3.11 Ursodeoxycholic acid (UDCA) may protect against intestinal injury in mouse models of peritonitis

Posted on January 18, 2018

M. Nguyen1, O. Escobar1, C. Gayer1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CALIFORNIA, USA

Introduction:
Ursodeoxycholic acid (UDCA) is a secondary bile acid that, unlike many other bile acids, is hydrophilic and has anti-inflammatory properties. We have previously demonstrated that UDCA inhibits intestinal epithelial cell proliferation and stimulates migration in in vitro through an EGFR-dependent mechanism involving downstream COX-2 upregulation. We hypothesize that UDCA will protect the intestinal epithelial barrier in an acute model of intestinal injury via a pathway that requires EGFR, ERK, and COX-2.

Methods:
Wild-type C57BL/6 or Velvet (EGFR dominant-negative) mice underwent intraperitoneal injections of 30 mg/kg lipopolysaccharide (LPS) and oral gavage of 175 mg/kg fluorescein isothiocyanate (FITC)-dextran, with or without 100 mg/kg UDCA . Mice were sacrificed at 16 hours, and small intestine and serum were harvested. Phosphorylated ERK and COX-2 were measured by Western blot and immunofluorescent (IF) staining of intestine.  FITC-dextran levels were measured by fluorimetry of serum.

Results:
Mice treated with UDCA demonstrated decreased villus height loss and serum FITC-dextran levels, as previously reported. Western blot revealed that small intestine mucosal scrapings of these same mice contained increased phosphorylated ERK. This effect was attenuated in mice treated with UDCA + LPS, and further attenuated in mice treated with LPS alone. As expected, small intestine mucosal scrapings of mice treated with LPS alone contained increased COX-2. Conversely, COX-2 was decreased in mice treated with UDCA and further decreased in mice treated with UDCA + LPS, though the differences were very slight. IF staining demonstrated increased COX-2 expression in mice treated with UDCA + LPS, though there was no appreciable difference in COX-2 expression among mice undergoing other treatments.

Conclusion:
These data suggest that UDCA protects the intestinal epithelial barrier in part through an EGFR- and ERK-dependent mechanism. In the context of our previously reported in vitro data showing that UDCA increases intestinal epithelial cell migration through a mechanism dependent on EGFR, COX-2, and ERK, the data presented here further support that UDCA could be used to protect the intestine from acute injury.
 

3.09 Nanofiber Scaffold-Skin Composite for Treatment of Excisional Wounds in Diabetic Rats

Posted on January 18, 2018

J. A. Ungar1, L. Fu2, S. Aravind1, J. Xie3, M. Carlson1  1University Of Nebraska College Of Medicine,College Of Medicine,Omaha, NE, USA 2University of Nebraska Medical Center,Regenerative Medicine,Omaha, NE, USA 3University of Nebraska Medical Center,Surgery – Transplant,Omaha, NE, USA

Introduction: For dermal defects in diabetic subjects, both split thickness skin grafting (STSG) and dermal replacement products are minimally effective in preventing wound contraction. We hypothesized that treatment of full-thickness skin wounds in diabetic rats with a composite of autologous skin islands embedded in a nanofiber scaffold (NFS) would produce less wound contraction compared to standard skin grafting.

Methods: NFS were electrospun from PCL and gas-expanded to produce a 2mm thick microporous matrix arrayed with 1mm wells (skin island placement) spaced 3mm apart. Wistar rats (3mo/300g, N=35) with STZ-induced diabetes underwent dermal wounding (2cm circular excision, two per dorsum) with an encircling ring tattooed around each wound. Subjects were randomized to receive immediate application of a wound treatment: (1) gauze only; (2) meshed STSG; (3) NFS only; (4) 1mm skin islands, or SI, every 3mm; (5) NFS+SI. Gross wound area (WA) and tattoo area (TA) at days 0, 14, 28 and microscopic cross-sectional granulation tissue area (GTA) at days 14, 28 were determined with digital planimetry.

Results: WA and TA on day 0 were not different. At d14, WA in the SI group was less compared to the other groups (Table 1). At both d14 & 28, wounds treated with any NFS had greater GTA compared to other groups. At d28, wounds were 90+% closed, based on WA & GTA. The NFS+SI group demonstrated the least wound contraction at d28, having the greatest TA and the smallest change in WA. However, the NFS+SI group also had the greatest GTA at d28. On d28 H&E histology, the skin islands in the NFS+SI groups were viable, but the NFS was only minimally incorporated into the underlying wound.

Conclusion: Dermal excisional wounds in diabetic rats treated with a NFS-autologous skin composite had less wound contraction compared to other treatments, including STSG. However, the mechanism for this effect may involve physical splinting or some other effect, since the NFS-treated rats only had minimal incorporation of the synthetic construct into the wound, while having greater area of granulation tissue. In order to obtain a dermal replacement which both prevents wound contraction and strongly incorporates into the wound, NFS modification likely will be necessary.

 

3.10 Decreased Inflammation in Scarless Fetal Skin Wound Healing is Regulated by miR-146a

Posted on January 18, 2018

L. C. Dewberry1, M. M. Hodges1, C. Zgheib1, J. Xu1, S. A. Hilton1, J. Hu1, K. W. Liechty1,2  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2Children’s Hospital Colorado,Pediatric Surgery,Aurora, CO, USA

Introduction:
We have previously shown fetal skin wounds heal without scar formation, however, if fetal wound size increases there is a transition from scarless regeneration to scar formation, similar to scar formation seen in the adult.  This transition from scarless regeneration to scar formation was also associated with an increased inflammatory response. Inflammation is regulated, in part, through gene expression of proinflammatory cytokines.  Previous work from our laboratory has demonstrated increased gene expression of these proinflammatory cytokines (IL-6, IL-8). An additional layer of regulation is provided by micrRNAs.  Specifically, miRNA-146a has been shown to act as a “molecular brake” on inflammation by inhibiting proinflammatory gene expression (specifically IL-6 and IL-8).  We hypothesized that the transition from scarless regeneration to scar formation is associated with increased inflammation, increased proinflammatory signaling, and decreased miRNA-146a expression.

Methods:
To test this hypothesis 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep (70-75 days gestation). Wounds were harvested at 3 and 7 days post-wounding, total RNA extracted, and gene expression of miR-146a analyzed with real-time PCR.

Results:
Large wounds were associated with increased inflammation and increased expression of proinflammatory cytokine expression.  In addition, miRNA-146a expression was significantly upregulated in day three small wounds compared to day three large wounds (Figure 1; p=0.03). There was no difference in gene expression between small and large wounds at day 7. 

