58.19 Inhibition of Metastasis With Celecoxib in Ewing Sarcoma is Not Dependent on Beta-catenin Signaling

Posted on December 22, 2014

C. Behr1,2,3, A. Hesketh1,2, B. Steinberg1, M. Symons1, S. Soffer1,2,3  1The Feinstein Institute For Medical Research,Manhasset, NY, USA 2Cohen Children’s Medical Center,Division Of Pediatric Surgery,New Hyde Park, NY, USA 3Hofstra North Shore – LIJ School Of Medicine,Hempstead, NY, USA

Introduction:  
Ewing Sarcoma (ES) is an aggressive childhood solid tumor in which 30% of cases are metastatic at presentation, and subsequently carry a poor prognosis. We have previously shown that treatment with the COX-2 inhibitor celecoxib significantly reduces invasion and metastasis of ES cells in a COX-2 independent fashion, suggesting that an off-target of celecoxib is likely responsible for this effect. Celecoxib is known to downregulate beta-catenin independently of COX-2, and beta-catenin upregulation is considered a poor prognostic sign in a host of metastatic and aggressive human cancers. Thus we hypothesized that celecoxib’s antimetastatic effect in ES acts via modulation of the beta-catenin signaling pathway.

Methods:
Human ES cells (SK-NEP1) were transfected with siRNA oligomers targeting beta-catenin. Non-targeting siRNAs were used as controls. Knockdown was verified using western blotting. A portion of the samples were also treated with celecoxib. An in vitro invasion assay was performed using standard Boyden chambers by layering a suspension of ES cells within basement membrane extract. After 72 hours, cells were fixed in the wells and stained with crystal violet. The number of invading cells was assessed using light microscopy. Non-transfected SK-NEP1 cells were additionally treated with celecoxib, and beta-catenin levels and activity were assessed using western blotting, qPCR, and immunocytochemistry.

Results:
Reduction of beta-catenin by at least 75% with two different beta-catenin siRNA oligomers did not reduce the ability of the cells to invade compared to their siRNA negative controls (mean of 161 and 176 cells invaded respectively for each siRNA oligomer versus 153 for the non-targeting siRNA control). As expected from our previous experiments, treatment with celecoxib resulted in decreased invasion and this effect was not dependent on the presence of beta-catenin in the cells. Furthermore, western blotting and qPCR failed to demonstrate any difference in beta-catenin protein and RNA levels within the cells after celecoxib treatment, and immunocytochemistry showed no change in the gross amount of beta-catenin nor in its cellular location compared to untreated tumor cells. 

Conclusion:
Celecoxib’s inhibition of Ewing Sarcoma invasion is independent of beta-catenin signaling. Decreasing beta-catenin levels in SK-NEP1 cells does not affect their ability to invade, ruling out beta-catenin as a potential mediator of celecoxib’s anti-metastatic properties. Another COX-2 independent target is likely responsible for celecoxib’s anti-metastatic effect.
 

58.20 Anti-Cancer Activity of a p53-Derived Peptide in a Colon Cancer Peritoneal Carcinomatosis Model

Posted on December 22, 2014

B. D. Babcock1, M. F. Shaikh1, K. Davitt1, Y. Piazza2, T. Meckmongkol1, V. Purohit1, R. Patel1, M. Estioko1, E. Gleeson1, M. R. Pincus3, W. B. Bowne1  1Drexel University College Of Medicine,Surgery,Philadelphia, Pa, USA 2Drexel University College Of Medicine,Pathology,Philadelphia, Pa, USA 3New York Harbor Healthcare System VAMC,Pathology,New York, NY, USA

Introduction:  PNC-27 is a p53-derived peptide construct from the MDM-2 binding domain that possesses anti-cancer activity while sparing normal untransformed cells. To further elucidate anti-cancer activity and mechanism we tested PNC-27 in a murine colon cancer (MCC) peritoneal carcinomatosis (PC) model.  

Methods:  1 x 104 CT-26 MCC cells and untransformed colonic fibroblasts (CF) were treated with PNC-27 and control peptide. Cellular proliferation (MTT), necrosis (LDH), and apoptosis (caspase-3) were measured.  Quantification of MDM-2 in CT-26 and CF was performed using western blot analysis.  Confocal microscopy localized PNC-27/MDM-2 interaction.  Nu/Nu mice with established PC determined by in-vivo bioluminescent imaging (IVIS-BLI) were treated by intra-peritoneal peptide injection for 14 days.  BLI, tumor volume, and histology were studied.

Results:  PNC-27 showed no elevation of pro-apoptotic proteins but induced rapid (4h) dose-dependent CT-26 specific cellular necrosis with 3-fold increase in LDH release (p<0.001) and 92.3% decrease in proliferation (p<0.001). This observed anti-cancer activity may, in part, be due to increased expression of MDM-2 detected in CT-26 plasma membranes compared to CF. Moreover, confocal microscopy demonstrated specific co-localization of PNC-27 and MDM-2 along CT-26 plasma membrane.  Importantly, tumor volume decreased by nearly 40% in PNC-27 treated mice (versus controls; p = 0.1) which was in accordance to observed reduction in BLI and histologically confirmed tumor specific coagulation necrosis.  No toxicity was observed in PNC-27 treated mice. 

Conclusion:  Our results suggest a mechanism of PNC-27 binding to MDM-2 on the CT-26 plasma membrane, leading to rapid targeted necrosis with a potential role in the treatment of colon cancer PC.

 

59.01 Enhanced Anti-fibrosis Effects of Novel Oridonin Derivative CYD0682 for Hepatic Fibrosis In Vitro

Posted on December 22, 2014

F. J. Bohanon1, X. Wang1, B. M. Graham1, C. Ding2, Y. Ding2, C. Rastellini1, J. Zhou2, R. S. Radhakrishnan1  1University Of Texas Medical Branch,Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Pharmacology And Toxicology,Galveston, TX, USA

Introduction:  Activated hepatic stellate cells (HSCs) are responsible for excess extracellular matrix (ECM) protein deposition in liver fibrosis. Our group reported previously that the natural compound oridonin induces apoptosis, inhibits cell proliferation, as well as down-regulates ECM proteins in activated HSCs. In this study, we investigate the anti-fibrosis effects of oridonin derivative CYD0682 on the activated human and rat HSC cell lines LX-2 and HSC-T6. 

Methods:  Cell proliferation was measured by Alamar Blue assay. Apoptosis was detected by cell death ELISA and staining of Yo-Pro-1/propidium iodide. Cell cycle was determined by flow cytometry. Immunoblot and Immunofluorescence staining were performed for cellular protein expression.

