25.10 Metabolic Derangement of Coagulation: A Likely Suspect For Post Operative Bleeding

Posted on December 22, 2014

G. D. Wiener1, H. B. Moore1, P. Lawson1, E. Gonzalez1, M. P. Chapman1, A. P. Morton1, A. Sauaia1, A. Banerjee1, E. E. Moore2  1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA

Introduction: Recently it has been appreciated that bile acids can promote degradation of fibrin sealant.  The concept of metabolites changing coagulation may explain persistent localized bleeding from the liver despite normalization of systemic coagulation.  We hypothesize that bile acid impairs whole blood clot formation and promotes fibrinolysis.

Methods: Blood was collected from healthy volunteers (n=6) into citrated tubes.  Taurcholic acid (TUCA) was titrated in vitro into whole blood with a range from 250 µM to 1000 µM.  This range was selected as previous literature indicated that systemic TUCA levels could exceed 600 µM.  Whole blood mixtures were assayed using thrombelastography (TEG) to quantify clot strength (MA) and degree of fibrinolysis (LY30).  Tranexamic acid (TXA) was used to block plasmin mediated fibrinolysis.  Statistical analysis used SPSS software.  Correlations between TEG parameters and concentration of TUCA were analyzed using Spearman's Rho Test, and the Wilcoxon test was used for pair-wise comparisons.

Results: Clot strength had a negative correlation to dose of TUCA (Spearman’s Rho = -0.677, p<0.001).  Median whole blood MA was 56.25 (IQR 53.25-58.50), which decreased to a median MA of 42.00 (IQR 38.00-48.50) at the highest dose of TUCA.  Ly30 had a positive correlation to dose of TUCA (Spearman’s Rho = 0.702, p<0.001).  Median whole blood Ly30 was 1.35 (IQR 0.8-1.73), which increased to a median Ly30 of 4.0 (IQR 2.85-10.3) at the highest dose of TUCA.  At a dose of 750 µM TUCA, TXA reduced Ly30 from 7.7 (IQR 5.7-14.8) to 2.9 (IQR 1.9-3.3), p=0.028, but did not have a significant effect on MA (p=0.249).  At the highest dose of 1000 µM TUCA, TXA did not significantly reduce Ly30 (p=0.658) and MA was unchanged by TXA at this dose (p=0.144).

Conclusion: Bile acid has a dose response in reducing clot strength and promoting fibrinolysis.  This is consistent with previous literature that bile has clot degradation properties.  The reduction in clot strength also suggests platelet inhibition, which is not correctable by TXA.  This metabolic effect on coagulation warrants further investigation, as localized areas of the body, particularly the liver, with high levels of bile acid may be at risk for post-operative bleeding.

26.01 Dipeptidyl peptidase-4 Inhibition Promotes Wound Healing in Murine Models of Type 1 and 2 Diabetes

Posted on December 22, 2014

A. J. Whittam1, Z. N. Maan1, D. Duscher1, L. H. Fischer1, N. Ho1, M. Rodrigues1, M. S. Hu1, G. G. Walmsley1, M. Januszyk1, J. Barrera1, A. J. Whitmore1, G. C. Gurtner1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction:
Chronic diabetic wounds are thought to result from impairments in the cellular and molecular mechanisms of wound repair in diabetic patients. Clinically, these wounds can result in significant disability, amputation, and increased mortality. Poor neovascularization in response to ischemia has been discovered to be fundamental to this problem, resulting from a high glucose induced defect in the transactivation of hypoxia-inducible factor-1α (HIF-1α). This leads to impairment of HIF-1α mediated expression of vascular endothelial growth factor (VEGF) and stromal-derived factor-1 (SDF-1). In particular, diabetic patients are known to be deficient in SDF-1, and it has been shown that in non-diabetic cells inhibiting DPP-4, which cleaves SDF-1, enhances the chemotaxis of murine and human HSCs and hematopoietic progenitor cells in vitro via SDF-1 induction. To explore the potential therapeutic benefits of DPP-4 inhibition for diabetic wounds, we utilized the DPP-4 inhibitor MK0626 and tested its effect on wound healing and cell behavior. 

