuptake transporter (SERT) inactivates 5-HT. Knockout or pharmacologic inhibition of SERT results in enhanced 5-HT signaling. P-chlorophenylalanine (PCPA) is an inhibitor of 5-HT biosynthesis. With institutional approval, C57Bl/6 wild-type (WT), SERT knockout (SERTKO), and selective serotonin reuptake inhibitor (SSRI)-treated WT mice were used for experiments. Four small bowel segments were harvested from proximal jejunum to distal ileum. Histologic sections were H&E-stained and villus height (VH), crypt depth (CD) and crypt proliferation index (CPI) were measured (n=40 per parameter). Values were compared using Student’s t-test and significance assumed when p≤0.05.

Results:

At baseline, morphometric (VH/CD) and proliferative (CPI) parameters varied from jejunum to ileum. Enhanced 5-HT signaling resulted in significant increases in morphometric and proliferative parameters for nearly every region of the small intestine (see table). Inhibition of 5-HT synthesis with PCPA resulted in reversal of the 5-HT-mediated effects, confirming the role of 5-HT in stimulating mucosal growth.

Conclusion:

Morphometric and proliferative parameters vary throughout the small intestine of WT mice. Enhanced 5-HT signaling resulted in taller villi, deeper crypts and increased crypt proliferation throughout the small bowel. The ability of this model to stimulate mucosal growth from the jejunum to the ileum suggests serotonergic signaling is a viable target for therapies aimed at increasing mucosal surface area in the residual bowel of SBS patients.

62.02 H2S Improves Intestinal Perfusion and Integrity After Ischemia by Nitric Oxide Dependent Pathways

Posted on January 26, 2017

A. Jensen1, N. Drucker1, S. Khaneki1, T. Markel1  1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction: Hydrogen Sulfide (H2S) is an endogenous gasotransmitter that has recently been found to exert beneficial vasodilatory properties. Therefore, hydrogen sulfide may be a novel therapeutic option in the setting of intestinal ischemia and reperfusion injury (I/R). We hypothesized that: 1) H2S would improve post-ischemic mesenteric perfusion and preserve intestinal histological architecture compared to vehicle following intestinal I/R, and 2) the benefits of H2S therapy would be mediated through nitric oxide dependent pathways.

Methods: Adult male C57Bl6J wild type (WT) and eNOS KO (eNOS KO) mice were anesthetized with isoflurane and a midline laparotomy was performed. The intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using Laser Doppler Imaging. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 minutes with a non-crushing vascular clamp. Following ischemia, the clamp was removed and the intestines were allowed to recover. Prior to abdominal closure, 250ul of 200nM sodium hydrosulfide (NaHS; an H2S donor) or 250ul of PBS vehicle was injected into the peritoneum.  Animals were allowed to recover for 24 hours after which time they were reanesthetized and their mesenteric perfusion reassessed. Perfusion was expressed as percentage of baseline. Following perfusion analysis, animals were sacrificed and intestines explanted and preserved in 4% paraformaldehyde. Intestinal segments were paraffin embedded, sectioned, and stained with H&E.  Sections were then graded based on a previously reported histologic injury scale.  Perfusion and histology scores were compared using Mann-Whitney test. P-values less than 0.05 were significant. 

Results:Hydrogen sulfide improved mesenteric perfusion (WT I/R + NaHS 71.0±6.6%, WT I/R + Vehicle 25.6±6.0%, p<0.01 (A)) following intestinal ischemia and reperfusion injury. In the setting of eNOS ablation, there was no improvement in mesenteric perfusion (eNOS KO I/R + 200nM NaHS 24.7±5.4%, eNOS KO I/R + Vehicle 32.5±8.1%, p=0.59, (A)). Histology injury scores were markedly improved in hydrogen sulfide treated WT animals (WT I/R + 200nM NaHS  1.1±0.3 vs. WT I/R + Vehicle 3.5±0.4, p<0.01(B)). Additionally there was no improvement in histology injury scores in the setting of eNOS ablation (eNOS I/R + 200nM NaHS 2.7±0.2 vs. eNOS KO I/R + Vehicle 2.7±0.3, p=0.84(B)). 

Conclusion:The use of hydrogen sulfide following intestinal I/R improves mesenteric perfusion and intestinal histology.  The benefits of H2S therapy appear to be mediated through nitric oxide dependent pathways.  Further studies are needed to define what other downstream mediators may be involved with hydrogen sulfide therapy prior to widespread clinical implementation.   

 

62.01 Evaluating the Efficacy of Different Types of Stem Cells in Preserving Gut Barrier Function in NEC

Posted on January 26, 2017

C. McCulloh1, J. Olson1, Y. Wang1, J. Vu1, S. Gartner1, G. E. Besner1  1Nationwide Children’s Hospital,Columbus, OH, USA

Introduction:
Necrotizing enterocolitis (NEC) is a leading cause of gastrointestinal morbidity and mortality in premature infants.  Central to NEC is the increased permeability of intestinal mucosa, leading to bacterial translocation and fulminant infection.  We have previously shown that administration of stem cells (SC) can reduce the incidence and severity of NEC, however no study has compared the ability of different stem cells to restore mucosal integrity.   Our goal was to investigate the efficacy of four different types of stem cells in preservation of gut barrier function during NEC.

Methods:
The following types of stem cells were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) bone marrow-derived mesenchymal SC (BM-MSC); (3) amniotic fluid-derived neural SC (AF-NSC); and (4) enteric neural SC (E-NSC).  SC lines were derived from Lewis rats, cultured, and verified via flow cytometry.  MSC multipotency was confirmed via adipogenic and osteogenic differentiation.  Sprague Dawley rat pups were delivered prematurely via C-section.  Each pup received an intraperitoneal (IP) injection of 2×106 SC in PBS.  Control pups received the same volume of PBS IP.  Pups were subjected to repeated stress (hypoxia and hypothermia three times daily, hypercaloric gavage feeds every 4h, and a single enteral dose of LPS on day one) to induce experimental NEC.  Control pups were breastfed and not subjected to experimental NEC. After 48h all animals received a single enteral dose of fluorescein isothiocyanante-labeled dextran (FD70, molecular weight 70,000, 750mg/ml) and sacrificed 4h later.  Serum was collected and the concentration of FD70 measured in a fluorescent plate reader with filter cutoffs 492/518nm.