Results: CYD0682 treatment significantly inhibited LX-2 cells proliferation in a dose-and time-dependent manner with an IC50 value of ~0.6 μM for 48 hours, ~10–fold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, 2.5 μM of CYD0682 treatment had no significant effects on proliferation of the human hepatocyte cell line C3A. CYD0682 treatment induced LX-2 cell apoptosis and S-phase cell cycle arrest, and was associated with activation of p53, p21, p16, and cleaved-caspase-3. The myofibroblast marker protein α-smooth muscle actin and major ECM proteins type I collagen (Fig. 1) and fibronectin were markedly suppressed in a time-and dose-dependent fashion by CYD0682. Furthermore, pre-treatment with CYD0682, blocked TGF-β-induced Smad2/3 phosphorylation, nuclear translocation and DNA binding, as well as TGF-β-induced type I collagen and fibronectin production in LX-2 cells. 

Conclusion: In comparison with oridonin, its novel derivative CYD0682 may be a more potent anti-hepatic fibrosis agent.

 

59.02 Luteolin Attenuates Fibrogenesis and Affects the Cell Cycle in Activated Hepatic Stellate Cells

Posted on December 22, 2014

A. J. Kandathiparampil1, X. Wang1, F. J. Bohanon1, C. Rastellini1, J. Zhou2, R. S. Radhakrishnan1  1University Of Texas Medical Branch,Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Pharmacology And Toxicology,Galveston, TX, USA

Introduction:  Luteolin is an important member of the flavonoid family and is present in fruits, vegetables, and plant-derived beverages. Luteolin is known to have anti-tumorigenic, anti-oxidant, and anti-inflammatory properties. A recent study revealed the therapeutic effect of luteolin on a mouse model of liver fibrosis. However, its molecular mechanisms of action remains unclear. In the present study, we investigated the effect of luteolin on activated hepatic stellate cells (HSC), the major source for excessive extracellular matrix (ECM) deposition in hepatic fibrosis.

Methods:  Alamar Blue assay was used for proliferation analysis of human activated HSC cell line LX-2. Cellular proteins were measured by immunoblot and immunofluorescence. Apoptosis was determined with the staining of Yo-Pro-1 and propidium iodide. Cell cycle was assessed by Flow Cytometry.

Results: Luteolin treatment induced LX-2 cells apoptosis and potent growth inhibition in a time-dependent fashion. Luteolin induced G1 and S phases cell cycle arrest, and correlated with increased p53 activity, and decreased expression of cyclinB1, cyclinD1, cyclinE2, CDK2, CDK9, and mini-chromosome maintenance 2 (MCM2). In addition, the HSC activation marker α-smooth muscle actin, as well as major ECM proteins collagen type I and fibronectin were markedly down-regulated by luteolin in a time-and dose-dependent manner (Fig. 1A). TGF-β is a potent profibrogenic cytokine. Our data showed that pretreatment with luteolin blocked TGF-β-induced fibronectin, collagen type I, collagen type III and phospho-Smad2/3 expression (Fig. 1B).

Conclusion: Luteolin treatment inhibited HSC proliferation, suppressed endogenous and TGF-β-induced ECMs expression, while impairing cell cycle and TGF-β signaling in LX-2 cells. Luteolin may be a promising agent for reducing liver fibrosis.

 

59.03 Immunostimulatory Peptide Biomaterials as Scaffolds for Defect Repair

Posted on December 22, 2014

Y. Vigneswaran1, H. Han1, J. Handley1, T. Sun1, J. Collier1  1University Of Chicago,Department Of Surgery,Chicago, IL, USA

Introduction: Peptide nanofibers have received significant interest as scaffolds for tissue repair, wound healing, and regeneration. These self-assembling peptide systems have been exploited for their modularity, ease of synthesis, ability to incorporate multiple signaling components, and structural similarity to native extracellular matrix. Recently, we have observed that these materials can be formulated to raise strong immune responses by incorporating specific T cell and B cell epitopes, yet they elicit no detectable inflammation at sites of delivery.   We sought to determine whether this nanofiber immunogenicity precluded the use of the materials in wound healing contexts, hypothesizing that some antibody responses may be consistent with the performance of the materials in tissue defects.

Methods: In mice, an excisional dermal wound healing model was used to study the effects of active immune responses at the site of healing.  Our worked used the self-assembling peptide Q11 (QQKFQFQFEQQ) and the OT-II antigen from the protein ovalbumin (containing a T cell and B cell epitope). Mice were either immunized with dilute solutions of the epitope-bearing nanofibers or not immunized. After vigorous anti-peptide immune responses were established, full thickness dermal wounds were created.  Immunized mice received the epitope-bearing nanofiber scaffold in the wound (n=6), whereas non-immunized mice received either the nanofiber scaffold (n=6) or phosphate-buffered saline (n=6) in their wounds.  Mice were sacrificed at 7 days and 16 days post wounding.  Wounds were harvested, fixed, paraffin embedded, and examined histologically.

Results: Immunized mice raised high antibody titers against the peptide scaffold.  However, their wound healing did not differ significantly differ from control groups with respect to epithelial gap or granulation tissue formation at day 7 (p=0.95, p=0.25) or day 16 (p=0.72, p=0.62), even when additional immunostimulating peptide nanofibers were placed in the wound.  Additionally, immunohistochemistry confirmed the presence of leukocytes at the site of the wound and an active immune response.

Conclusion: We conclude that the phenotype of the immune response raised by peptide nanofibers is compatible with using such materials within healing defects.  This finding is consistent with previous studies from our group showing that even vigorous antibody responses raised by the materials are not associated with any measurable inflammation at their sites of delivery, but it runs counter to the generally held view to avoid anti-biomaterial immune responses in tissue engineering applications. Further basic investigation may help clarify whether the non-inflammatory character of these materials exerts itself via a passive or active mechanism.

59.04 Activation of HIF by small molecule inhibitors of PHD2 accelerates wound healing in vivo

Posted on December 22, 2014

M. S. Hu1,2, W. Hong1, M. Xie3, S. Tang3, R. Rennert1, G. Walmsley1, Z. Maan1, A. Zimmermann1, G. Gurtner1, A. Giaccia4, H. P. Lorenz1, S. Ding3, M. Longaker1  1Stanford University,Surgery,Palo Alto, CA, USA 2University Of Hawaii,Surgery,Honolulu, HI, USA 3University Of California – San Francisco,Gladstone Institutes,San Francisco, CA, USA 4Stanford University,Radiation Oncology,Palo Alto, CA, USA

Introduction:
Impaired wound healing, particularly in diabetic and vasculopathic patients, represents a significant clinical challenge. Prior studies have revealed that an important prognostic determinant of wound repair is the presence of hypoxia. Hypoxia-inducible factor (HIF), master regulator of cellular response to hypoxia, is critical for enhancing the appropriate inflammatory and angiogenic responses that promote wound healing. Herein, we examine the effect of small molecule activators of the HIF pathway on wound healing.