Methods:
Wild-type mice were either treated with streptozocin or fed a high fat diet to induce type 1 and 2 diabetes, respectively, which was confirmed via glucose tolerance test. Type 1 and type 2 diabetic mice were treated for 6 weeks with either: vehicle (high fat chow), glipizide (hypoglycemic agent), or MK0626. Humanized excisional wounds were subsequently created on the dorsum of treated type 1 and type 2 diabetic mice, with wounds being photographed and assessed at two-day intervals. Tissue was harvested for histology and qRT-PCR. 

Results:
MK0626 significantly accelerated wound healing compared to both control and glipizide groups (*p= <0.05). This effect became evident by day 4, with significantly reduced mean wound area relative to original size (MK0626 = 79%, Control = 90%, Glipizide = 87%, *p= <0.05). qRT-PCR demonstrated increased transcription of SDF-1 (*p= <0.05), and vascular endothelial growth factor (VEGF) (*p= <0.05) in MK0626 treated v control mice. Immunohistochemistry studies demonstrated increased expression of SDF-1 (*p=0.008) and CD31 (*p=0.02) in MK0626 v control groups.

Conclusion:
DPP-4 inhibition has the potential to play a pivotal role in diabetic wound healing. The administration of MK0626 enhances wound healing more effectively than reducing hyperglycemia alone. Furthermore, a low concentration of MK0626 (1mg/kg/BW in chow) seems to be the optimal dosage for oral administration.
 

26.02 SDF-1 α attenuates diabetic wound inflammation through modulation of miR-146a expression

Posted on December 22, 2014

J. Xu1,2, C. Zgheib1,2, J. Hu1,2, K. W. Liechty1,2  1University Of Colorado Denver,Surgery,Aurora, CO, USA 2Children’s Hospital Colorado,Pediatric Surgery,Aurora, CO, USA

Introduction: Impaired wound healing represents a significant complication of diabetes.  The etiology of this wound healing impairment is multifactorial and includes an increased and chronic inflammatory response.  We have previously shown that murine diabetic wounds are deficient in SDF-1α and that treatment of diabetic wounds with SDF-1α can improve healing, however, the mechanisms of this correction are not well characterized.  MicroRNAs (miRNAs) are novel RNA molecules that regulate the translation of mRNAs at the post-transcriptional level.  In particular, microRNA-146a (miR-146a) has been shown to be a key repressor of the inflammatory response by targeting interleukin-1 receptor associated kinease (IRAK1) and tumor necrosis factor receptor associated factor 6 (TRAF6) and repressing NFκB.  We have recently shown that miR-146a expression is decreased in murine diabetic wounds. We hypothesized that the improved diabetic wound healing following treatment with SDF-1α is due, in part, to correction of the dysregulated miR-146a gene expression resulting in decreased inflammatory response.

Methods: To test this hypothesis, 8mm full-thickness wounds were created on the flank of diabetic (Db/Db) and non-diabetic (Db/+) mice with a dermal punch instrument.  At the time of wounding, the wounds were treated with an intradermal injection with either 108 plaque-forming units (PFU) of a lentivirus containing the SDF-1α or GFP transgene.  The wounds were harvested 7days after injury and processed for histology and isolation of total cellular RNA. In order to examine the role of the fibroblast in the dysregulation of the inflammatory response, we isolated diabetic and non-diabetic dermal fibroblasts and treated them with either 106 PFU of a lentivirus containing the SDF-1α or GFP transgene. Gene expression was analyzed using Real-time PCR analysis.

Results: At 7 days, diabetic wounds treated with lenti-SDF-1α exhibited a significant decrease in wound surface area compared to lenti-GFP treated wounds. Diabetic wounds treated with SDF-1α also demonstrated a significant increase in miR-146a expression, and a significant decrease in the miR-146a targets IRAK-1 and TRAF-6, compared to GFP treated diabetic wounds. At the cellular level, MiR-146a was significantly down-regulated in diabetic fibroblasts at baseline, compared to non-diabetic fibroblasts. In addition, SDF-1α treatment corrected the miR-146a gene expression to levels similar to the non-diabetic fibroblast. 

Conclusion: SDF-1α mediated correction of the diabetic wound healing impairment is due, in part, to correction of dysregulated microRNA-146a expression.  Increased expression of miR-146a may result in decreased inflammatory response and less oxidative stress resulting in improved healing.  Further studies are needed to define the role of abnormal miRNA regulation in the pathogenesis of the diabetic wound healing impairment.