Results:
Compared to breastfed, unstressed pups which had intact gut barrier function and normal intestinal permeability (serum FD70 concentration 2.22 ± 0.271 µg/mL), pups exposed to experimental NEC that received PBS had impaired gut barrier function with significantly increased intestinal permeability (18.6±4.25 µg/mL, p = 0.047) (Figure 1).  Compared to treatment with PBS, pups treated with SC had significantly reduced intestinal permeability: AF-MSC (9.45 ± 1.36 µg/mL, p = 0.017); BM-MSC (6.73 ± 2.74 µg/mL, p = 0.049); AF-NSC (8.052 ± 1.31 µg/mL, p = 0.0496); and E-NSC (6.60 ± 1.46 µg/mL, p = 0.033).  Values are shown as mean ± SEM.

Conclusion:
Stem cells improve gut barrier function in experimental NEC. Although all four types of stem cells reduce permeability equivalently, stem cells derived from amniotic fluid (AF) may be preferable due to the availability of AF at delivery and the ease of culture expansion, enhancing the potential for clinical translation.

61.10 Critical Intestinal Cells Originate From the Host in Enteroid-Derived Tissue Engineered Intestine

Posted on January 26, 2017

B. Cromeens1, Y. Wang1, Y. Liu1, J. Johnson2, G. E. Besner1  1Nationwide Children’s Hospital,Pediatric Surgery,Columbus, OH, USA 2Nanofiber Solutions, Inc.,Columbus, OH, USA

Introduction: Our lab has produced tissue engineered intestine (TEI) in mice from enteroids, a purely epithelial culture system, and identified the presence of intestinal subepithelial myofibroblasts (ISEMF) and smooth muscle cells (SMC) in the TEI produced. As these cell types are not present in the donor enteroids, these cells are either recruited from the host animal or transdifferentiated from donor epithelial lineages. Our current goal was to determine the origin of these cell types.

Methods: Crypts were harvested from pan-EGFP or LGR5-EGFP mice and used to culture enteroids. Enteroids were seeded onto scaffolds and implanted in the abdomen of NOD/SCID mice for four weeks. TEI grown from pan-EGFP enteroids was labeled for smooth muscle α-actin (SMA) to identify ISEMFs and SMCs and green fluorescent protein (GFP) to identify cells from the pan-EGFP enteroids. FISH directed at the Y-chromosome was applied to TEI grown from male LGR5-EGFP enteroids implanted in female NOD/SCID mice to determine which cells originated from the enteroids. To identify potential chemotactic effects of intestinal epithelium on ISEMFs, a Boyden chamber assay was performed. ISEMFs and enteroids from pan-EGFP mice were seeded on opposite sides of a membrane with 8 µm pores. Enteroids were seeded in Matrigel as they are in culture.  Wells with equivalent volume of Matrigel without enteroids and wells with media alone served as controls. Cells that crossed the membrane were stained and quantified from five fields at 20X magnification.

Results: Pan-EGFP enteroids implanted in NOD/SCID mice produced TEI in 6/13 (46%) scaffolds. Immunofluorescent labeling revealed GFP-positive epithelium with surrounding SMA positivity, the latter indicating the presence of ISEMFs and/or SMCs (Panel A). There was no co-localization of SMA and GFP. Male LGR5-EGFP enteroids implanted in female NOD/SCID mice produced TEI in 10/14 (71%) scaffolds. FISH revealed that only the epithelium was positive for the Y-chromosome (Panel B). The chemotaxis assay demonstrated significantly increased ISEMF migration in the presence of enteroids (983±133) compared to either Matrigel alone (357±36) or media alone (339±24) (p≤0.05) (Panel C).

Conclusion: Lack of co-localization of GFP with SMA suggests that ISEMFs and SMCs are derived from the host animal. This was confirmed by FISH which identified only epithelial cells as being male. All other cell types originated from host animals. The mechanism by which these cells are recruited is unknown, however, the chemotaxis assay indicates a direct chemotactic effect of intestinal epithelial cells on ISEMFs. Understanding the biology of these cell types is important as we optimize TEI for translational application.

61.09 Using Small Molecule Screens to Identify New IRF6 Pathways and Mitigate Orofacial Cleft Pathogenesis

Posted on January 26, 2017

E. Li1,2,3, B. Garrity1,2,3, D. Truong1,2,3, K. Mukherjee1,2,3, E. C. Liao1,2,3  1Massachusetts General Hospital,Boston, MA, USA 2Harvard Medical School,Boston, MA, USA 3Harvard Stem Cell Institute,Cambridge, MA, USA

Introduction:

Mutations in the transcription factor IRF6 represent the most common genetic determinant of both syndromic and nonsyndromic cleft lip and/or palate (CL/P). We hypothesized that the IRF6 gene regulatory network contains pharmacological targets that could prevent CL/P in utero, much like the dramatic effect of prenatal folate supplementation on decreasing the incidence of spina bifida.

CRISPR genome editing was used to disrupt irf6 in zebrafish. All mutant embryos displayed an embryonic epithelium (periderm) rupture phenotype, which represents a sensitive platform for high-throughput chemical screening to identify small molecules that could modulate irf6 regulatory pathways. Using our irf6 mutant model, we present a screen of known bioactive compounds for modulation of Irf6 activity in zebrafish, and identification of pathways that could play a role in palate development.

Methods:

Mutant irf6-/- embryos were dispensed 10 embryos/well in 96-well plates and incubated in media containing propidium iodide (renders ruptured embryos fluorescent). The ICCB known-bioactives library consisting of 480 FDA-approved drugs with well-characterized biological targets was screened. Mutant embryos treated with DMSO were used as solvent controls. Wildtype embryos treated with drugs were used as toxicity controls. Timelapse images of the wells were captured by automated brightfield and fluorescence microscopy and analyzed with ImageJ software. Molecular pathways associated with the positive hits were identified through the library index and analyzed using computational modeling programs.

Results:

65 of the 480 small molecules screened reached statistical significance in delaying periderm rupture compared to DMSO-treated controls without causing developmental delays in wildtype embryos. The molecular targets of the small molecule hits were analyzed by Gene Ontology and revealed not only molecular pathways previously known to play crucial roles in palate development such as PDGF and FGF, but also novel pathway connections between IRF6 and retinoic acid, aryl hydrocarbon, and adenosine pathways among others. Furthermore, when these pathways were aberrantly modulated in wildtype zebrafish embryos, craniofacial defects were observed.