Methods:
We generated 25 small molecule analogue inhibitors of PHD2, designated GPHD-1 through GPHD-25. A high throughput HRE-luciferase assay was performed on NIH 3T3 fibroblasts on 96-well plates to identify GPHD compounds that would achieve the greatest increase in the HIF pathway. The best three compounds were tested in vivo using a murine model of wound healing with splinted 6 mm full thickness excisional wounds. The compound was delivered every other day at a concentration of 10 uM. Photographs were taken every other day and the rate of wound healing was analyzed.

Results:
Using the HRE-luciferase assay, we identified compounds GPHD-11, -14, and -15 for upregulating HIF activity 4.01-, 4.38-, and 4.08-fold, respectively (*p<0.05). Full thickness excisional wounds treated with GPHD-11, -14, and -15 completely healed on days 11.5, 11.4, and 11.8, respectively. Wounds treated with saline control healed on day 13.4 (*p<0.05).

Conclusion:
Our results validate the ability of small molecule analogue inhibitors of PHD2 to activate the HIF pathway in vitro. In vivo, we demonstrate accelerated wound healing with topical application of our compounds. With further studies, the small molecule activators of HIF may prove to be a novel therapeutic to stimulate wound healing.
 

59.06 Bile Acid-Induced Modulation of Intestinal Cell Proliferation is Regulated by FXR and EGFR Signaling.

Posted on December 22, 2014

A. Y. Dossa1, A. Roberts1, H. R. Ford1, M. R. Frey1, C. P. Gayer1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction: Intestinal bacteria can convert taurine-conjugated cholic acid (TCA), a primary bile acid, into the secondary bile acid deoxycholic acid (DCA). Previous work suggests that TCA stimulates cell proliferation while DCA inhibits it. Bile acids can interact with the epidermal growth factor receptor (EGFR) and the farnesoid-X receptor (FXR), which may explain the differential effects of bile acids on cell proliferation. EGFR may promote proliferation through a mechanism involving Src and downstream modulation of the extracellular signal-regulated kinase (ERK), while FXR may inhibit proliferation. Therefore, we hypothesize that TCA and DCA differentially regulate intestinal epithelial cell proliferation by activation of EGFR and FXR, respectively.

Methods: Cell proliferation was measured using nucleic acid incorporation (EdU). Rat intestinal epithelial cells (IEC-6) were treated with TCA with or without the Src kinase inhibitor PP2, the EGFR inhibitor AG1478, the ERK inhibitor PD98059, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or the Akt inhibitor IV. The effect of EGFR inactivation was assessed in EGFR-null mouse colon epithelial cells (EGFR-/- MCE) transfected with an empty vector, wild-type human EGFR or kinase inactive EGFR. FXR was either blocked pharmacologically (z-guggulsterone) or decreased with FXR-specific siRNA knockdown prior to treatment with DCA. Src, EGFR and ERK 1/2 phosphorylation was assessed by immunoblotting.

Results: TCA induced intestinal epithelial cell proliferation in a dose-dependent manner, while DCA led to a decrease in proliferation. TCA-induced stimulation was blocked with Src, EGFR or ERK inhibition. Interestingly, blockade of PI3K or Akt had no effect on TCA-induced cell proliferation. TCA treatment of EGFR-/- MCE cells expressing wild-type human EGFR resulted in increased proliferation (48.6% ± 14.7 above baseline). However, EGFR-/- MCE cells expressing an empty vector or kinase inactive EGFR did not respond to TCA administration. TCA treatment resulted in Src, EGFR and ERK 1/2 phosphorylation in a time-dependent fashion. Pre-treatment with EGFR inhibitor abolished TCA-induced phosphorylation of ERK. In DCA-treated cells, pharmacologic inactivation and protein knockdown by siRNA specific to FXR abolished DCA-induced inhibition of proliferation.

Conclusions: These results suggest that TCA-induced intestinal cell proliferation requires EGFR, Src and ERK activation, independent of the PI3K/Akt pathway. This pathway may involve first Src activation with subsequent transactivation of EGFR. In contrast, DCA inhibits proliferation, possibly via FXR activation and inactivation of the Src/EGFR/ERK pathway. Since elevated secondary bile acids levels are the results of specific bacterial modification, this may provide a mechanism by which an altered microbiome contributes to intestinal diseases.

59.07 Reducing Adult Scar Formation Through Targeted Inhibition of an Embryonic Fibroblast Lineage

Posted on December 22, 2014

G. G. Walmsley1,2, Y. Rinkevich2, M. S. Hu1, Z. N. Maan1, J. M. Tsai2, D. Duscher1, E. R. Zielins1, D. D. Lo1, G. C. Gurtner1, H. P. Lorenz1, I. L. Weissman2, M. T. Longaker1,2  1Stanford University School Of Medicine,Department Of Surgery, Division Of Plastic & Reconstructive Surgery,Stanford, CA, USA 2Stanford University School Of Medicine,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA

Introduction:
Wound healing involves a complex series of molecular and cellular events that culminates in fibrosis and scarring. However, wounds heal without a scar during early fetal development in many mammals. Prior studies have offered possible explanations for this phenomenon, but the critical events that trigger the transition from scarless regeneration to fibrotic repair during embryogenesis remain poorly characterized. We have previously identified a fibroblast lineage using Engrailed-1Cre (En1Cre); R26mTmG transgenic mice, prospectively isolated this lineage using surface markers (CD26), and found that this lineage is responsible for depositing the bulk of connective tissue during dermal development, wound healing, radiation fibrosis, and cancer stroma formation in the dorsal skin.

Methods:
An established model of scarless fetal wound healing allowed for investigation of the role of En1-derived cells in the transition from scarless to fibrotic healing during embryonic development from E16.5 to E18.5. RNA sequencing analysis of E16.5 and E18.5 En1-derived fetal fibroblasts was performed to identify cell intrinsic changes in the En1 fibroblast lineage that may explain the transition from scarless healing to fibrotic repair. En1Cre; R26mTmG mice were crossed to R26tm1(HBEGF)Awai mice resulting in triple positive offspring that both traced En1-derived fibroblasts and rendered them susceptible to ablation with diphtheria toxin. Finally, a small molecule was used to inhibit CD26 enzymatic activity on the surface of En1-derived fibroblasts during wound healing in adult wild type mice.

Results:
A fetal model of scarless wound healing revealed that En1-derived fibroblasts are responsible for the bulk of scar deposition in wounds made at E18.5. RNA sequencing analysis of E16.5 vs. E18.5 En1-derived fibroblasts revealed activation of a ‘fibrotic program’ in E18.5 fibroblasts that may explain the transition from scarless healing to scarring at this developmental stage. DTR-based ablation of En1-derived fibroblasts during adult wound healing lead to a slower rate of healing and a significant reduction in connective tissue deposition by En1-derived fibroblasts. Small molecule-based inhibition of CD26 also lead to a slower rate of wound healing, a reduction in gross scar size, and reduced connective tissue deposition.