Conclusion:
We conclude that inflammation and inflammatory signaling plays a role in scarless wound healing. In particular, upregulation of miRNA-146a in the smaller wounds may create an environment conducive to regeneration by decreasing the inflammatory response.   More studies are needed to further elucidate the mechanisms of inflammatory regulation in fetal regeneration.
 

3.07 Intestinal Epithelial Cell-Specific mTORC1 Regulates Food Intake

Posted on January 18, 2018

S. Tay2, J. Guo1, B. W. Warner1  1Washington University,Pediatric Surgery,St. Louis, MO, USA 2Washington University,School Of Medicine,St. Louis, MO, USA

Introduction:
Intestinal epithelial cell (IEC)-specific mammalian target of rapamycin complex 1 (i-mTORC1) regulates IEC cell growth and proliferation. Recently, we found that intestine-specific blockade of i-mTORC1 activity by disrupting Raptor gene expression after small bowel resection in mice led to significant loss of body weight. The purpose of this study was to determine a mechanism for this weight loss.

Methods:
Raptor knockout mice (Villin-CreER(+); Raptor(flox/flox) ) and wild-type littermate mice underwent intraperitoneal injection of tamoxifen to inducibly delete Raptor protein expression selectively in the intestinal epithelium of adult mice. We then placed 8 Raptor knockout and 8 control mice on high fat diet for 5 weeks and measured body composition, food intake, fasting blood glucose, lipid metabolism, and white and brown adipose tissue stores. 

Results:
Compared to control mice, Raptor-deficient mice had 46% less white adipose tissue, 30% lower total cholesterol, and 34% lower direct HDL (T test, P < 0.05). Further, the Raptor-deficient mice had 40% less food intake and gained only 14% of baseline weight over 5 weeks, compared to 40% weight gain in control mice. Interestingly, fecal fat content, fasting blood glucose, and brown adipose tissue were not affected. Our results suggest that the decreased body weight gain following i-mTORC1 inactivation is not due to glucose response or lipid absorption or excretion, instead it is strongly associated with reduced food intake. 

Conclusion:
The finding of reduced food intake due to an intestinal epithelial cell-specific gene disruption is novel. Prior studies have looked at satiety and adiposity signals such as cholecystokinin, insulin, and leptin, but the drastic decrease in food intake coupled with normal glucose response and lipid absorption observed in Raptor knockout mice could not be explained by either of these signals. While gut microbiota has been found to modulate gut-brain interaction and food intake, and we cannot rule out the effects of commensal bacteria, the remarkable suppression of food intake seen in Raptor knockout mice suggests that an internal pathway may underlie such phenomenon. Future studies are planned to investigate this pathway.
 

3.08 MiR-146a-Conjugated Cerium Oxide Nanoparticles Accelerate Diabetic Wound Healing by Promoting Angiogenesis

Posted on January 18, 2018

C. Zgheib1, J. Xu1, M. M. Hodges1, J. Hu1, L. C. Dewberry1, S. A. Hilton1, S. Seal2, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Department Of Surgery, School Of Medicine, University Of Colorado Denver – Anschutz Medical Campus And Colorado Children’s Hospital,Aurora, CO, USA 2Advanced Materials Processing And Analysis Centre, Nanoscience Technology Center (NSTC), Mechanical Materials Aerospace Eng, University Of Central Florida,Orlando, FL, USA

Introduction: Delayed wound healing is one of the most common complications associated with diabetes. These wounds do not heal properly due to chronic inflammation and impaired angiogenesis. We have previously shown that local delivery of the anti-inflammatory miR-146a using cerium oxide nanoparticles carrying miR-146a (CNPs+miR-146a) accelerates diabetic wound healing by promoting an anti-inflammatory M2 macrophage phenotype and reducing the inflammatory response. M2 macrophages have been shown to stimulate angiogenesis through the production of pro-angiogenic growth factors such as VEGF. Thus, we hypothesize that in addition to normalizing the inflammatory response, CNPs+miR-146a enhance diabetic wound healing through the correction of impaired angiogenesis.

Methods: To test this hypothesis, 8 mm full-thickness excisional wounds were created on the dorsal skin of diabetic mice and immediately treated with 10 uM of CNPs+miR-146a or PBS. Digital images of wounds were taken for calculation of wound surface area. Subsets of these wounds were harvested 7 days after wounding and miR-146a, miR-15b, VEGF, and BCL2 gene expression were analyzed.

Results: Our data show that at 7 days post-wounding, CNPs+miR-146a treatment resulted in improvement in wound closure over PBS-treated control wounds in diabetic mice. In addition, CNPs+miR-146a treatment rectified the compromised angiogenesis by increasing the levels of VEGF and BCL2. This is partly due to the downregulation of the levels of miR-15b, an anti-angiogenesis microRNA.

Conclusion: Our findings demonstrate that local delivery of miR-146a via conjugation with CNPs accelerates healing of diabetic wounds by normalizing inflammation and promoting angiogenesis. This CNP-based platform for miR-146a delivery is a promising effective adjunct therapy and warrants further studies prior to future clinical translation.

3.06 Hyperglycemia reduces long non-coding RNA Lethe expression through the ribosomal binding protein HuR

Posted on January 18, 2018

J. Xu1, J. Hu1, C. Zgheib1, M. M. Hodges1, K. W. Liechty1  1University Of Colorado-Anschutz Medical Campus,Department Of Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA

Introduction: Recent studies reveal that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. We have previously shown that the lncRNA Lethe is down-regulated in diabetic wounds and that Lethe is involved in the regulation of Reactive oxygen species (ROS) production in macrophages, indicating a potential role for Lethe in the pathogenesis of diabetic wounds. RNA binding protein HuR stabilizes mRNA by binding to 3’UTR of mRNA and inhibiting microRNA binding. We hypothesize that hyperglycemia reduces Lethe expression by alteration of HuR binding.

Methods: To test our hypothesis, we incubated the murine macrophage cell line RAW264.7 with media containing 5 mM glucose (low glucose), or 25 mM glucose (high glucose) for 24 hours. RNA immunoprecipitation (RIP) was used to analyze HuR binding and Real-time PCR used to quantify relative gene expression.

Results: Lethe was significantly down-regulated in high glucose conditions.  Under low glucose conditions, the level of lethe in the anti-HuR immunoprecipitated lysate was significantly higher than in the IgG control group.  Under high glucose conditions, the level of lethe was not significantly different between the anti-HuR group and IgG control group, and was significantly lower compared to anti-HuR group under low glucose conditions.