Conclusion:

Zebrafish irf6-/- embryos represent a robust platform for high-throughput small molecule screens to identify modulators of IRF6 capable of potentially mitigating cleft pathogenesis. The results identified several critical developmental pathways, some previously reported as essential in palate development, while others are not yet characterized. These novel pathways could represent unexplored regulatory mechanisms of palate development and novel nodes of pharmacological intervention for orofacial clefting.

61.08 IL-10 Regulates the Perivascular Hyaluronan Metabolism to Improve Pulmonary Hypertension in CDH

Posted on January 26, 2017

S. Balaji1, M. Shah2, X. Wang1, M. Phillips2, M. Fahrenholtz1, C. M. Moles1, M. Rae1, S. G. Keswani1, S. E. Mclean2  1Texas Children’s Hospital And Baylor College Of Medicine,Division Of Pediatric Surgery,Houston, TX, USA 2University Of North Carolina At Chapel Hill School Of Medicine,Division Of Pediatric Surgery,Chapel Hill, NC, USA

Introduction: The management of pulmonary arterial hypertension(PAH) associated with congenital diaphragmatic hernia(CDH) is challenging. 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. Slit3-/- mice demonstrate CDH at birth and develop PAH over time with associated alterations in hyaluronan(HA) metabolism in perivascular matrix in the lungs. Inhaled IL-10 decreases or reverses the development of PAH in Slit3-/- mice with CDH. The role of IL-10 in regulating HA in pulmonary artery smooth muscle cells(PASMC) and pulmonary fibroblasts(PFB) has not been examined.

Methods: Primary PASMC and PFB isolated from C57BL/6J wildtype mice were cultured under static and mechanical tension and were treated with murine IL-10(200ng/ml). To determine the effect of IL-10 on regulation of pulmonary perivascular HA in CDH, 2-3m old Slit3-/- mice were treated twice with IL-10 mixed with HA-hydrogel, administered intranasally 7d apart. Lungs (n=3-4/group) were harvested and embedded. RNA was isolated from cell cultures and frozen lung samples, and hyaluronan synthases(HAS1-3) and hyaluronidases(Hyal1-2) were measured (qPCR). HA expression and localization(HA-binding protein) and leucocyte(CD45) and macrophage(CD206) infiltration was determined histologically in paraffin sections. Data represented as mean+/-SD; p<0.05 denotes significance; t-test
 

Results: PASMC expressed increased IL-6(2.8 fold; p<0.05), HAS1(31.4 fold; p<0.005) and MMP9(15.5 fold; p<0.05), but expressed lower collagen 1 and 3(1.75 fold; p<0.05) and MMP2(3.3 fold; p=0.005) under mechanical stress, which may partly explain the dysregulation of the HA in the perivascular matrix in CDH lungs. IL-10 over expression did not alter the HAS1, 2 or 3 expression of the PASMC under normal culture conditions. In PFB, however, IL-10 treatment significantly increased HAS1(2.5 fold; p<0.05) and decreased Hyal1(1.5 fold; p<0.05), suggesting a differential regulation of HA metabolism by IL-10 in the key cell types responsible for perivascular matrix turnover. Slit3-/- mice that developed PAH demonstrated significantly more HA deposition in the perivasculature, but exhibited a less dense, dysregulated matrix structure. Inhaled IL-10 hydrogel treatment for 2wk resulted in a more cohesive and dense distribution of HA. IL-10 treatment also significantly reduced inflammatory response in Slit3-/- murine lungs: CD45 infiltration(3.2 fold decrease; p<0.05) and macrophage infiltration(1.5 fold decerase; p<0.05)

Conclusion: These data provide evidence for a possible role for IL-10 in the regulation of altered HA metabolism, along with its anti-inflammatory role in the attenuation of PAH in CDH. Targeting the ECM of the pulmonary vasculature would represent a paradigm shift in treatment of PAH in CDH patients and the possible development of novel therapeutics.

 

61.07 Placement of Tissue Engineered Small Intestine in Newborn Pig

Posted on January 26, 2017

L. Y. Martin1, M. Ladd1, H. Jia1, C. Sodhi1, A. Werts1, Y. Yamaguchi1, D. Nino1, P. Lu1, Q. Zhou1, J. Sung1, W. Fulton1, T. Prindle1, E. Banfield1, D. Hackam1  1Johns Hopkins University School Of Medicine,Surgery,Baltimore, MD, USA

Introduction:
Short bowel syndrome is a leading cause of morbidity and mortality in children for which treatment options are limited. Several investigators have reported successful techniques for growing intestinal stem cells (“enteroids”) in vitro, and we recently reported successful mucosal growth on a novel scaffold in dogs. We now propose to test the feasibility and functionality of implantation of scaffold with auto-transplanted intestinal stem cells in a porcine model that resembles the human disease in infants.

Methods:
Synthetic scaffolds with a 3D architecture that resembles the human intestine were prepared with a serial fabrication technique, using laser indentation in an agarose gel to create microvilli. On day of life 3, piglets (1500±50g, n=5) underwent laparotomy and small bowel resection (10 cm.) Intestinal stem cells (ISCs) were isolated by mucosal stripping and cellular disaggregation from the resected specimen, and cultured on collagen-coated scaffold with serum at 37oC. Immunohistochemistry was used to confirm growth and differentiation of intestinal tissue on the scaffold. At 8 days of age, 4 piglets underwent laparotomy with auto-transplantation of enteroid-seeded scaffold into the omentum. Four control conditions were utilized to account for variables: no operation (n=1), small bowel resection alone (n=2), small bowel resection and empty scaffold implantation in single operation (n=1), small bowel resection with delayed implantation of empty scaffold on DOL 8 (n=1). Repeat laparotomy was performed at age 26d (n=2) and 31 (n=1), and the scaffold was evaluated grossly for vascularization. H&E and IHC staining were used to evaluate cellular ingrowth and vascularization.