Conclusion:
Here we demonstrate that the identification and characterization of a scar-forming lineage of fibroblasts allows for therapeutic modulation of wound healing. Furthermore, these results demonstrate that deciphering the mechanisms of fetal scarless wound healing holds value for developing anti-scarring therapies in adults.

59.08 Bile composition affects diclofenac induced anastomotic leak rates in rats

Posted on December 22, 2014

S. Yauw1, R. Lomme1, H. Van Goor1  1Radboud University Medical Center,Department of Surgery,Nijmegen, Gelderland, Netherlands

Introduction:
Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with anastomotic leakage in humans and animals, but pathophysiological mechanisms are unknown. Diclofenac causes leakage of anastomoses in the ileum and proximal colon, but not in the distal colon of the rat, suggesting a role for the ileal and proximal colonic content in NSAID induced leakage. Other studies suggest an increase of bile toxicity by specific NSAIDs, causing small intestinal damage. In this experiment we determined if changes in bile composition are relevant in diclofenac induced leakage.

Methods:
An anastomosis was constructed in the ileum of four groups of male Wistar rats in which all bile was diverted and replaced either by ‘control bile’(B-) or ‘diclofenac bile’(B+) from donor rats. Two groups received diclofenac (3mg/kg/day) orally (O) from day 0 until sacrifice on day 3: group O+B+ (n=11) and O+B- (n=8), and two groups did not receive oral diclofenac: O-B+ (n=10) and O-B- (n=11). Primary outcomes were anastomotic leakage and leak severity score (0=clean, 1= small abscess, 2=pus, 3= dehiscence/peritonitis). Secondary outcomes were bursting pressure and breaking strength. Excretion of diclofenac in bile was determined with HLPC.

Results:
Leak rates were equally high in group O+B+ (10/11), group O+B- (7/8) and group O-B+ (9/10), and lower in group O-B- (3/11; p=0.020 vs. O+B+). Leak severity score decreased orderly from group O+B+ (2.4±1.0) to O+B- (1.9±1.1; p=0.625*) to O-B+ (1.2±0.8; p=0.021*) to O-B- (0.3±0.5;p<0.001*) (*compared to O+B+ with ANOVA). Bursting pressure results corresponded with leak severity scores, breaking strength was similar among groups. Diclofenac molecules were detected with HPLC in bile starting from 1 hour after administration.

Conclusion:
Diclofenac (metabolites) in bile seems to increase anastomotic leakage rates. The effect appears inferior to the effect of oral diclofenac, though it may explain the reduced effect of diclofenac on distal colon anastomoses in the rat.
 

59.09 Stromal-derived factor-1α (SDF-1) Treatment of Diabetic Wounds Decreases NOX-2 Expression

Posted on December 22, 2014

S. Deeney1,2, C. Zgheib1,2, J. Xu1,2, J. Hu1,2, T. M. Crombleholme1,2, K. W. Liechty1,2  1University Of Colorado Denver,Center For Fetal And Regenerative Biology,Aurora, CO, USA 2Children’s Hospital Colorado,Department Of Surgery,Aurora, CO, USA

Introduction:
Diabetic impaired wound healing occurs in part due to high levels of oxidative stress. NADPH oxidase 2 (NOX-2) is an inflammatory enzyme involved in superoxide generation; levels of NOX-2 are indicative of tissue levels of oxidative stress. We previously showed that Stromal-derived factor-1α (SDF-1α) levels are decreased in diabetic wounds, that inhibition of SDF-1α increases time to diabetic wound healing, and that treatment of diabetic wounds with SDF-1 α reduces time to epithelialization.  The mechanism by which SDF-1α enhances diabetic wound healing has yet to be fully elucidated. We will test the hypothesis that SDF-1α treatment enhances diabetic wound healing by reducing the high oxidative stress associated with diabetic impaired wound healing, as measured by wound tissue NOX-2 levels.

Methods:
Single dorsal full-thickness wounds were created with an 8-mm punch biopsy in C57BKS.Cg-m/Leprdb Db/Db diabetic (n=6) and Db/+ non-diabetic (n=10) mice. Subsets of diabetic and non-diabetic wounds were treated with a lenti-virus expressing SDF-1α (Db/Db n=3; Db/+ n=5) or GFP control (Db/Db n=3; Db/+ n=5). Wounds were harvested 7 days following injury. NOX-2 levels expressed in the wounds were assessed by quantitative real time PCR.

Results:
Wounds in diabetic mice demonstrated significantly elevated levels of NOX-2 compared to those in non-diabetic mice 7 days following wounding (p=0.03). When treated with SDF-1α, wounds of diabetic mice expressed significantly decreased levels of NOX-2 compared to GFP treated wounds of diabetic mice (p=0.04). This indicates that diabetic wounds carry a higher burden of oxidative stress one week after injury than non-diabetic wounds, and that treatment with SDF-1α leads to a decrease in oxidative stress.

Conclusion:
These findings provide evidence that the oxidative stress observed in diabetic wounds can be significantly reduced by SDF-1α treatment as measured by the decrease in NOX-2 levels. This may be a mechanism by which SDF-1α enhances wound healing. Further studies in this area are warranted to elucidate the mechanisms by which SDF-1α reduces NOX-2 expression.

59.10 Autologous Platelet-Rich Plasma (PRP) Augments Ventral Hernia Repair with Polyester Mesh in Rats

Posted on December 22, 2014

J. L. Van Eps1,2, J. S. Fernandez-Moure1,2, F. J. Cabrera2, A. Chaudhry7, S. Shajudeen7, R. Righetti7, W. Ellsworth1,3, B. J. Dunkin1,4,5, E. Tasciotti2, B. K. Weiner2,5,6  1Houston Methodist Hospital,Department Of Surgery,Houston, TX, USA 2Houston Methodist Research Institute,Department Of Nanomedicine, Surgical Advanced Technology Lab,Houston, TX, USA 3Houston Methodist Hospital,Department Of Surgery, Division Of Plastic Surgery,Houston, TX, USA 4Houston Methodist Research Institute,Methodist Institute For Technology, Innovation And Education (MITIE),Houston, TX, USA 5Weill Cornell Medical College,New York, NY, USA 6Houston Methodist Hospital,Department of Orthopedics & Sports Medicine,Houston, TX, USA 7Texas A & M University,Department Of Electrical & Computer Engineering,College Station, TX, USA

 

Introduction:

In the U.S. today, approximately 350,000 ventral hernia repairs (VHR) are performed yearly, making it one of the most common operations overall and an important target for improvement using novel techniques and materials. Although mesh repair has improved recurrence rates vs. suture repair, as many as 5% may still recur and other complications such as seroma or SSI are increased in this group. Literature demonstrates that improved metrics of early wound healing and tissue ingrowth can enhance repair strength and diminish infection; and PRP is a universally-available source of growth factors that may improve such metrics, but has never been applied to soft tissue healing and hernia repair. We hypothesized that the addition of PRP would cause greater early neovascularization within implanted polyester mesh and thus, overall repair strength and quality.    