 

26.03 Accelerated Myocutaneous Revascularization Following Graded-Ischemia in db/db Mice

Posted on December 22, 2014

R. M. Clark1, B. Coffman1, D. A. Ramirez2, S. Godoy2, S. A. Meyers2, T. McGregor2, S. Krishna2, P. G. McGuire3, T. R. Howdieshell1  1University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 2Skinfrared LLC,Albuquerque, NM, USA 3University Of New Mexico HSC,Cell Biology And Physiology,Albuquerque, NM, USA

Introduction: Murine models have provided valuable insight into the pathogenesis of both diabetes and chronic wounds.  The db/db mouse possesses a spontaneous mutation in the leptin receptor gene resulting in obesity, hyperglycemia, hyperinsulinemia, hypercholesterolemia, and insulin resistance.  This strain is characterized by impaired epithelialization of excisional wounds, with little known about wound neovascularization.

Methods: A cranial-based, peninsular-shaped myocutaneous flap was surgically created on the dorsum of C57BL6 (wild-type) and db/db mice (n=16 total; 5 mice per operative group and 3 unoperated mice per group as controls). Planimetric analysis of serial digital photographic images was utilized to determine flap viability in wild-type and db/db mice. Real-time myocutaneous flap perfusion and surface temperature were determined by laser speckle contrast and thermal infrared imaging respectively. Mice with surgical flaps were sacrificed on postoperative day 10. Image analysis of CD-31 immunostained sections confirmed flap microvascular density and anatomy. Quantitative RT-PCR was performed on nonoperative back skin and postoperative flap tissue specimens to determine local gene expression.

Results: Day 10 planimetric analysis revealed a mean flap viability of 95% in db/db mice compared to 79% in wild-type mice, and 60% of wild-type mice developed distal flap dehiscence not evident in db/db mice. Over 10 days, laser speckle contrast imaging documented markedly increased perfusion at all times points (p<0.001) with functional revascularization to supranormal perfusion in db/db flaps. In contrast, wild-type flaps displayed expected graded flap ischemia with failure to return to baseline perfusion during the postoperative period. Thermal infrared imaging documented complementary spatiotemporal assessment of thermal-metabolic tissue state. Immunostaining confirmed significant differences in preoperative and postoperative microvascular density (mean post-op distal vessel count 60 ± 8 versus 80 ± 15 vessels/mm2 for wild-type and db/db mice respectively, p=0.018; mean post-op distal vascular surface area 4108 ± 95 versus 8250 ± 795 µm2/mm2 for wild-type and db/db mice respectively, p<0.001). Finally, quantitative RT-PCR demonstrated statistically significant differences in angiogenic gene expression between wild-type and db/db mice at baseline (unoperated) and at day 10.

Conclusion: In a graded-ischemia wound healing model, accelerated myocutaneous revascularization and improved wound healing were evident in db/db mice compared to wild-type controls. Gene expression analysis reveals the db/db mouse may be “primed” for wound neovascularization and warrants further investigation into the role of the leptin-leptin receptor axis in wound angiogenesis.

 

 

26.04 Effect of Stretch On Extracellular Matrix And Morphology Of Fibroblasts in Regenerative Wound Healing

Posted on December 22, 2014

S. Balaji1, N. Hann1, R. Ranjan1, C. Moles1, A. F. Shaaban1, T. M. Crombleholme1,3, P. Bollyky2, S. G. Keswani1  1Cincinnati Children’s Hospital Medical Center,Pediatric General Thoracic And Fetal Surgery,Cincinnati, OH, USA 2Stanford School Of Medicine,Infectious Diseases, Department Of Medicine,Palo Alto, CA, USA 3Children’s Hospital Colorado,Center For Children’s Surgery,Aurora, CO, USA

Introduction: Mid gestation fetal skin heals without scar and is characterized by negligible resting tension and distinct extracellular matrix (ECM) with elevated levels of hyaluronan (HA) produced by fetal fibroblasts. In contrast, adult skin is characterized by relatively low levels of HA, and much higher resting tension and scar formation. Of note, wounds of a critical size even in the fetus heal with a scar suggesting a role for biomechanical forces in the regulation of the fetal regenerative phenotype. There is little data that examined the interaction of mechanical stress and regulation of the ECM, specifically HA synthesis. Taken together, we hypothesize that higher mechanical tension may alter the fibroblast regulation of HA synthases (HAS1-3).