Results:
The operative model was well-tolerated. Weight gain was appropriate with 100-150% increase by 2 weeks. Immunohistochemistry revealed proliferation of the stem cells on the scaffold villi with staining for Ki67, BRDU, and PCNA, and differentiation via staining for Muc2a, SI, Lysozyme, and chromogranin.  After implantion into the piglet omentum, subsequent laparotomy revealed that the scaffold was encapsulated by 15 days, with gross evidence of vascularization as revealed by fluorescein angiography. Confocal microscopic evaluation confirmed the expression of vascular markers PECAM and Endothelin on the scaffold, as well as smooth muscle markers actin and vimentin

Conclusion:
We have demonstrated successful growth intestinal tissue on a scaffold, and establishment of a blood supply in a large animal model resembling the human condition. These findings raise the possibility of tissue engineering for intestinal stem cells in the management of children with short bowel syndrome

 

 

61.06 Feasibility and Scalability of Spring Parameters in Distraction Enterogenesis in a Murine Model

Posted on January 26, 2017

N. Huynh1, J. D. Rouch1, G. Dubrovsky1, A. Scott1, M. Stelzner2, S. Shekherdimian1, J. Dunn1  1University Of California – Los Angeles,Division Of Pediatric Surgery, Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA 2University Of California – Los Angeles,Division Of General Surgery, Department Of Surgery,Los Angeles, CA, USA

Introduction:
Distraction enterogenesis has been investigated as a novel treatment for patients with short bowel syndrome (SBS). Given the variability of intestinal sizes, it is critical to determine safe, translatable spring characteristics in different sized animal models prior to clinical use. Self-expanding nitinol springs have been shown to lengthen intestines in rats and pigs. Here we show spring-mediated intestinal lengthening is scalable and feasible in a murine model.

Methods:
A 10 mm nitinol spring was compressed to 3 mm and placed in a 5 mm intestinal segment isolated from continuity in mice. A non-compressed nitinol spring placed in a 5 mm isolated intestinal segment served as a control. Spring parameters were proportionally extrapolated from previous rodent and porcine spring parameters to accommodate the smaller size of murine intestines. After 1 to 2 weeks, the intestinal segments were examined for size and retrieved for histological analysis.

Results:
Intestinal segments with compressed spring constants between 0.2 to 1.4 N/m lengthened from 5 to 9.3 ± 1.0 mm (Figure), whereas control segments lengthened from 5 to 6.0 ± 0.5 mm (p<0.01). Diameter of both groups increased similarly, 1.5 to 3.1 ± 0.1 mm and 1.5 to 3.1 ± 0.8 mm. Perforation of the isolated intestinal segment was noted in two animals when the spring constant was greater than 2 N/m. Histologically, lengthened segments had increased smooth muscle thickness and crypt depth in comparison to normal small intestine.

Conclusion:
Self-expanding nitinol springs, which exert up to 1.4 N/m, can safely yield 2-fold distraction enterogenesis in length and diameter in a scalable mouse model. Not only does this study derive the safe ranges and translatable spring characteristics in a scalable murine model for patients with SBS, it also demonstrates the feasibility of spring-mediated intestinal lengthening in a mouse, which can be used to study this technique in transgenic mice in the future.
 

61.05 Characterization of Scar Forming Fibroblasts

Posted on January 26, 2017

A. L. Moore1,2, C. D. Marshall2, U. Litzenburger2, E. A. Brett2, L. A. Barnes2, R. C. Ransom2, M. S. Hu2,3, G. Walmsley2, T. Leavitt2,4, H. Y. Chang2, H. P. Lorenz2, M. T. Longaker2  1Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 2Stanford University School Of Medicine,Department Of Surgery,Palo Alto, CA, USA 3University Of Hawaii , John A. Burns School Of Medicine,Department Of Surgery,Honolulu, HI, USA 4Boston University School Of Medicine,Boston, MA, USA

Introduction:

Scarring of the skin poses a major burden to the US healthcare system. While scarring is the expected outcome of tissue damage, it may lead to several negative outcomes including painful contractures, keloids, and hypertrophic scars. Skin wounds in early gestational age heal without a scar, but factors allowing for this scarless phenotype are still unknown. We have shown that in murine dorsal skin, Engrailed positive fibroblasts (EPFs) appear in the dermis at the same time that the transition from a scarless to a scarring phenotype occurs. Additionally, EPFs are responsible for the formation of dermal scar in adult mice. We hypothesize that EPFs are responsible for the transition from scarless to scar-forming phenotype in the late gestational fetus, and that the cells are functionally distinct.

Methods:
Dorsal dermal fibroblasts from En1Cre; R26mTmG mice were isolated at gestational ages E10, E16, E18, P1 and P30. EPFs and ENFs from these time points were sorted using fluorescence-activated cell sorting (FACS) and were analyzed using assay for transposase-accessible chromatin using sequencing (ATAC-seq). This assay compared the accessibility of segments of genomic DNA. 

Results:

Preliminary ATAC-seq data comparing EPFs to ENFs reveals a total of 3008 genes more accessible in EPFs than in ENFs. For example, vimentin and α -SMA are both expressed in EPFs and ENFs, however the accessibility is significantly higher in EPFs. Genetic segments more accessible in EPFs are correlated with fibrosis, mesenchymal tumors, systemic scleroderma, pulmonary fibrosis, and endometriosis.

Additionally, male and female EPFs have several gender specific differences. Males tend to have more open domains in cell and germline differentiation, whereas female mice have a predominance of genes implicated in leukocyte development, as well as diseases associated with a dysfunctional immune system. 

Conclusion:

ATAC-seq analysis of EPFs and ENFs confirms that they are functionally distinct cells, consistent with our prior published data. Additionally, male and female EPFs and ENFs possess different genetic signatures in areas associated with gender specific disorders. This may indicate that fibroblasts function in gender biased ways. Alternatively, the gender differences may demonstrate the plasticity and responsiveness of fibroblasts to external signals such as androgens.

Based on these data, we will perform reciprocal transplantation experiments in which EPFs and ENFs isolated from the dorsal skin of postnatal mice are transplanted into fetal skin and vice versa. These experiments will determine whether EPFs intrinsically carry the ability to form scar, or whether scar-forming behavior depends on local factors. 