Methods:

Chronic ventral abdominal hernias were created in 16 male Lewis rats by incising the musculofascial abdominal wall at the linea alba, closing the overlying skin and allowing them to mature for > 28 days. Rats were randomly divided into two surgical groups – a control (C) group that would receive standard of care repair with Parietex® polyester mesh, and an experimental (E) group with mesh repair augmented by adding autologous PRP. All repairs were performed in an underlay fashion with mesh secured with eight circumferential interrupted prolene sutures. Autologous PRP was isolated by double-centrifugation of whole blood, platelets were quantified and concentrated to a standard 1×106 platelets/μL, and 200μL of PRP was applied directly to the mesh repair site at the time of implantation. Rats were sacrificed after 6 weeks, 3D ultrasound elastography analysis performed, and the specimens processed histologically with H&E and α-smooth muscle actin (α SMA) antibody IHC. Blinded observers then quantified samples for neovascularity and staining intensity.  

Results:

No rats suffered complication requiring euthanasia or exclusion from study, but one rat in the group E suffered a postop seroma compared to 3 in group C. Rats treated with PRP had an average of 223.7 (+ 49) neovessels on histological analysis compared to 73.7 (+ 11.5) – a 3-fold difference. These vessels were subjectively larger and more mature as well. Staining with α SMA was significantly increased in Group E rats as well. On ultrasound analysis, the average elastic modulus at the tissue-mesh interface in experimental rats 46.5kPa (+ 5.3) was significantly higher (p<0.05) than control rats at 1.3kPa (+ 0.2).  

Analysis:

Early neovascularization is known to enhance wound healing and mature tissue integration with mesh in VHR, and our data suggests that VHR can be improved with the addition of autologous PRP. This neovascularization seems to translate to improved overall repair strength and quality. Further investigation is warranted regarding the clinical utility of PRP in VHR. 

59.11 Synthetic Resorbable vs. Cellulose Bandage for Minor Hemorrhage in a Porcine Model

Posted on December 22, 2014

U. R. Yanala1,2, S. Noriega3, R. Spretz3, J. Ragusa3, L. Nunez3, G. Larsen3,4, M. A. Carlson1,2  1University Of Nebraska Medical Center,Surgery,Omaha, NE, USA 2Veteran Affairs Medical Center,Surgery,Omaha, NE, USA 3LNK Chemsolutions,Lincoln, NE, USA 4University Of Nebraska,Chemical & Biomolecular Engineering,Lincoln, NE, USA

Introduction:  Commercially-available topical hemostats for minor hemorrhage incurred during elective surgical procedures are relatively expensive. We believe that more economical synthetic hemostats could be produced. Our objective here was to compare the efficacy and toxicity of a synthetic resorbable hemostatic bandage vs. an analogous commercial product in a porcine model of minor hemorrhage.

Methods:  For the nonsurvival efficacy study, anesthetized domestic swine (boars, 3 months, 29-40 kg) underwent arterial/venous line placement and splenectomy. A 1 x 8 cm section of liver was resected from the edge of the left lateral lobe, and test bandage (macroporous polycaprolactone mesh, PCL; N = 10) or oxidized regenerated cellulose (ORC; Surgicel®, Ethicon®; N = 10) was applied with manual pressure for 5 minutes. Resuscitation then was performed with warm LR (target MAP = 80% of preinjury), and blood loss was measured 60 min after injury. For the survival toxicity study, a similar resection technique was employed (N = 6 for each material), and necropsy was performed at 30 days to evaluate for bandage toxicity (subject growth, serum chemistry, histology). 

Results: Pre-injury weight, VS, and laboratory testing did not differ among groups. Resection mortality was zero. In the efficacy study, there were no differences between the PCL vs. ORC groups in blood loss or other post-injury variables (Table), except that the resuscitation fluid volume in the ORC group was greater. Other results from the efficacy study not shown in the Table include platelet counts and coagulation testing (no significant differences). Other than minor granuloma formation at the implantation site with both PCL and ORC, the survival study did not reveal any measurable toxicity. 

Conclusion: The efficacy and toxicity of the PCL test bandage vs. the ORC comparator were not different in a porcine model of minor hepatic hemorrhage. Based on projected costs of production (not shown), the PCL bandage could represent a lower-cost alternative to ORC for the treatment of minor surgical bleeding.

 

59.12 Need for Better Animal Experiments on Intestinal Anastomotic Healing

Posted on December 22, 2014

S. Yauw1, K. Wever1,2, A. Hoesseini1, H. Van Goor1  1Radboud University Medical Center,Department of Surgery,Nijmegen, GELDERLAND, Netherlands 2Radboud University,2Systematic Review Center For Laboratory Animal Experimentation (SYRCLE),Nijmegen, Gelderland, Netherlands

Introduction:
Anastomotic leak rates have failed to diminish despite extensive experimental research in laboratory animals. This warrants a review of animal experiments on anastomotic healing to provide directions for improving animal research.

Methods:
Animal studies on intestinal anastomotic healing were retrieved by a systematic search in Pubmed and Embase databases. Conference abstracts, posters, reviews, studies in human, studies on other types of anastomoses (e.g. esophagojejunal) and intestinal transplants, studies with other outcomes (e.g. on cancer recurrence) and studies using enterotomy instead of anastomosis were excluded. Full text retrieval was attempted by online searches, by consulting Dutch and German university libraries and by E-mail to the corresponding author. All languages but Japanese were included. Study objective, study conclusion, and animal model were recorded. Reporting quality score (adapted from ARRIVE guidelines) and risk of bias (randomization and blinding) were assessed.

Results:
1342 studies were included, of which 245 were conducted in the US. The number of animal experiments on intestinal anastomosis increases, reaching an average of 53 published per year in the last decade. Most studies were therapeutic trials (n=914; 65%) and risk factor identification studies (n=385; 28%). A positive effect on anastomotic healing was reported in 293 of 345 (85%) articles on ‘experimental therapeutics’. Animal models for healing varied widely in terms of animal species, compromise of healing, intestinal segment and outcome measures. The use of rats has increased in contrast to the use of dogs. Methods to compromise healing are used in 32% and vary between different regimens of chemotherapy, ischemia, radiotherapy, immunosuppressants and more. On average 45% relevant study items, in particular anastomotic complications (31%), the use of antibiotics (76%), sterile surgery (83%) and postoperative analgesics (91%), were not reported. Randomization and blinding of primary outcome assessment increased in the last two decades but is still insufficient in the current decade at 62% and 8% respectively.