Conclusion: These findings demonstrate a novel mechanism in the regulation of the effects of lncRNA Lethe expression, with HuR binding to lncRNA Lethe and hyperglycemia reduces HuR binding to Lethe to de-stabilize Lethe. Furthermore, these results may represent a potential novel therapeutic target to correct the impaired diabetic wound healing response.

 

3.04 The Role of microRNA-21 in the Regulation of Inflammation and Macrophage Polarization

Posted on January 18, 2018

C. E. Liechty1, J. Hu1, C. Zgheib1, K. W. Liechty1, J. Xu1  1University Of Colorado-Anschutz Medical Campus,Department Of Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA

Introduction: The diabetic wound healing impairment has been shown to be multifactorial. A central feature of diabetic wounds is the persistence of chronic inflammation, partly due to the prolonged presence of pro-inflammatory (M1) macrophages in diabetic wounds.  Persistence of the M1 macrophage phenotype, and failure to transition to the regenerative or pro-remodeling (M2) macrophage phenotype plays an indispensable role in diabetic wound impairment; however, the mechanism underlying this relationship remains unclear. Recently, microRNAs have also been shown to provide an additional layer of regulation of gene expression.  In particular, microRNA-21 (miR-21) is essential for inflammatory response. We hypothesized that miR-21 plays a role in regulating inflammation through promoting M1 macrophage polarization and the production of pro-inflammatory cytokines.

Methods: To test our hypothesis, we treated the mouse macrophage cell line RAW264.7 with LPS (10 pg/ml) and (20 ng/ml) for 24 h to generate M1 macrophages, or IL-4 (20 ng/ml) for 24 h to generate M2 macrophages after overnight serum starvation. MiR-21 overexpression was achieved by transfection of miR-21 mimic.  In order to examine the effect of inflammation on miR-21 expression, we treated the mouse macrophage cell line RAW264.7 with LPS; a known inducer of inflammation.  Cells were treated with 1, 10, or 100ng/ml LPS for 6 hours or 100ng/ml for 2, 4, 6 hours.  Real-time PCR analysis to quantify relative gene expression, using GAPDH or U6 as internal control for mRNA or miRNA expression.

Results: M1 polarized macrophages exhibited an upregulation of miR-21, as well as the M1 and pro-inflammatory markers IL-1b, TNFa, iNos, IL-6, and IL-8. Cells exposed to M2 conditions exhibited an upregulation of M2 markers Mrc1 and Arg1, and no significantly change of miR-21.  LPS stimulation of inflammation resulted in upregulation of miR-21, as well as the inflammatory markers TNFa and IL-6 in a dose and time dependent manner.  Overexpression of miR-21 in RAW264.7 macrophage cells resulted in an upregulation of miR-21 and also increased expression of the M1 markers IL-1b, TNFa, iNos, and IL-6.

Conclusion: These findings provide the first evidence that miR-21 is involved in the regulation of inflammation and promoting M1 macrophage polarization. Dysregulation of miR-21 in diabetic wounds may explain the abnormal inflammation and persistent M1 macrophage polarization seen in diabetic wounds, and may represent a potential role of miR-21 as a therapeutic target to counteract the impaired wound healing response in diabetic wounds.

 

3.05 Inflammatory Properties of Ileal Fluid from Patients with IBD

Posted on January 18, 2018

Y. Liu1, F. Kuehn1, R. Vasan1, E. Liu1, F. Adiliaghdam1, E. Samarbafzadeh1, R. Hodin1  1Massachusetts General Hospital,General And GI Surgery,Boston, MA, USA

Introduction:
Luminal contents play a crucial role in the induction and maintenance of intestinal inflammation. Patients with Crohn´s disease benefit from diversion of the fecal stream, with immediate recurrence of inflammation after restoration of intestinal continuity. Furthermore, pouchitis after ileo-anal anastomosis for ulcerative colitis does not occur prior to ostomy closure. These observations support the premise that exposure to factors within the fecal stream is a critical component in inciting phenotypic expression of inflammatory bowel disease (IBD). And, yet, the components within the fecal milieu that play a role in activating the inflammatory pathways still remain unknown. Recent data has demonstrated that levels of the anti-inflammatory mucosal defense factor Intestinal alkaline phosphatase (IAP) are reduced in colon biopsies of patients with IBD. The objectives are to examine the inflammatory properties of ileal fluid from patients with and without IBD. Our central hypothesis is that pro-inflammatory factors in intestinal contents activate inflammatory cascades in the intestinal epithelial lining and that targeting these factors/pathways can present novel therapeutic approaches to treat IBD.

Methods:
Ileal fluid samples from 46 ileostomy patients with and without IBD were collected in the surgical clinic at Massachusetts General Hospital, Boston. IAP Activity was measured using the para-Nitrophenylphosphate (pNPP) assay. The effluent was centrifuged and the supernatant assayed by ELISA for key pro-inflammatory cytokines. Human THP1 macrophages were exposed to the fluid from 23 consecutive patients and assayed for cytokine expression. 

Results: 

Ileal fluid from 46 patients (28 IBD, 18 non-inflammatory controls) with a median age of 58 years (range; 23-94) was collected and assayed. TNF-a levels were significantly higher in ileal fluid of IBD patients than in controls (33.4 ± 62.9pg/ml vs. 9.7 ± 7.6pg/ml; p < 0.05). IAP activity was significantly lower in patients with IBD compared to patients without underlying inflammatory disease (16.9 ± 6.1 U/mg protein vs. 21.0 ± 6.7 U/mg protein; p<0.05). The inflammatory response of THP1 cells exposed to ileal fluid supernatant showed an individual cytokine profile for each patient and did not correlate with the cytokine levels in the original sample or the underlying disease. 

Conclusion:
Analysis of Ileal luminal contents showed significantly higher TNFa levels and lower IAP activity in patients with IBD. The individual inflammatory response profile of each patient could serve as a basis for determining the risk for recurring disease or pouchitis in stoma patients with IBD. 
 

3.02 Intestinal Alkaline Phosphatase Deficiency Confers Susceptibility to T1DM in STZ Mouse Model

Posted on January 18, 2018

R. Vasan1, F. Kuehn1, J. Ramirez1, F. Adiliaghdam1, E. Liu1, Y. Liu1, M. Farber1, R. Pepen1, C. Freguia2, M. Kaleko2, R. A. Hodin1  1Massachusetts General Hospital,General Surgery,Boston, MA, USA 2Synthetic Biologics,Rockville, MD, USA

Introduction:

Intestinal alkaline phosphatase (IAP) maintains intestinal barrier integrity, prevents translocation of bacterially-derived products into the bloodstream, and diminishes low-grade systemic inflammation thought to contribute to diabetes mellitus pathogenesis. We have recently described the expression of IAP within pancreatic islets themselves. We aimed to determine resilience of IAP-KO animals to β-cell damage.