Methods: Primary murine adult fibroblasts (AFb) and fetal fibroblasts (FFb) were cultured with +/- mechanical stretch for 24 hours. Static tension at 58 KPa (resting tension of adult mouse skin) was applied using flexcell apparatus to induce tonic stretch. Stretch induced phenotypic changes in FFb and AFb were assessed by changes in PCM (particle exclusion assay), HAS1-3 synthases (qPCR) and cell morphology and cytoskeleton changes (a-SMA Immunohistochemistry). Data presented as average+/-SD, n=4/group, p values by t-Test or ANOVA.

Results:AFb and FFb demonstrate differential cell responses to biomechanical stretch. Under static condition, AFb had significantly lower HAS1 (AFb 1.4+/-0.4 vs FFB 4.6+/-1.1, p<0.01) and HAS2 (AFb 1.2+/-0.25 vs FFB 2.7+/-0.8, p<0.01) gene expression that encodes for fetal-like high molecular weight hyaluronan, and smaller PCM (AFb 1.84+/-0.08 vs FFb 2.78+/-0.14; p<0.001). AFb also demonstrated thicker a-SMA fibers compared to FFb, but the fiber orientation appeared random in both cell types. 24 hours of mechanical stretching resulted in the loss of FFb phenotype with a significant decrease in HAS1 (FFb+stretch 1.0+/-0.35 vs FFb 4.6+/-1.1, p<0.01) and HAS2 (FFb+stretch 1.0+/-.2 vs FFb 2.7+/-.8, p<0.01) gene expression and a significant reduction in PCM formation (FFb+stretch 1.98+/-.15 vs FFb 2.78+/-.14, p<0.01) in FFb, to levels similar to the AFb under static condition. Stretching increased HAS1 expression in AFb (AFb+stretch 3.0+/-.6 vs AFb 1.4+/-0.4, p<.05), but had no effect on HAS2. Interestingly a-SMA staining demonstrated that stretching resulted in thickening of the stress fibers in FFb, but not AFb, and a trend toward reorientation of actin fibers in the strained cells perpendicular to the direction of stretch in both AFb and FFb.

Conclusion:Our data suggest that biomechanical forces may have a significant role in influencing the dermal fibroblasts’ morphology and their regulation of the ECM in the fetal and adult wound healing phenotype. Understanding the contribution of mechanical environment via morphological and phenotypic alterations may yield novel therapeutic targets in recapitulating fetal regenerative healing in postnatal tissues.

 

26.05 GelE/sprE are critical for Enterococcus faecalis-induced anastomotic leak in a rat model

Posted on December 22, 2014

J. N. Luo1, B. A. Shakhsheer1, R. Klabbers2, A. Zaborin1, N. Belogortseva1, O. Zaborina1, J. C. Alverdy1  1The University Of Chicago Pritzker School Of Medicine,Department Of Surgery,Chicago, IL, USA 2Radboud University Nijimegen Medical Centre,Department Of Surgery,Nijimegen, GELDERLAND, Netherlands

Introduction:  Anastomotic leak following colorectal surgery is a dreaded complication of which the cause remains unknown. Our laboratory has recently established that high collagenase producing strains of Enterococcus faecalis (E. faecalis) “bloom” in the gut following anastomotic injury and are both necessary and sufficient to cause anastomotic leak in rats.  Here we performed a mutational analysis to define the roles of gelE and sprE – co-regulated genes that control: adherence, penetration and collagenase production – on anastomotic leak.  We used a double knockout mutant lacking gelE and sprE derived from E. faecalis V583 (a vancomycin-resistant clinical isolate). We hypothesize that mutants deficient in both genes (ΔgelEΔsprE) would be attenuated in their capacity to cause leak in rats. 