Conclusion:
Animal experiments on anastomotic leakage are increasing, contrary to the aim of politics, science and the public to reduce animal research. Reporting and methodological quality must improve to refine animal experiments and possibly increase clinical impact. Selecting the most reliable and reproducible models that have translational value is the next step of our research.
 

58.07 Validating Novel p53-Derived Anti-Cancer Peptide Activity Against a Human Colon Cancer Cell Line

Posted on December 22, 2014

M. F. Shaikh1, B. Babcock1, E. Gleeson1, K. Davitt1, P. Love1, A. Desai1, D. Zimmerman1, M. R. Pincus2, W. B. Bowne1  1Drexel University College Of Medicine,Philadelphia, Pa, USA 2New York Harbor Healthcare System VAMC,New York, NY, USA

Introduction:  PNC-27 is an anti-cancer peptide derived from the MDM-2 binding domain of p53, residues 12-26, attached to a membrane residency peptide. Previously, this peptide has been shown to be effective against murine colon cancer. We now test PNC-27 against a human colon cancer cell line, HCT-116, to further characterize anti-cancer activity and mechanism.

Methods: 1 x 104 HCT-116 cells and untransformed colonic fibroblasts (CF) were treated with PNC-27 and control peptide. Anti-cancer activity was assessed using the MTT cell proliferation assay. Mechanism of cancer cell death (necrosis vs. apoptosis) was assessed using the LDH and caspase-3 assays, respectively. Western blot analysis determined HDM2 expression in both HCT-116 and CF. PNC-27/HDM-2 interaction was studied using immunofluorescent staining and confocal microscopy.

Results: PNC-27 demonstrated specific anti-cancer effects in HCT-116 cells with a greater than 80% reduction in cell proliferation after treatment (p<.007). Mechanism studies revealed no elevation of pro-apoptotic proteins but did reveal rapid cell death by 5-fold increase in LDH release (p<.0001), signifying necrosis. Western blot analysis showed increased expression in HCT-116 cells as compared to untransformed CF cells. Moreover, confocal microscopy demonstrated specific co-localization of PNC-27 and MDM-2 along the cancer plasma membrane.

Conclusion: Our results suggest that PNC-27 targets HDM-2 on the HCT-116 cancer cell membrane, leading to specific anti-cancer necrosis. This peptide shows promise in the treatment of colon cancer.

58.08 Improvements to a Murine Colon Cancer Model for Cancer Progression and the Tumor Microenvironment

Posted on December 22, 2014

K. P. Terracina1, T. Aoyagi3, W. Huang1, A. Yamada4, M. Nagahashi2, K. Takabe1  1Virginia Commonwealth University,Surgical Oncology,Richmond, VA, USA 2Nigata University,Surgery,Nigata, , Japan 3Chiba University,Surgery,Chiba, , Japan 4Yokohama City University,Surgery,Yokohama, , Japan

Introduction:  Over the past several decades, it has become clear that the murine xenograft models, which implant human cancer cells into immune-deficient nude mice, used in the majority of preclinical studies, poorly predict the outcome of human clinical trials. The emerging understanding of the important role of immune responses in the tumor microenvironment for cancer progression necessitates animal models that do not preclude the immune response. As new targets for cancer treatment are discovered and investigated, preclinical studies should be conducted with murine models that include intact immune responses, and that allow real time monitoring of cancer progression to provide a closer correlation with human cancer. In our laboratory, we have recently developed a modified syngeneic orthotopic murine colon cancer model that mimics human colon cancer progression with consistent results.

Methods:  We have genetically engineered a murine colon adenocarcinoma cell line, CT26, to overexpress the firefly luciferase gene (CT26-luc1), which allowed real time in vivo monitoring of tumor burden when the substrate, D-luciferin, was injected intraperitoneally using In Vivo Imaging System (IVIS). We have established a syngeneic orthotopic colon cancer model using CT26-luc1 cells suspended in matrigel and either injected submucosally into the cecum wall under direct visualization, or directly injected into the cecum wall as a control. 

Results: Our syngeneic orthotopic colon cancer submucosal injection model has demonstrated consistent implantation in the cecum compared to the direct injection technique. In contrast, the direct injection model demonstrated complications such as premature peritoneal carcinomatosis prior to development of lymph node metastasis. In vivo bioluminescence allowed real time monitoring of total tumor burden. We have also found that perioperative care has a significant impact on reproducibility of the model. Finally, we found that total tumor burden quantified with bioluminescence enabled us to estimate lymph node metastasis in vivo. 

Conclusion: Our newly established method that maintains intact immune response in the tumor microenvironment is expected to provide an invaluable murine metastatic colon cancer model particularly in preclinical studies for drug development targeting those mechanisms. 

 

58.09 Establishment of a Xenogenic Model of Hepatic Oligo- and Polymetastases of Colorectal Cancer

Posted on December 22, 2014

G. Oshima1,2, S. C. Wightman1, A. Uppal1, J. Oskvarek2, M. Stack1, X. Huang2, T. E. Darga2, M. C. Posner1, N. Khodarev2, R. R. Weichselbaum2  1University Of Chicago,Department Of Surgery,Chicago, IL, USA 2University Of Chicago,Department Of Radiation & Cellular Oncology, Ludwig Center For Metastasis Research,Chicago, IL, USA

Introduction: Patients with colorectal cancer (CRC) often present with liver metastases, which, despite systemic treatment, frequently results in a fatal outcome. Between 5 to 20% of patients with limited numbers of hepatic metastases and slow rates of progression/recurrence (oligometastases) are successfully treated with local treatment approaches, with or without systemic therapy. Yet, little is known about molecular basis of oligometastatic disease. Animal models are critical to study pathophysiological mechanisms and new treatments for patients with metastatic CRC. We propose here a xenogenic animal model of hepatic metastasis of CRC that simulate oligo- and polymetastases,

Methods: We generated a panel of monoclonal HCT116-derived cell lines double-labeled with luciferase and tdTomato protein using lentiviral-based gene delivery. Hepatic tumors were generated by injecting 2 million cells into spleens in mice. Five minutes post-injection splenectomies were performed to avoid carcinomatous peritonitis and residual growth in spleen. Bioluminescent signals were measured weekly using the Xenogen IVIS 200 (MA, United States) imaging system. Two to 4 weeks after injections, livers were harvested and ex vivo fluorescent intensities of liver tumors were quantified. The number and size of liver tumors were directly measured to see the correlation between fluorescent intensities and macroscopic disease. Twenty individual monoclonal cell lines were tested to select candidates for oligo- and polymetastatic phenotype. Diffuse Luminescent Imaging Tomography (DLIT) was performed for evaluation of tumor burden and organ distribution using real-time 3D reconstruction of bioluminescence signals.