Methods:

A 5-day low-dose 40mg/kg/day, intraperitoneal streptozotocin (STZ) injection protocol was utilized as a model of T1DM β-cell damage. 12-week-old C57BL/6J Wild Type and IAP-KO mice received i.p. STZ or vehicle (4 groups, n=5). Study duration was 5 weeks. Measurements included periodic blood glucose, weekly food intake and weights, and daily water intake. Serum insulin and c-peptide were measured at sacrifice. Pancreatic islets were harvested for histology and insulin staining.

Results:

Blood glucose was significantly increased at 5 weeks in IAP-KO mice injected with STZ compared to their WT counterparts (554.2+/-37.2 vs. 296.4+/-11.6 mg/dL, p<0.01), as was food (47.8 vs. 23.5 g/week, p<0.05) and water intake (14.2 vs. 5.8ml/day, p<0.01). Serum insulin and c-peptide levels were markedly reduced – (21.1 vs. 75.4 µU/ml, p<0.01) and (0.43 vs. 1.22 ng/ml, p<0.01) respectively.

Conclusion:

IAP-KO mice in this study were more susceptible to STZ-induced β-cell damage and developed significantly worse diabetes compared to their WT counterparts. These results suggest that in addition to its well-recognized role in promoting gut barrier function, IAP may also serve a protective function within the pancreatic islets.

3.03 Notch is Protective in Spleen Against Apoptosis During Endotoxemia via TLR4/Myd88/iNOS/TACE Pathway

Posted on January 18, 2018

C. Yang1,2, M. Deng2, M. Scott2, T. Billiar2  1Tsinghua University,Medical School,Beijing, BEIJING, China 2University Of Pittsburg,Pittsburgh, PA, USA

Introduction:
Notch is a highly conserved transmembrane receptor well-known to regulate cell to cell communication as well as cell fate decisions. In recent years, Notch signaling has been implicated in inflammation, yet the regulation and roles of Notch in sepsis are unknown. The sheddase, TNFα-converting enzyme (TACE/ADAM17) participates in cleavage and activation of Notch and we have previously shown that TACE is activated via the TLR4-MyD88-iNOS-NO pathway in hepatocytes during endotoxemia. Thus, we hypothesized that Notch signaling pathway is regulated by TLR4/Myd88/iNOS/NO/TACE pathway in spleen during endotoxemia.

Methods:
To test our hypothesis, WT (C57BL/6), Myd88-/-, TLR4-/-, iNOS-/-mice were injected saline or 5mg/kg LPS i.p, spleen and serum were harvested 8 hours after injection. In vitro, primary mice splenocytes were exposed to LPS and treated with or without iNOS inhibitors and NO donors. Activation of Notch and cell death in splenocytes was assessed.

Results:
The Notch signaling pathway was activated in the spleen and in splenocytes after LPS challenge in a time-dependent manner, indicated by increased cleaved intracellular notch measure as notch intracellular domain (NICD) by western. The NICD levels peaked at 8 hours and 4 hours after LPS in the spleen and in the splenocytes, respectively. Inhibition of Notch activation using DAPT (5 mg/kg), 3 hours prior to LPS injection, resulted in a significantly increased level of cleaved-caspase-3, indicating more apoptosis in the absence of Notch signaling. LPS-induced Notch activation was significantly lower in Myd88-/- mice and  TLR4-/- mice as well as iNOS-/- after LPS challenge and this we associated with increased levels of cleaved-caspase3. Similar results were also obtained in vitro in splenocytes treated with an iNOS inhibitor 1400w (2 uM). Moreover, Notch activation was induced in splenocytes by using SNAP (200 uM) as an NO donor or directly by the cGMP analog 8-pCPT-cGMP(400 uM). Inhibition of TACE with TAPI-II (800 nM) greatly reduced LPS-induced Notch activation in splenocytes and resulting in increased cleaved-caspase3. 

Conclusion:
Our data suggests that Notch signaling pathway is activated during endotoxemia under the regulation of iNOS/NO and TACE and plays a critical role in regulating splenic cell survival and homeostasis.
 

3.01 Serotonin Mediated Neuro-Intestinal Regulation of Immune Development

Posted on January 18, 2018

J. H. Neilson1, K. Brawner1, S. Dees1, A. Chen1, J. Bibb1, C. Martin1  1University Of Alabama At Birmingham,Department Of Surgery,Birmingham, AL, USA

Introduction: Intestinal epithelial barrier function is critical for appropriate immunity and disease protection. Dysregulation of neural hormone serotonin can affect intestinal subsepratibility to inflammation, However the mechanisms regulating these findings are poorly understood. Serotonin is synthesized through the conversion of tryptophan being rate limited by the enzyme tryptophan hydroxylase (TH). Tryptophan is metabolized into a variety of different molecules, some of which are ligands to the aryl-hydrocarbon Receptor (AhR). AhR is important in intestinal immune function. We hypothesized that depleting serotonin by inhibiting TH would limit inflammation and increase the protective barrier of the gut. One mechanism of protection could be through increased AhR stimulation.

Methods:  8-week-old C57/B6 mice were treated for 5 consecutive days with a 150 mg/kg dose of the TH inhibitor 4-Chloro-L-phenylalanine (PCPA). After 5 days, drug efficacy was measured by quantifying serum serotonin levels. Immune function was measured by quantifying fecal IgA by ELISA. To assess barrier function and bacterial translocation, mesenteric lymph node (MLN) samples were homogenized and plated on Schaedler agar in aerobic conditions. Colonies were counted after 3 days of incubation. AhR ligand availability was measured using a cell-based luciferase reporter assay. HCT 116 cells were used that has been stably transfected with the DRE-driven firefly luciferase construct.

Results: Serotonin was depleted in the PCPA treated mice from an average of 7800 ng/mL (n=3) to 4300 ng/mL (n=3) to a significant degree within the intestine (P=0.004). IgA in the stool showed no difference with pretreated mice averaging 36 µg/mL and an average of 37 µg/mL after treatment (n=6, P=0.89). There was a significant decrease in bacterial translocation in treated mice. Treated mice averaged 4.5 colonies per plate (n=11) while controls averaged 1057 colonies per plate (n=6, P=0.013). The AhR luciferase assay also showed a significant increase in AhR activity in the stool showing a light intensity with an average of 2815 (n=3) for control and 5454 (n=3) for treated mice (P=0.013).