61.04 Improving the Efficacy of Liposomal-Mediated Vascular Gene Therapy via Lipid Surface Modifications

Posted on January 26, 2017

R. K. Fisher1, S. I. Mattern-Schain2, M. D. Best2, S. S. Kirkpatrick1, M. B. Freeman1, O. H. Grandas1, D. J. Mountain1  1University Of Tennessee Graduate School Of Medicine,Department Of Surgery,Knoxville, TN, USA 2University Of Tennessee,Department Of Chemistry,Knoxville, TN, USA

Introduction: Molecular gene therapy shows promise in the management of vascular disease, and the development of biocompatible nanocarriers provides alternative non-viral delivery methods with translational potential. Cationic liposomes (CLs) have been shown to be effective nanocarriers for gene therapeutics in vitro. However, in vivo success has been hampered by CL-induced cytotoxicity resulting from their electrostatic interactions with mammalian cell membranes. Recently, neutral liposomes (NLs) formed with natural lipid constituents have been modified with cell-penetrating peptides (CPPs) with the goal of enhancing cellular uptake. Here we aim to establish CPP-modified neutral liposomes (CPP-NLs) as efficient molecular nanocarriers in vascular smooth muscle cells (VSMCs), with reduced cytotoxicity and enhanced siRNA delivery.

Methods: Unmodified NLs, CPP-NLs, and CLs were formed with 30% cholesterol +/- PEGylation for enhanced membrane stability. Using phosphatidylcholine (PC) as the bulk lipid, CPP-NLs were formed by anchoring stearylated-octaarginine (STR-R8) in the liposome bilayer, confirmed by MALDI-TOF. Liposomes were loaded with GAPDH siRNA via ethanol injection, extruded at 100nm, and purified via dialysis. Narrow size distribution at ~100nm and neutral/positive charge was confirmed with dynamic light scattering and electrophoretic mobility, respectively. Encapsulation was measured via RiboGreen assay, and VSMCs were transfected at 100nM or 200nM siRNA. Non-transfected VSMCs served as the control. Live/dead dual stain was used to measure cytotoxicity. Cy3 labeled siRNA was used to quantify liposomal:cell interaction via fluorometry. GAPDH gene silencing was measured by qPCR.

Results:VSMC cytotoxicity was increased by CL-mediated transfection (12±2 and 13±4%; P<0.05 vs. control; A). VSMCs transfected with CLs exhibited higher fluorescence units (FAU; B), but effective GAPDH silencing was not significantly different among any group (P=NS; C). 

Conclusion:NLs and CPP-NLs demonstrated virtually no cytotoxic effect in VSMCs, while CLs were significantly more cytotoxic. While CLs induced greater liposomal:cell interaction, NL and CPP-NL-mediated delivery demonstrated equivalent gene knockdown. The cytotoxic nature of CLs compromises its future as a translatable drug delivery system. Considering the efficacy demonstrated here, NLs and CPP-NLs show promise as alternative non-viral, non-toxic nanocarriers in the delivery of vascular gene therapeutics. Future studies will aim to optimize transfection capacity of CPP-NLs in vascular cells via further lipid/surface modifications, with the goal of developing a molecular drug delivery platform for future in vivo application in vascular injury models of disease. 

 

61.03 Decellularized Intestinal Scaffolds Support Co-culture of Smooth Muscle and Neural Progenitor Cells

Posted on January 26, 2017

D. M. Schwartz1, N. Nagy1, L. S. Cheng1,2, K. Kitano1, H. C. Ott1, A. M. Goldstein1  1Massachusetts General Hospital,Boston, MA, USA 2University Of California – San Francisco,San Francisco, CA, USA

Introduction:
Patients with short bowel syndrome suffer extensive morbidity from reliance on parenteral nutrition. Small bowel transplant is unfortunately complicated by frequent graft failure. Intestinal tissue engineering aims to use patient-specific cells to create bioengineered small bowel. Regenerated intestine must have peristaltic function, and isolated enteric neural progenitor cells and smooth muscle cells are promising sources for creating neo-innervated and contractile bowel. We hypothesized that decellularized intestine, composed of only native extracellular matrix, could function as a permissive environment to support growth and development of these cell types.

Methods:
Intestinal scaffolds were generated from Sprague-Dawley rats using perfusion decellularization of whole jejunal segments. Colonic smooth muscle cells (SMCs) were isolated from 7-week old C57BL/6 mice, passaged in culture, and suspension-seeded onto the serosa of scaffolds (n=6). Neural stem cells were isolated from the intestine of 3-week old Actb-DsRed mice and propagated in culture as floating neurospheres. Secondary neurospheres were transplanted to the submucosa of decellularized scaffolds and cultured for 6 days (n=3). EdU was added as a marker of cell proliferation. To examine cell interactions, neurospheres were transplanted to the submucosa of scaffolds on which SMCs had been cultured for 3 weeks. This SMC and neurosphere co-culture was sustained for an additional week (n=2). All scaffolds were fixed, processed for immunohistochemistry, and analyzed for cell survival, migration, proliferation and maturation.

Results:
Smooth muscle cells transplanted onto decellularized intestinal segments survived in the host environment and organized into a four-cell-thick layer. The recellularized SMCs maintained immunoreactivity to calponin, a marker of differentiated smooth muscle. Enteric neural stem cells transplanted to the scaffolds survived and migrated an average maximal distance of 642 μm without additional stimulus. These cells maintained immunoreactivity to the neural crest lineage marker p75, and formed plexuses containing differentiated neurons, as evidenced by expression of PGP9.5 and NOS. A subpopulation of cells differentiated into enteric glia, expressing S100 and GFAP. Neural stem cells continued to proliferate throughout the culture period. In scaffolds co-cultured with SMC and neurospheres, the neural stem cells migrated along the smooth muscle layer and both cell types maintained their identities while establishing physical contact with each other.

Conclusion:
Decellularized intestinal scaffolds support the growth, differentiation, and proliferation of both neural stem cells and enteric smooth muscle cells in co-culture. These results support the achievability of regenerating an innervated muscularis externa, an essential step towards the ultimate goal of engineering small bowel with peristaltic function on a platform of decellularized extracellular matrix.