Results: Monoclonal cell lines varied in the ability to form liver metastases with 2 cell lines demonstrating oligometastatic phenotype (O1 and O2) and 2 other cell lines-polymetastatic phenotype (P1 and P2). Bioluminescent intensities of P1 and P2 were higher than O1 and O2 at 1 to 3 weeks after injection. Mean tumor number of P1 was higher than P2, O1 and O2 (p < 0.05). Mean tumor size of P2 was larger than P1, O1 and O2 (p < 0.05). Tumor burden estimated by tumor number and size correlated with bioluminescent intensity and fluorescent intensity (R = 0.99). DLIT showed tumor distribution and size dependent signal intensity comparable to macroscopic findings.

Conclusion: An animal model of metastatic CRC to the liver can reproducibly mimic oligo- and polymetastatic CRC in humans. Cell lines double-labeled with luciferase and tdTomato protein can be used as a reliable method to evaluate tumor burden using live imaging which enable sequential monitoring in the same mice and detailed information about tumor size and distribution in secondary site(s). This animal model can be used to improve pre-clinical testing of novel therapies. It may also facilitate evaluation of new biological mechanisms discriminating oligo-and polymetastatic disease in liver.

58.10 CD44 is Associated with Pathologic Response after Neoadjuvant Chemoradiation in Rectal Cancer

Posted on December 22, 2014

L. Thai1,2, J. DeVecchio2, G. Karagkounis1,2, L. C. Duraes1,2, A. Mace1,2, G. Gantt1,2, K. F. Matthew1,2  1Cleveland Clinic,Colorectal Surgery, Digestive Disease Institute,Cleveland, OH, USA 2Cleveland Clinic,Department Of Stem Cell And Regenerative Medicine,Cleveland, OH, USA

Introduction:

Response to neoadjuvant chemoradiation (CRT) improves oncologic outcomes in rectal cancer, however, a subset of patients still develop recurrence.  Colorectal cancer stem cells (CSCs) are relatively resistant to CRT, and could contribute to poor response or recurrent disease.  We have previously shown in vitro that the proportion of CD44+ (marker for colorectal CSCS) cells is increased after chemoradiation. As an extension to study of human tissues, we hypothesized that rectal cancers with high CD44+ cells would be most resistant to chemoradiation.     

Methods:

Thirty-five patients with rectal cancer underwent standard neoadjuvant CRT, and post-treatment responses were evaluated pathologically based on the American Joint Committee on Cancer (AJCC) criteria.  Total tumor mRNAs were isolated from pretreatment specimens.  CD44 mRNA expression was measured using real-time reverse-transcription polymerase chain reaction (RT-PCR) and protein expression was examined by immunofluorescence.  Gene expressions were compared between responders (AJCC 0) and non-responders (AJCC 1-3).  Associations between gene expression with nodal status and tumor recurrence were also evaluated. 

Results:
Pretreatment CD44 mRNA expression was significantly lower in responders (AJCC 0) compared to non-responders (AJCC 1-3) (p=0.02).  CD44 protein expression was also significantly lower in AJCC 0 patients as measured by immunofluorescence.  Conversely, CD44 was significantly higher in nonresponders (AJCC 3) (p=0.018).  When evaluating mRNA expression with stage and outcome, lower levels of CD44 expression significantly correlated with pathologic N0 nodal status, whereas higher levels of CD44 were associated with N1 and N2 nodal status (p=0.008).  There was no significant correlation between CD44 levels and tumor recurrence.  

Conclusion:

Low pretreatment CD44 expression levels in rectal cancers correlate with better response to neoadjuvant chemoradiation and are associated with pathologic N0 nodal status.  CD44, as a marker for CSC, may be a predictive biomarker for identifying rectal cancer patients who may derive the most or least benefit from neoadjuvant chemoradiation.  Further characterization may potentially lead towards more individualized treatment decisions.

 

 

 

58.11 BMP signaling within glioblastoma mediates GSC quiescence and treatment resistance.

Posted on December 22, 2014

S. Das1,2, M. Wu2, M. Srikanth6,7, H. Kim4, A. Celebre2, D. Brat5, J. Kessler6, J. Karamchandani2,3, M. Bredel5  1St. Michael’s Hospital, University Of Toronto,Neurosurgery/Surgery,Toronto, ON, Canada 2Ann And Arthur Labatt Brain Tumour Centre, Hospital For SickKids,Cell Biology,Toronto, ON, Canada 3Montreal Neurological Institute,Laboratory Medicine,Montreal, QC, Canada 4University Of Alabama,Radiation Oncology,Birmingham, Alabama, USA 5Emory University School Of Medicine,Pathology,Atlanta, GA, USA 6Northwestern University,Chicago, IL, USA 7Harvard School Of Medicine,Brookline, MA, USA

Introduction:  The role of intratumoral heterogeneity as a determinant of treatment failure and disease recurrence has become increasingly appreciated.  In glioblastoma, intratumoral heterogeneity—on the functional, genetic, and transcriptional level—likely accounts for the inability of conventional and targeted therapies to achieve long-term remission. In the neural stem cell (NSC) niche, NSCs have been shown to shuttle between a quiescent and activated state. This dynamic has also been shown to be present in the hair follicle stem cell niche, where oscillating levels of bone morphogenetic protein (BMP) and TGF-β2 expression regulate activation of hair follicle stem cell proliferation following physiologic quiescence. We postulated that glioma stem cells (GSCs) could also shuttle between a quiescent and activated state, and that these fate choices could be directed by TGF-β and BMP signaling within the tumor microenvironment.  Further, we postulated that TGF-β and BMP might construe cell identities in glioblastoma that could be relevant to glioblastoma treatment resistance, disease evolution and disease progression.

Methods: We performed immunohistochemical analysis for in situ evidence of TGF-β and BMP signaling in glioblastoma using surgical specimens, then confirmed our findings using in vitro stem cell assay systems. Using knockdown and overexpression stuides, we characterized the quiescent and activated GSC states. We then confirmed the basis for our phenomonological hypothesis in mouse glioblastoma long-term label retaining and chemoradiation treatment models.

Results: We demonstrate that TGF-β and BMP signaling are active in the glioblastoma microenvironment, and regulate the shuttling of GSCs from an activated to a quiescent state through their effects on p21, Stat3 and EGFR. In vivo, BMPhi GSCs are long-term retaining cells, consistent with the quiescent phenotype. BMP-mediated quiescence protects GSCs from genotoxic stress and treatment-associated DNA damage. Further, BMPhi GSCs serve as a cellular reservoir for tumour recurrence following chemotherapy. 