Conclusion: Serotonin depletion augments intestinal barrier function resulting in less bacterial translocation by MLN culture, but has no effect on IgA secretion. This could be due to increased AhR activity causing a variety of effects of the intestinal barrier. AhR signaling is critical for intestinal immune development. One mechanism to explain this finding could be differences in immune cell development allowing for decreased bacterial translocation in serotonin depleted mice. Targeted serotonin regulation may be a way to regulate bacterial translocation in patients at risk for infectious intestinal diseases.

2.20 Preclinical Evaluation of Novel Retinoic Acid Derivatives in Neuroblastoma

Posted on January 18, 2018

A. J. Lazenby1, A. P. Williams1, L. L. Stafman1, J. Aye1, V. R. Atigadda2, J. Stewart1, D. D. Muccio4, C. Grubbs3, E. A. Beierle1  2University Of Alabama at Birmingham,Dermatology,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Chemistry,Birmingham, Alabama, USA 5University Of Alabama at Birmingham,Pharmacology And Toxicology,Birmingham, Alabama, USA 6University Of Alabama at Birmingham,Pediatrics,Birmingham, Alabama, USA 1University Of Alabama at Birmingham,Pediatric Surgery,Birmingham, Alabama, USA

Introduction:  Neuroblastoma (NB), a tumor derived from neural crest cells, is the most common extracranial solid tumor in children. 13-cis-retinoic acid (RA) is a differentiating agent currently utilized in therapy for high risk NB, but its use is limited by toxicities related to cholesterol and lipid metabolism. A synthetic rexinoid, 9-cis-UAB30 (UAB30), has been developed which has a favorable toxicity profile, demonstrating no significant toxicities in animal or human studies. Our lab has shown that UAB30 decreased NB cell proliferation and tumor growth in murine NB models. While UAB30 is promising, a reduction in drug dosage would be ideal to decrease the risk of potential side effects. Therefore, the aim of this project was to initiate pre-clinical evaluation of 9 new, synthetic rexinoid compounds (UAB111, UAB113, UAB114, UAB115, UAB116, UAB125, UAB126, 5-Me-UAB30 and 7-Me-UAB30) designed to have greater potency than that of UAB30 or RA. We hypothesized that these compounds would affect NB survival and differentiation in a manner comparable to RA and UAB30.

Methods:  Four NB cell lines were utilized: 2 MYCN non-amplified (SK-N-AS, SH-EP) and 2 MYCN amplified [SK-N-BE(2), WAC2]. The effect of the rexinoids on cell viability and differentiation were evaluated using alamarBlue® assay and assessment of neurite outgrowth, respectively. Cells were treated for 72 hours at increasing concentrations ranging from 0 to 100 µM. Data were compared using Student’s t-test or ANOVA as appropriate and reported as mean ± SEM with p≤0.05 considered statistically significant.

Results: The 9 new rexinoids were tested against compounds previously studied, RA and UAB30, in all four NB cell lines. The SK-N-AS cell line was the most sensitive to the novel compounds, showing significantly decreased viability after treatment with 5 of them (UAB111, UAB113, UAB114, UAB116, 7-Me-UAB30) compared to RA or UAB30 (Figure, upper panel).  The remaining cell lines, SK-N-BE(2), SH-EP and WAC2 demonstrated results similar to each other and had the most significant changes in viability with UAB116 and 7-Me-UAB30 (Figure, lower panel), so these 2 rexinoids were utilized for further studies. Neurite outgrowths are a measure of NB cell differentiation. Treatment with UAB116 or 7-Me-UAB30, resulted in a statistically significant increase in neurite outgrowths in both the SH-EP and SK-N-AS cell lines, indicating differentiation.

Conclusion: Treatment with these synthetic rexinoids, particularly UAB116 and 7-Me-UAB30, decreased NB cell viability significantly more than RA or UAB30. In addition, they led to NB cell differentiation. These data indicate that UAB116 and 7-Me-UAB30 should be further evaluated as potential novel therapeutics for NB.

2.18 Is Gastroschisis A Lymphatic Disease? Study In A Fetal Rabbit Model

Posted on January 18, 2018

F. Scorletti1, M. Oria1, F. Y. Lim1, J. L. Peiro1  1Cincinnati Children’s Hospital Medical Center,The Center For Fetal, Cellular And Molecular Therapy,Cincinnati, OH, USA

Introduction: Gastroschisis (GS) patients have high morbidity secondary to intestinal dysmotility and malabsorption that require parenteral nutrition for a long period.  Main alterations in the extruded bowel include edema and inflammation caused by amniotic fluid contact. Sufficient lymphatic clearance plays a crucial role in clearing the inflammation process, while lymphatic failure is believed to intensify the intestinal edema. Our aim was to investigate the intestinal lymphatic system alteration in a rabbit GS experimental model.

Methods: IACUC#2013-0294. Gastroschisis was surgically created on fetal New-Zealand rabbits at E24 and harvested on day E30 (term=31days). The siblings were collected as controls (n=10 per group). Body and intestinal weights (BW, IW), their ratio (IBR) and dry test were performed. RNA was isolated and pellets were partially re-extracted, precipitated, DNAseI-digested and cleaned. A 1-µg cDNA sample was used to set up RT-qPCR using primers for VEGF-C, VEGFR3 and GAPDH. Histology, immunofluorescence (IF) and western blotting (WB) were assessed to VEGF-C (Vascular Endothelial Growth Factor), VEGFR-3 (receptors), SMA (Smooth Muscle Actin) in GS eviscerated bowel and controls.

Results: IW, IBR and water (in dry test) were significantly increased (p<0.005) in the exposed GS intestine [1.391 (±0.208) versus 1.029 (±0.156); 0.041 (±0.004) versus 0.036 (±0.003); 96.08 (±1.13) versus 80.36 (±6.26), respectively]. VEGF-C and VEGFR3 gene expression were down-regulated in exposed GS especially at early stages respect time matched controls (p<0.05). Whereas, VEGFR-3 and SMA were increased (p<0.05) in GS pups versus controls. VEGFR-3 staining showed a different distribution of lymphatic vessel receptors decreasing on muscular and subserosa layers to be increased on intestinal villi.