61.02 Anti-sFlt-1 Antibody Treatment Prevents Respiratory Disease in Bronchopulmonary Dysplasia Rat Model

Posted on January 26, 2017

B. Wallace1,2, G. Seedorf1, A. Peisl1, S. H. Abman1  1University Of Colorado Denver,Pediatric Heart Lung Center, Department Of Pediatrics,Aurora, CO, USA 2University Of Colorado Denver,Department Of General Surgery,Aurora, CO, USA

Introduction:
Epidemiologic studies have shown the risk for bronchopulmonary dysplasia (BPD) is increased in pregnancies complicated by preeclampsia (PE). However, mechanisms linking PE with the pathogenesis of BPD and therapies for prevention remain unknown. Past clinical studies of PE have consistently reported high levels of soluble fms-like tyrosine kinase 1 (sFlt-1), an endogenous antagonist to vascular endothelial growth factor, in maternal blood and amniotic fluid. Furthermore, cord blood levels of sFlt-1 are increased in preterm neonates who subsequently develop BPD. We have previously shown intra-amniotic (IA) sFlt-1 exposure in infant rats causes sustained abnormalities of lung growth and pulmonary hypertension (PH) that mimic clinical BPD. Therefore, we hypothesize that administration of an anti-sFlt-1 monoclonal antibody (Mab) would prevent the abnormal lung structure and PH in experimental BPD caused by IA sFlt-1 exposure. We further hypothesize that antenatal Mab treatment may be a more effective strategy than postnatal therapy.

Methods:
We studied the effects of anti-sFlt-1 Mab treatment in infant Sprague-Dawley rats after IA sFlt-1 (1 μg/sac) at 20 days gestation (E20; term 22 days). We compared 3 different Mab treatment strategies:  1) IA injection (1.5 μg/sac) at E20 alone; 2) postnatal injections (1 mg/kg) on alternate days up to 2 weeks; and 3) antenatal maternal uterine artery injection (1 mg/kg) at E20 alone. Pups were delivered on E22 by c-section and observed for 14 days after delivery. Pups were then killed and the lungs inflated and fixed with 4% paraformaldehyde for histology. Alveolarization was assessed by radial alveolar counts (RAC) and evidence of PH measured by right ventricular hypertrophy (RVH) as reflected by the weight ratio of right ventricle to left ventricle plus septum.

Results:
Antenatal sFlt-1 reduced RAC by 37% and increased RVH by 64% versus controls (p<0.0001, respectively). Anti-sFlt-1 Mab treatment preserved lung growth as assessed by RAC when compared to sFlt-1 alone for all treatment strategies (Figure 1). Antenatal IA anti-sFlt-1 Mab treatment decreased RVH by 20% compared to sFlt-1 exposed pups (p<0.003), whereas postnatal and maternal anti-sFlt-1 Mab treatment of sFlt-1 exposed pups completely prevented RVH (Figure 1).

Conclusion:
We conclude that anti-sFlt-1 Mab preserves lung growth in infant rats after IA exposure to sFlt-1. Furthermore, whereas antenatal IA treatment effectively protected lung structure, only postnatal and maternal treatment strategies additionally completely prevented PH throughout infancy. We speculate that anti-sFlt-1 Mab therapy may provide a novel strategy for the prevention of BPD in preterm infants who were born from pregnancies complicated by PE.

61.01 THE DEVELOPMENT OF A NANOCOMPOSITE AURICULAR IMPLANT FOR CLINICAL TRANSLATION

Posted on January 26, 2017

M. F. Griffin1,2, D. Kalaskar1, A. Seifalian1, P. Butler1,2  1University College London,Surgery,London, UK, United Kingdom 2Royal Free Hospital,Plastic Surgery,London, , United Kingdom

 

Introduction: Current surgical reconstruction of auricular defects involves harvesting rib cartilage and carving a new cartilaginous framework. However, this ‘gold standard’ technique causes pain, donor site morbidity and is restricted by cartilage availability. The use of alternative synthetic materials is also limited due to the complications relating to poor tissue integration and angiogenesis, leading to extrusion and infection. We aim to meet this clinical challenge by creating a new material for auricular reconstruction using a novel nanocomposite polymer called polyhedral oligomeric silsesquioxane poly(carbonate urea) urethane (POSS-PCU), which has already been successfully taken to the clinical benchside as a replacement trachea, tear duct and vascular by-pass graft.

Method: The chemical, structural and mechanical properties of POSS-PCU has been optimised and characterized to ensure it is suitable for auricular reconstruction. The biocompatibility of POSS-PCU has been tested with several human cell lines and levels of angiogenesis, tissue integration and immune response has been explored using in vivo animal models.

Result: POSS-PCU was found to support cell adhesion, proliferation and extracellular matrix formation of human dermal fibroblasts, endothelial cells, chondrocytes and adipose stem cells, using QT-PCR and immunocytochemistry. The subcutaneous implantation of POSS-PCU in rodent models demonstrated good tissue integration and vessel formation over 3 months without infection and extrusion. 

Conclusion: POSS-PCU has shown to be a promising material for auricular reconstruction. Preclinical testing will be completed in the near future prior to embarking on a UK multicenter clinical trial.

60.09 NF-kB in Stroma Promotes Cancer Growth by Protecting Against Immune Mediated Cytotoxicity

Posted on January 26, 2017

B. Giri1, B. Garg1, S. Modi1, V. Sethi1, S. Ramakrishnan1, S. Banerjee1, A. Saluja1, V. Dudeja1  1University Of Miami,Miami, FL, USA

Introduction: While the role of NFκB in tumor cells in cancer cell growth and metastases is well established, the impact of NFκB in tumor stroma on pancreatic cancer growth is unknown. We sought to understand the effect of depletion of p50 subunit of NFκB in the stromal cells on pancreatic cancer growth and metastases.  

Methods: Pancreatic cancer cells isolated from spontaneous tumors developing in Pdx1-Cre;K-Ras+/LSLG12D;p53R172H/+ (KPC) mice were surgically co-injected with either wild-type (WT) or p50-/- (thus lacking p50 subunit of NFκB) pancreatic stellate cells (PSC). The impact of lack of p50 subunit of NFκB on tumor growth and metastases was measured. This model was repeated in athymic nude mice lacking a functional immune system to assess the effect of p50-/- PSC on tumor cells in the absence of adaptive immunity. The impact of deletion of p50 subunit of NFκB in stroma cells on growth of melanoma (B16-F10) and Lung Cancer (Lewis Lung Cancer) was also evaluated. Immune infiltration in the ex-vivo tumor samples were analyzed by flow cytometry.