Conclusion: Our findings demonstrate a role for BMP-mediated quiescence in glioblastoma disease resistance and recurrence, and suggest that targeted inhibition of BMP during chemoradiation and TGF-β following its conclusion could favorably alter the natural history of this disease.

 

58.12 A novel steroidal lactone targets head and neck cancer stem cells blocking migration and EMT.

Posted on December 22, 2014

P. T. White1, C. Subramanian1, P. T. Grogan1, E. Brandes1, H. Zhang2, R. Gallagher2, B. N. Timmermann2, M. S. Cohen1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Kansas,Department Of Medicinal Chemistry,Lawrence, KS, USA

Introduction: Head and neck squamous cell carcinoma (HNSCC) survival rates have been stagnant for the last four decades highlighting the need for novel therapeutic approaches and a better understanding of the disease biology. Recent advances indicate that cancer stem cells (CSCs) in the tumor are responsible for recurrence and metastasis, and that standard cisplatin treatment enriches CSC numbers via BMI-1 upregulation. Withanolides are 28-carbon steroidal lactones that have been shown by our group and others to have potent anticancer activity through inhibition of HSP90-chaperoned kinases. These selectively target key proliferative pathways in cancers (including notch, β-catenin, and NF-κB) that also play a critical role in CSC maintenance. We hypothesize that a novel withanolide, withalongolide A (WGA) and its triacetate derivative (WGA-TA) will prevent tumor growth and invasion through inhibition of CSC epithelial to mesenchymal transition (EMT) and cell migration.

Methods:   Validated human HNSCC cell lines (JMAR, MDA1986,UMSCC-11B) were grown in 2D culture and treated with 0.1 to 5μM WGA or WGA-TA for 24h. Proteins involved in the maintenance of CSCs and EMT were analyzed by Western blot (WB) and matrigel invasion assays were performed post treatment to evaluate migration. Orosphere formation assay was conducted to determine self-renewal after treatment. The percentage of apoptotic CSCs was determined by flow cytometry (FC) and confirmed by WB.

Results:  Treatment of HNSCC cells with WGA-TA demonstrated a dose dependent increase in inhibition of CSC markers compared to WGA, including BMI-1, CD44, EZH2, notch1 and other proteins involved in CSC maintenance. 5 µM WGA-TA treatment had the highest inhibition on CSC regulatory proteins and markers (80% for EZH2, 45%BMI-1, 60% for CD44) which was significant (p<0.01)vs. 5 µM WGA (30% inhibition for EZH2, 35% for CD44, and none for BMI-1) and vs. controls (p<0.01). Similarly 5 µM WGA-TA significantly inhibited levels of Akt (95%), p-Akt (80%), and p-GSK3β (90%) compared to WGA (95%, 80% and 30%, respectively, p<0.001 vs controls). 5 µM WGA-TA treatment also decreased the EMT protein vimentin by 60%. In the migration assay, treatment with 5μM WGA-TA showed greater than 80% suppression of migration compared to control (p<0.001), as opposed to 20-30% for 5 µM WGA treatment (p<0.01 vs WGA-TA). An increase in apoptotic CSCs by >50% vs controls, and decrease in orosphere formation occured in a dose-dependent manner with WGA-TA.

Conclusions: WGA-TA represents a novel therapy for HNSCC targeting key pathways and kinases implicated in the maintenance of CSCs, EMT, and invasion. These withanolides target BMI-1, EZH2 and its downstream effectors in HNSCC CSCs resulting in decreased EMT and tumor migration. Further in vivo translation is needed to define the role of this CSC inhibition on tumor growth kinetics, invasion, and metastatic spread.

 

58.13 Evaluating the activity of Tasquinimod (ABR-215050) in head and neck squamous cell carcinoma (HNSCC)

Posted on December 22, 2014

M. B. Burch1, J. M. Warram1, N. G. Patel1, T. M. Zimmermann2, E. L. Rosenthal1  1University Of Alabama At Birmingham,Division Of Otolaryngology, Department Of Surgery,Birmingham, AL, USA 2Mayo Clinic,Department Of Otorhinolaryngology,Rochester, MN, USA

Introduction:
S100A9, a subunit of the protein calprotectin, is released during tissue damage and has been implicated in the progression of inflammation and cancer metastasis. Recent studies suggest that extracellular matrix metalloproteinase inducer (EMMPRIN) can act as a cell-surface receptor for S100A9. EMMPRIN is a well-characterized factor in the progression of many head and neck tumors. Therefore, evidence of an S100A9-EMMPRIN interaction would serve to further demonstrate a possible role for S100A9 in local tumor invasion and disease progression.

Methods:
Human SCC1-Luc+ HNSCC cells were injected into the unilateral flank of nude athymic mice. Tumors were grown for 1 week before exposure to trial compounds. Studies incorporated exposure of these tumors to the S100A9 inhibitor Tasquinimod (TASQ) via drinking water at a dose of 10 mg/kg/day, as well as anti-EMMPRIN monoclonal antibody (CNTO3899). Groups included exposure to TASQ (n=5), anti-EMMPRIN (n=5), TASQ + anti-EMMPRIN concurrently (n=5), and untreated (n=5). Microcalipers and bioluminescence imaging via luciferase were used to quantify tumor size. Tumors were resected, sectioned, and mounted. Immunohistochemistry was performed using primary antibodies to EMMPRIN and S100A9. Corresponding fluorescently-labeled secondary antibodies and fluorescence microscopy were used to assess localized expression. Scores were assigned descriptively using a modified Allred scoring method by assigning a relative expression of 0 to 5 based on fluorescence intensity and 0 to 5 based on fluorescence distribution within each sample. Aggregated scores were then used for statistical analysis.

Results:
Upon study termination, tumors exposed to TASQ alone and exposed to TASQ + anti-EMMPRIN were significantly larger than untreated tumors (p= 0.015 and p=0.009, respectively). In vivo analysis revealed that mice administered TASQ alone exhibited an increase in tumor size 110.77% over untreated tumors, whereas tumors exposed to both TASQ and anti-EMMPRIN concurrently demonstrated a 113.04% increase in tumor size over untreated tumors during this same period. Tumors exposed only to anti-EMMPRIN decreased 78.26% in size compared to untreated tumors. Tumors exposed to TASQ demonstrated a significant decrease in EMMPRIN expression compared to untreated tumors (p=0.015).

Conclusion:
Inhibition of S100A9 activity by TASQ in HNSCC leads to accelerated tumor growth, suggesting a possible protective role for this molecule in squamous cell tumors. The concurrent inhibition of S100A9 and EMMPRIN contributes to enhanced disease progression in HNSCC. Inhibition of EMMPRIN alone decreases HNSCC progression. Inhibition of S100A9 by TASQ leads to a significant decrease in cellular EMMPRIN expression.
 

« Previous Page
Next Page »