Conclusion: Exposed bowel in GS showed more water content suggesting edema. VEGF-3/VEGFR-3 is the primary trophic signal for the proper development and maintenance of the intestinal lymphatic vessels. We hypothesize that these changes in expression and structure of this pathway can be intent to decrease the permeability of lymphatic vessels to canalize the excess of edema. Those disturbances could alter the integrity of the intestinal lymphatic vessel network. The failure to adequately clear lymph from the intestine wall may sustain and intensify intestinal inflammation and edema. Additionally, impaired lymphatic system development may also explain the poor absorption in babies with gastroschisis.

 

2.19 The Role of Intestinal TLR4 in the Development of Small Bowel Resection Associated Metabolic Syndrome

Posted on January 18, 2018

C. M. Courtney1,2, L. K. Barron1,2, E. J. Onufer1,2, J. Guo1, B. W. Warner1,2  1Washington University,Pediatric Surgery,St. Louis, MO, USA 2St Louis Children’s Hospital,Pediatric Surgery,St Louis, MO, USA

Introduction:  Toll-like receptor 4 (TLR4) signaling plays a crucial role in inflammatory cytokine responses and the metabolic consequences of massive small bowel resection (SBR). Our laboratory has pioneered a murine model that demonstrates altered body composition, impaired glucose tolerance, and hepatic steatosis after 50% SBR. Interestingly global TLR-4 knockout mice do not develop this metabolic phenotype after SBR. While the role of hepatic TLR4 has been studied in unoperated mice with similar phenotypes, the role of intestinal epithelial TLR4 is presently unknown. Our aim was to determine if a direct relationship exists between intestinal TLR4 expression and the novel resection associated metabolic phenotype.

Methods:  Wild type (WT) and intestinal TLR4 knock out (iTLR4-KO) mice underwent 50% proximal small bowel resection and were maintained on standard liquid diet for 10 weeks. Weights were obtained weekly. At 10 weeks, mice were fasted overnight and glucose tolerance testing (GTT) was performed. Body composition was analyzed via echo magnetic resonance. At the time of sacrifice, liver was fixed in formalin, embedded paraffin and stained with hematoxylin and eosin. Slides were analyzed at 40x magnification (5 random fields/animal) using NIS Elements V4.3 software. Lipid area was calculated as measured lipid/ total parenchymal area. Body weights and glucose tolerance were compared using 2-way repeated measures ANOVA.  Fasting blood glucose, body composition, and hepatic steatosis were compared using student’s t-test. 

Results: When compared to wild type (WT) mice, iTLR4-KO mice demonstrated similar weight loss and recovery after SBR. Body composition between the groups was also similar at 10 weeks after operation with WT mice demonstrating 15% ± 0.02 fat mass and 84% ± 0.02 lean mass and iTLR4-KO mice with 17% ± 0.002 fat mass and 82% ± 0.002 lean mass (n=4 WT, n=8 iTLR4-KO). When comparing fasting blood glucose (FBG), the groups are not significantly different with WT FBG measuring 121.8 ± 9.077 (n=4) and iTLR4-KO FBG measuring 108.1 ± 5.894 (n=7). After 2mg/g intraperitoneal glucose load, iTLR4-KO glucose tolerance was not significantly different from WT mice (Figure 1A). Additionally, hepatic steatosis was not significantly different between the groups at 10 weeks post operatively (Figure 1B).

Conclusion: Specifically knocking out intestinal TLR 4 does not appear to alter the metabolic phenotype follow SBR. Future studies isolating other locations of TLR4 signaling are needed to help gain an understanding of the link between massive loss of small bowel and the resulting metabolic syndrome. 

 

2.16 Etoposide Loaded Silk Wafers Slow Neuroblastoma Tumor Growth

Posted on January 18, 2018

J. C. Harris3, B. Yavuz1, J. Zeki2, J. Coburn4, N. Ikegaki2, D. Levitin1, D. Kaplan1, B. Chiu2,3  1Tufts University,Biomedical Engineering,Boston, MA, USA 2University Of Illinois Chicago,Pediatric Surgery,Chicago, IL, USA 3Rush University Medical Center,Surgery,Chicago, IL, USA 4Worcester Polytechnic Institute,Biomedical Engineering,Worcester, MA, USA

Introduction: Etoposide is used to treat high-risk neuroblastoma patients. In this study we created sustained release, etoposide-loaded silk wafers to treat murine orthotopic neuroblastoma tumors.  We hypothesized that intra-tumoral implantation of etoposide wafers could decrease tumor growth.

Methods: Silk wafers were made using previously described techniques and loaded with 100 μg etoposide. In vitro testing was performed to evaluate the etoposide release profile from the wafer, and the dose-dependent toxicity was determined using human neuroblastoma KELLY cells. KELLY cells were used to create orthotopic tumor in mice. When tumor volume reached >100mm3 on ultrasound (1) Etoposide 3% uncoated (EtoU-3%), (2) Etoposide 6% uncoated (EtoU-6%), (3) Etoposide 6% glycerin coated (Eto20x-6%) or (4) control wafer was implanted into the tumor. Tumor size was longitudinally measured using ultrasound following implantation. Paraffin-embedded tumor sections were stained with H&E.

Results: Etoposide killed 50% of the KELLY cells in vitro at 1 μg/mL. EtoU-3% released 72% of the loaded drug in 24 hours and 92.6% in 2 days. Drug release from EtoU-6% and Eto20x-6% continued for 30 days and 45 days, respectively. Tumors treated with EtoU-6% took 8.6 ± 1.7 days to reach 500mm3, EtoU-3% took 8.1 ± 1.7 days, 6% control wafers took 5.7 ± 1.0 days, and 3% control wafers 4.4 ± 1.0 days. Significantly slower tumor growth was seen with EtoU-6% and EtoU-3% compared to their respective controls (p=0.01, p=0.02), but no difference between EtoU-6% and EtoU-3% (p=0.63). Tumors treated with EtoU-6% took 8.2 ± 3.0 days to reach 500mm3, Eto20x-6% took 5.9 ± 1.9 days, and control wafers 1.6 ± 0.6 days. EtoU-6% and Eto20x-6% significantly slowed tumor growth compared to control wafer (p=0.01, p=0.02) but no difference in tumor growth between EtoU-6% and Eto20x-6% (p=0.23). H&E staining demonstrated tumor necrosis adjacent to the wafer in tumors implanted with EtoU-3%.

Conclusions: Silk wafers can be loaded with etoposide and used for intra-tumoral treatment of neuroblastoma. All formulations of etoposide-loaded silk wafer slowed tumor growth compared to controls, and the additional silk coating provided extended release. Silk wafer is a versatile implantable drug delivery system for neuroblastoma treatment. 