Results:Cancer cells co-injected with p50-/- stellate cells formed smaller tumors compared to WT PSC. This was also observed in B16-F10 as well as the Lewis Lung Cancer model. On analysis by flow cytometry, tumors co-injected with p50-/- stellate cells had a higher infiltration of CD8+ cells. Interestingly, the tumor inhibitory effect of p50-/- stellate cells was lost when KPC cells were grown with p50-/- PSC in immuno-deficient athymic nude mice suggesting that stromal cells in the presence of p50 -/- may function to protect cancer cells from an immune mediated attack.

Conclusion:Tumor stroma protects cancer cells from immune-mediated cytotoxicity in NFκB dependent fashion. Developing strategies to downregulate NFκB in tumor stroma may lead to an anti-tumor effect, alone or in combination with immunomodulatory strategies. 

 

60.10 The prognostic value of pseudomyxoma peritonei tumor infiltrating T lymphocytes

Posted on January 26, 2017

C. Boutros1,2, M. Bedra2, J. Emel2, O. Ioffe3, N. Hanna1, N. Espat4, S. Katz4  1University Of Maryland School Of Medicine,Surgery,Baltimore, MD, USA 2University Of Maryland Baltimore Washington Medical Center,Tate Cancer Center,Glen Burnie, MD, USA 3University Of Maryland School Of Medicine,Pathology,Baltimore, MD, USA 4Roger Williams Medical Center,Surgery,Providence, RI, USA

Introduction: Tumor infiltrating lymphocytes (TIL) are independent predictors of survival for numerous metastatic solid tumors. The biologic significance of TIL in pseudomyxoma peritonei (PMP) has yet to be elucidated.

Methods: We accessed our tissue bank for surgical patients after cytoreductive surgery and heated chemotherapy for PMP. Immunohistochemical staining (IHC)for CD3, CD4, CD8 and FOXP3 was correlated with clinicopathologic factors including preoperative CEA and PCI. Markers were correlated with survival using the median cell count as the cutoff point for LOW and HIGH groups.

Results: Of 50 patients, 31 had suitable tissue for IHC analysis. The mean age was 59, preoperative CEA 44.0 and PCI score 17. Median survival was 45 months. CD3, CD4, and CD8 density did not correlate with patient survival. Similarly, preoperative CEA or PCI score alone were not significant predictors of survival. FOXP3, a marker of suppressive regulatory T cells (Treg), was a strong predictor of 5-year survival (82% FOXP3LOW vs 28% FOXP3HI , p=0.001).  FOXP3/CD4 ratio predicted of 30 and 60-month survival (80.5% for LOW vs 31% for HIGH, p=0.038 at 30 months, p=0.004 at 60 months). Similarly, FOXP3/CD8 ratio was found to be a predictor of 60 months survival (77% for LOW, vs 33% for HIGH;p=0.023). Low CD8/CD4 ratio correlated with high preoperative CEA and high PCI score (p=0.01). Patients with the combination of low CEA, low PCI score, and high CD8/CD4 ratio had longer survival times (p=0.01  )

Conclusion: TIL proved to be significant predictors of outcome in this small group of patients. A high number of tumor infiltrating suppressive FOXP3+ Treg predicted shorter survival time, suggesting that immunotherapeutic approaches may benefit PMP-PC patients.

 

60.08 Inhibition of Apelin Receptor Signaling Decreases Cholangiocarcinoma Growth in a Xenograft Model

Posted on January 26, 2017

C. Hall1, L. Ehrlich2, T. Shepperd2, A. O’Brien2, G. Alpini2, S. Glaser2, T. C. Lairmore1  1Scott & White Healthcare,Temple, Texas, USA 2Texas A & M Health Science Center College Of Medicine,Temple, TX, USA

Introduction:
Cholangiocarcinoma (CCA) is a malignancy of the intrahepatic and extrahepatic biliary epithelium that is associated with low five-year survival despite multidisciplinary treatment strategies. Tumor angiogenesis correlates with CCA progression, metastases, and patient survival. The apelin receptor (APLNR), which is activated by the apelin peptide, is a G-protein coupled receptor that has been implicated in the growth and angiogenesis of other malignancies, such as colon, breast, prostate and hepatocellular carcinoma, but has not been studied in CCA. The purpose of this study is to quantify APLNR expression in CCA, characterize the proliferative and angiogenic effects of receptor activation, and determine if inhibition of the APLNR axis can inhibit tumor growth in a murine xenograft model. 

Methods:
In vitro, CCA cell lines (CCLP, HuH-28, HuCCT-1, SG231, TFK-1 and Mz-ChA-1) and benign cholangiocytes (H69) were used to measure the expression of apelin and the APLNR via flow cytometry, ELISA and immunofluorescence. Immunohistochemistry (IHC) and qPCR was used to measure APLNR expression in human CCA tissues. Mz-ChA-1 cells were treated with increasing concentrations of apelin and ML221, an APLNR antagonist. Expression of proliferative (Ki-67 and PCNA) and angiogenic (VEGF-A, ANG1, ANG2) genes were measured via qPCR. Phosphorylation of the ERK1/2 pathway, a known pathway for cholangiocyte proliferation, was measured using flow cytometry and immunoblots. In vivo, Mz-ChA-1 cells were injected into the flanks of NU/NU immunocompromised mice, which were treated with ML221 (150 ?g/kg) via tail vein injection for 4 weeks. 

Results:
APLNR expression and apelin secretion was upregulated in human CCA cells and tissues compared to benign controls. In vitro, treatment of Mz-ChA-1 cells with apelin increased proliferation and angiogenesis via activation of the ERK1/2 pathway in a dose-dependent response, whereas, ML221 inhibited these affects. Treatment of Mz-ChA-1 cells with apelin also increased expression of the apelin gene, suggesting an autocrine/paracrine mechanism of receptor activation. Treatment of CCA tumors in NU/NU mice with ML221 significantly decreased tumor growth in the xenograft model (Figure 1). 

Conclusion:
APLNR is increased in CCA tissues and the autocrine/paracrine effects of APLNR receptor signaling regulate tumor growth and angiogenesis, both in vitro and in vivo. Inhibition of the APLNR axis decreases tumor growth in our xenograft CCA model. Targeting APLNR signaling has the potential to serve as a novel, tumor directed therapy for CCA by inhibiting cell proliferation and angiogenesis.  
 