2.17 Impact of Toll-like Receptor 4 Stimulation on Human Neonatal Neutrophil Transcriptomic Response

Posted on January 18, 2018

S. L. Raymond1, R. B. Hawkins1, T. J. Murphy1, J. C. Rincon1, J. A. Stortz1, M. Lopez2, R. Ungaro1, H. V. Baker2, J. L. Wynn3, L. L. Moldawer1, S. D. Larson1  1University Of Florida College Of Medicine,Department Of Surgery,Gainesville, FL, USA 2University Of Florida College Of Medicine,Department Of Molecular Genetics And Microbiology,Gainesville, FL, USA 3University Of Florida College Of Medicine,Department Of Pediatrics,Gainesville, FL, USA

Introduction: Neonates rely predominantly on the innate immune system to recognize and combat life-threatening bacterial infections. Neutrophils, a key component of the innate immune system, therefore play a crucial role in neonatal survival. In neonatal murine models, toll-like receptor (TLR) agonists have shown promise in improving survival to polymicrobial sepsis by enhancing neutrophil recruitment. To better understand the transcriptomic response in human neonates to TLR 4 stimulation, we measured neutrophil transcriptomics using microfluidic approaches that require less than 0.5 ml of blood.

Methods: Whole blood samples from six young adults (21-45 years old), six term neonates (gestational age >37 weeks), and six preterm neonates (gestational age <37 weeks) were collected and incubated for 2 hours with and without TLR 4 stimulation by lipopolysaccharide (final concentration of 100 ng/ml).  CD66b+ cells were subsequently captured by positive selection on antibody coated microfluidic devices, and genome-wide expression analyses were performed. Ingenuity Pathway Analysis software was utilized to predict biological functions of significant genes. Significant pathways were determined using a –log p-value greater than 1.3 and a z-score greater than 2 or less than -2.

Results: Supervised analysis identified 1517 unique genes whose expression significantly differed between age groups in response to TLR 4 stimulation (p<0.001, FC>1.5). Biological function predictions suggest that both adults and neonates had a significant overexpression of genes involved in immune response of cells, cell-mediated response, and inflammatory response following TLR 4 stimulation. Adults and term neonates also had overexpression of genes associated with antimicrobial and antiviral response. Preterm neonates failed to upregulate these same pathways but had a significant overexpression of genes involved in the recruitment and response of neutrophils which was not observed in adults.

Conclusion: Microfluidic techniques were utilized on whole blood to identify unique neutrophil transcriptomic profiles among different age groups in response to TLR 4 stimulation. These novel data provide important insights into the neonatal neutrophil response.

 

2.14 Generation of Synthetic Intestinal Bioscaffold with Similarities to Native Mammalian Small Intestine

Posted on January 18, 2018

M. R. Ladd1, C. Costello2, B. Johnson1, C. Gosztyla1, A. Werts1, L. Martin1, W. Fulton1, P. Lu1, H. Jia1, E. Banfield1, J. Sung1, S. Wang1, T. Prindle1, Y. Yamaguchi1, C. Sodhi1, J. C. March2, D. J. Hackam1  1Johns Hopkins University School Of Medicine,General Surgery,Baltimore, MD, USA 2Cornell University,Ithaca, NY, USA

Introduction: Short bowel syndrome is a devastating disease with limited treatment options. The development of an artificial intestine offers a potential solution, however, the ability to develop functional small intestine has been limited in part due to scaffolds with inadequate mechanical properties to promote intact intestinal tissue formation. The goal of this study is to develop and evaluate intestinal-like scaffolds that mimic the mechanical properties of native small intestine and thus to cross a technical hurdle in the generation of an artificial intestine.

Methods:  Intestinal-like scaffolds were fabricated from poly(glycerolsebacate) using a serial fabrication technique with laser indentation into agarose gel to create microvilli that mimic the height and width of native intestinal villi. The pore size and crosslinker were varied from the villus portion to the basal portion to further approximate the native bowel. We evaluated the tensile properties of these synthetic scaffolds (n=10, in triplicate) and compared them to porcine intestine from 3-week-old piglets (n=9, in triplicate). The in vitro degradation of the scaffolds in media and media plus digestive enzymes (to mimic the intestinal environment) was characterized by mass loss (n=3 per condition) and scanning electron microscopy. Scaffolds were coated with matrigel and seeded with murine intestinal stem cell cultures (n=3) harvested to evaluate for cell attachment. Young's modulus, a measure of stiffness, was calculated as the slope of the linear portion of each stress-strain curve.

Results: Our novel scaffolds demonstrated projections which approximated villi seen in intestine. The Young’s modulus of the scaffolds was 5.6 MPa vs. 1.03 MPa for small intestine. The ultimate tensile strength and maximum load of the scaffolds were 1.45 MPa and 3.2 N compared to 1.11 MPa and 1.3 N for the small intestine. Strain at failure was higher in the intestine (175% vs. 77%). In vitro degradation studies demonstrated 42% mass loss at 5 weeks yet the villus structures were still present, consistent with that seen in the native mammalian state. When seeded with murine intestinal stem cells, the scaffolds demonstrated good cell attachment by confocal microscopy and scanning electron microscopy (SEM).

Conclusion: We have successfully developed synthetic scaffolds with mechanical properties that approximate those of native piglet small intestine and allow for attachment of stem cells suggesting they may be suitable for tissue engineered small intestine.  

 

2.12 Double Plication for Spring-Mediated In-Continuity Intestinal Lengthening in a Porcine Model

Posted on January 18, 2018

G. Dubrovsky1, N. Huynh2, A. Thomas2, S. Shekherdimian1, J. C. Dunn1,2  1University Of California – Los Angeles,Division Of Pediatric Surgery, Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA 2Stanford University,Division Of Pediatric Surgery, Department Of Surgery, Stanford University School Of Medicine,Palo Alto, CA, USA

Introduction:
Spring-mediated distraction enterogenesis has been shown to increase the length of an intestinal segment in rat and pig models by as much as 3-fold, and therefore has potential as a new therapy for patients with short bowel syndrome. However, this method for intestinal growth has required the spring to be confined within a segment of intestine that has two closed ends and that has been defuctionalized. The goal of this study is to use suture plication to confine a spring within a functional intestinal segment while maintaining the luminal patency of the intestine and allowing for the normal flow of GI contents.

Methods:
Juvenile mini-Yucatan pigs underwent placement of a nitinol spring within a functional segment of jejunum in continuity. A 20 French catheter was passed temporarily, and sutures were used to plicate and narrow the intestinal lumen to the diameter of the catheter around the encapsulated spring on both sides (FIGURE). Pigs were maintained on a liquid diet post-operatively. The intestine was re-examined 3 weeks after spring placement for lengthening and for histological changes.

Results:
All pigs tolerated a liquid diet with appropriate weight gain. In the absence of plication, springs passed through the intestine within a week. Double plication allowed the spring to stay in place within the jejunum for 3 weeks. Compared to uncompressed springs that showed no change in the length of plicated segments, compressed springs caused a nearly 1.5-fold increase in the length of plicated segments. Histology showed a significant increase in both the thickness of the muscularis propria from 230 µm to 450 µm and in the crypt depth from 210 µm to 420 µm in lengthened segments compared to normal segments of intestine.

Conclusion:
Intestinal plication is an effective method to confine endoluminal springs. The confined springs are able to lengthen intestine while allowing for normal GI function and growth of animals, making this a clinically relevant model. We also see histological changes consistent with intestinal growth at the cellular level. This approach may be useful to lengthen intestine in patients with short bowel syndrome.
 

2.13 The Effects of Gestational Psychological Stress on Neonatal Mouse Intestinal Development

Posted on January 18, 2018

J. Shah1,2, S. B. Deas2,3, J. H. Neilson2,3, C. Ren4, T. Jilling4, K. M. Brawner2, C. A. Martin2  1University Of Alabama at Birmingham,Department Of Clinical & Diagnostic Sciences,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Department Of Medicine,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Department Of Pediatrics,Birmingham, Alabama, USA

Introduction: Psychological stress during pregnancy has been shown to cause subsequent harm to the fetus and newborn.  Many studies focus on neurodevelopmental outcomes, but little is known about the effect of gestational stress on intestinal immunity and development. We have shown that psychological stress disrupts immune development, but its effect on intestinal development is not known. The purpose of this study was to determine the effect of psychological stress during pregnancy on the newborn’s intestinal architecture and growth.

Methods:  8-week-old C57BL/6 littermates underwent timed breeding. Pregnant dams were subjected to one hour of daily psychological stress by using a well-established restraint model during days E7-E14 of the gestational period. The distal ileum of 2-week-old offspring of stressed mothers and non-stressed controls was harvested for histological analysis. Slides were blinded and Axiovision Rel. 4.7 software was used to measure villus height and crypt depth. To determine the effect of excess stress hormones on intestinal proliferation, an explant model was used. 2 mm biopsies were taken from wild type non-stressed mice and treated with 100 mM of corticosterone for 24 hours. RNA was isolated and RT-PCR was performed to determine the effect of corticosterone on the intestinal stem cell marker Leucine-rich-repeat-containing G-protein-coupled Receptor 5 (LGR5) and growth factors Epidermal Growth Factor Receptor (EGFR) and Insulin Growth Factor-1 (IGF-1). A non-parametric T test was used to determine any significant differences between the groups. Results were expressed as the mean SEM.

Results: 7 mice were included in each group. The villus height was 126.4  5.6 m for control and 100.4  4.5 m for stress mice, p value <0.05. The crypt depth was 63.9  1.2 m for control and 53.7  2.3 m for the stressed group, p value <0.05. RT-PCR revealed that explants exposed to corticosterone had a 2.1-fold increase in LGR5 compared to controls, p value = 0.04. There was no significant difference in the IGF-1 and EGFR expression between control and treatment groups.

Conclusion: Here we establish that neonatal mice with mothers that were subjected to psychological stress during pregnancy have significantly shorter villi and crypts compared to controls.  In addition, pups from stressed mothers had higher expression levels of the intestinal stem cell marker LGR5, which may suggest a compensatory response to stress.  Future studies will further clarify the temporal relationship between intrauterine stress and intestinal development as well as the mechanisms of how excess stress hormones affect neonatal intestinal development. These findings will aid in determining the effect of gestational psychological stress on intestinal development and stem cell proliferation

2.10 FAK Inhibition Decreases Tumorigenicity in a PDX Model of Primary and Metastatic Wilms Tumor

Posted on January 18, 2018

J. Aye1, S. Mruthyunjayappa1, L. Stafman1, E. Garner1, J. Stewart1, E. Mroczek-Musulman1, K. Yoon1, K. Whelan1, E. Beierle1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction:  

Approximately 12% of patients with Wilms tumor (WT) will have metastatic disease at diagnosis and often have a grave prognosis.  We established a patient-derived xenograft (PDX) of metastatic WT, including a liver metastasis (COA 42), along with its matched isogenic primary renal tumor (COA 25).  Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in the tumorigenesis of pediatric renal tumors.  To date, the role of FAK in metastatic WT has not been investigated.  We hypothesized FAK inhibition would decrease tumorigenicity in this PDX model.

Methods:

Two human WT samples, primary renal tumor, COA 25, and hepatic metastasis, COA 42, were implanted in athymic nude mice.  H&E staining of the patient’s renal primary and liver metastasis and subsequent PDXs, COA 25 and COA 42, was performed.  FAK expression and phosphorylation was detected with immunohistochemical staining (IHC) of patient samples and by immunoblotting of PDX protein lysate.  COA 25 and COA 42 cells were treated with small molecule FAK inhibitors, PF-573228 (PF) and 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15), for 24 hours. Cell viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively.  Cell cycle analysis was performed by flow cytometry.  Results were compared with student’s t-test and p≤0.05 was considered significant.

Results:

H&E staining confirmed COA 25 and COA 42 recapitulated the patient’s primary and metastatic tumor, respectively, and IHC confirmed FAK expression and phosphorylation in these tissues.  Immunoblotting revealed FAK was present and phosphorylated in both PDXs.  FAK inhibition with PF (10 μM) significantly decreased cell viability by 63% and proliferation by 47% in the COA 25 cells (renal primary) compared to untreated cells.  Y15-induced FAK inhibition (10 μM) of COA 25 cells also significantly decreased cell survival by 61% and proliferation by 36%.  Treatment of the COA 42 cells (hepatic metastasis) with PF and Y15 (10 μM) also significantly reduced cell survival by 83% and 68%, respectively, and proliferation by 63% and 72%, respectively.  Flow cytometry revealed G1 cell cycle arrest for both COA 25 and COA 42 treated with PF and Y15 (10 μM) compared to untreated cells (Figure).

Conclusion:

FAK protein was expressed and phosphorylated in primary renal and metastatic WT PDXs.  FAK inhibition with two small molecules led to decreased tumor cell viability and proliferation along with cell cycle arrest in both the primary renal tumor and liver metastasis.  These findings suggest that further exploration of FAK as a therapeutic target for metastatic WT should be undertaken.  

 

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