60.07 Tumor Treating Fields are Effective in Temozolomide Resistant Glioblastoma Cancer Stem Cells

Posted on January 26, 2017

J. K. Strebe5, P. A. Clark5, C. Pasch2, D. A. Demings2,3,4,8, H. I. Robins2,3,6,7,8, J. S. Kuo5  2University Of Wisconsin School Of Medicine And Public Health,Carbone Cancer Center,Madison, WI, USA 3University Of Wisconsin School Of Medicine And Public Health,Division Of Hematology And Oncology, Department Of Medicine,Madison, WI, USA 4University Of Wisconsin School Of Medicine And Public Health,McArdle Laboratory For Cancer Research, Department Of Oncology,Madison, WI, USA 5University Of Wisconsin School Of Medicine And Public Heath,Department Of Neurological Surgery,Madison, WI, USA 6University Of Wisconsin School Of Medicine And Public Health,Department Of Neurology,Madison, WI, USA 7University Of Wisconsin School Of Medicine And Public Health,Department Of Human Oncology,Madison, WI, USA 8William S Middleton Memorial Veterans Hospital,Madison, WI, USA

Introduction:  Tumor Treating Fields (TTFs) are a novel antimitotic, non-invasive, externally applied cancer treatment being developed for use against many human solid cancers. Glioblastoma (GBM) is the most frequently diagnosed adult brain malignancy, with a median survival of less than 2 years after the standard therapeutic regimen of maximal surgery followed by radiation and temozolomide (TMZ) chemotherapy. In a Phase 3 clinical trial (NCT#00916409), addition of TTFs to current therapies significantly increased overall and progression free survival for newly diagnosed GBM. TTFs are FDA-approved for newly diagnosed and recurrent GBM. We report the first study of TTFs alone, or combined with TMZ, against GBM stem-like cells (GSC) expressing different levels of the therapy resistance DNA repair enzyme, O-6-methylguanine DNA methyl-transferase (MGMT), to test the hypothesis that TTF is effective against TMZ-resistant GSC.

Methods:  Effects of TTFs and TMZ were studied in the patient-derived 22 GSC (MGMT-expressing, TMZ resistant) and 33 GSC (non-MGMT-expressing, TMZ sensitive) cell lines with continuous application of in vitro TTFs at varying frequencies to GSC cultures using the Inovitro system (Novocure Ltd). The effects of varying doses or frequencies of TMZ, TTFs, and combined TMZ+TTFs on GSC proliferation and sphere-forming ability were analyzed. 

Results: We have previously determined in vitro that 200 kHz is the optimal TTF frequency to inhibit GSC proliferation, identical to the clinical trial frequency. At 200 kHz, TTFs significantly inhibited proliferation (22 GSC: 61±10.8%; 33 GSC 56±9.5%; p<0.05) and clonogenic tumor sphere formation (22 GSC: 38±2.6%; 33 GSC: 60±7.1%; p<0.05) in both TMZ-resistant and TMZ-sensitive GSC subtypes. In combination, TTFs and TMZ (at IC25, IC50, IC75 concentrations) showed an additive interaction.

Conclusion: This is the first study to directly demonstrate that TTFs can overcome TMZ-resistance in GBM cancer stem cells (GSC), and reports the effects of TTFs on GSC proliferation and clonogenic tumor sphere formation with equivalent effectiveness against both therapy resistant and sensitive GSC subtypes (+/- MGMT expression). The combination of TTFs and TMZ was additive, and supports the hypothesis that TTF acts via a mechanism independent of TMZ-mediated DNA alkylation. Further study of TTFs in additional human cancers and in potential combination with other therapeutics will be needed to optimize TTFs as a new therapy option for human cancer. 

60.06 Brachytherapy with concurrent chemotherapy treatment for anal cancer in transgenic mice

Posted on January 26, 2017

H. Sleiman1, L. Meske1, W. Culberson2, J. Micka2, E. Carchman1  1University Of Wisconsin,General Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Medical Physics,Madison, WI, USA

Introduction: Chemoradiotherapy is the current standard of care for patients with anal cancer1. The 5 year survival rates of patients with localized disease after chemoradiotherapy is approximately 65%, with 1/3 of these patients developing disease recurrence. The survival rates are significantly less in patients with locally advanced or metastatic disease treated with standard of care. Finally, the toxicity associated with chemoradiotherapy regardless of stage, is not insignificant with 30% of the patients requiring colostomy creation due to therapy related side-effects.  Unfortunately there have been no changes in the treatment for anal cancer over the last 4 decades, largely in part to a lack of preclinical models.  This project describes the first mouse preclinical model for anal cancer therapy with treatment responses mirroring those seen in humans.

Methods: K14E6/E7 (expressing HPV-16 oncoproteins in their epithelium) mice were utilized and anuses treated with 7,12-Dimethylbenz[a]anthracene (DMBA) topically until squamous cell carcinoma of the anus developed. The study contained four treatment groups: no-treatment controls (7 mice), radiotherapy alone-2 Gy (5 mice), chemotherapy alone- mitomycin C and 5-flourauracil (3 mice), and chemoradiotherapy (4 mice). The Xoft® Axxent eBx® system, consisting of an x-ray tube within a cone-shaped skin applicator, was used to irradiate anal tumors to a depth of 3mm at a known absorbed dose.  Anal tumors were measured at three dimensions (mm3) on a daily basis until mice died or met criteria to be removed from the study (22-day treatment time period). All mice tumor sizes were normalized to 0 at day 0 and changes in tumor size over time noted. Groups were then compared using one-way ANOVA.

Results:  Treatment Response: Within 16 days of initiating chemoradiotherapy there was noted to be a significant treatment response compared to no-treatment controls (p-value<0.05). This treatment response was noted, to a lesser degree, in mice that recieved chemotherapy alone.  Please refer to graph 1 for average tumor volume changes over time. Survival Times: Mice that received no treatment survived on average 30 days with signficant growth rates of anal tumors. Mice that received radiation alone survived on average 22 days without significant growth rates of anal tumors, but died of complications due to tumors outside of the anus. Mice that received chemotherapy or chemoradiotherapy survived to the 48 day endpoint.

Conclusion:We have generated a HPV mouse model of anal cancer treatment that mimics responses seen in the humans with chemoradiotherapy. This model can be utilized to test new therapies with the hope of improving response rates and decreasing toxicity in anal cancer treatment.

 

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