Methods:  Adult rats (n=24) underwent colon resection followed by recto-sigmoid anastomosis. Following anastomotic construction, 5cc of 107 CFU/ml of either the double mutant deficient in both gelE and sprE (ΔgelEΔsprE), or the mutant complemented with gelE and sprE (ΔgelEΔsprE/gelE+sprE- termed VT07) were administered via rectal enema to inoculate the anastomosis. On postoperative day six, all rats were sacrificed and evaluated for evidence of anastomotic leak via previously established criteria. Anastomotic tissues were harvested and separated into mucosal, serosal, and perianastomotic samples for bacterial species identification via culture and phenotype analysis (collagenase activity).
 

Results: Anastomotic leakage was observed to be significantly greater in rats rectally inoculated with the complemented strain VT07 that produces high collagenase compared to its matched double mutant ΔgelEΔsprE that produces no collagenase (p<0.01). Leaks were characterized by dense perianastomotic adhesions, perianastomotic abscesses, and occasional gross anastomotic dehiscence.  The most severe leaks were identified to be associated with high adherence of E. faecalis to the mucosa, penetration into the serosa, high collagenase activity, and culture positivity of perianastomotic tissues. Dense mucosal colonization alone was not associated with leak. 
 

Conclusion: High collagenase producing E. faecalis present at and within anastomotic tissues appears to play a critical role in the pathogenesis of anastomotic leak. Although various genes are likely to confer its pathogenic potential at healing anastomotic tissues, we show in this study that gene products and pathways regulated by the gelE/sprE system that control adherence, penetration, and collagenase/protease production are likely to play a key role.  Because E. faecalis is a ubiquitous commensal organism in humans, and only certain strains of it are associated with anastomotic leak, a more complete understanding of the microbial pathogenesis and phenotypic expression could enable us to better predict leak and thus improve patient management.

26.06 Adipose-Derived Stem Cell-Seeded Hydrogels Increase Progenitor Cell Recruitment and Functionality

Posted on December 22, 2014

R. Kosaraju1, R. Rennert1, J. Barrera1, A. J. Whittam1, Z. N. Maan1, M. Rodrigues1, D. Duscher1, M. Januszyk1, G. C. Gurtner1  1Stanford University,Department Of Surgery, Division Of Plastic Surgery,Palo Alto, CA, USA

Introduction:
Biomaterials that promote neovascularization represent a promising treatment of chronic wounds, particularly in combination with stem cells. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest, due to their pro-regenerative function and ease of harvest. Our laboratory has demonstrated the benefits of delivering ASCs seeded on a pullulan-collagen hydrogel bioscaffold, resulting in upregulation of genes related to stemness and neovascularization and accelerated wound closure in vivo. More recently, it has been shown that mesenchymal stem cells (MSCs) enhance the recruitment of endogenous progenitor cells, likely through cytokine secretion. Therefore, we investigated the recruitment of endogenous progenitor cells by ASCs seeded on our hydrogel bioscaffold.

Methods:
ASCs were either seeded into hydrogels or plated in standard growth medium. After 48 hours of incubation in hypoxia, conditioned medium (CM) from the ASC-seeded hydrogels and from the plated ASCs was harvested. The effects of CM from ASC-seeded hydrogels on the functionality of bone-marrow mesenchymal progenitor cells (BM-MPCs), a cell population defined by our laboratory to be crucial to neovascularization, were assessed using qPCR, ELISAs, immunocytochemistry to measure cell proliferation, a migration assay, and a tubulization assay. In vivo, we parabiosed green-fluorescent protein positive (GFP+) donor mice and wild-type recipient mice. An excisional wound model on the recipient mouse was treated with a control saline injection, injection of ASCs, or ASC-seeded hydrogels, and wounds were harvested four days post-wounding for fluorescence-activated cell sorting (FACS) and microfluidic single-cell analysis.

Results:
In vitro, we found that ASC-seeded hydrogel CM significantly upregulated the functionality of BM-MPCs. In particular, BM-MPCs exposed to ASC-seeded hydrogel CM displayed a significant increase of 44% in cell migration (p=0.019) and a fourfold increase in cell proliferation (p=0.0027). Furthermore, qPCR and ELISAs revealed the upregulation of several angiogenic genes, including Hgf and Mmp3, and a significant increase in tubulization of BM-MPCs exposed to ASC-seeded hydrogel CM (p=0.045). In vivo, microfluidic single-cell analysis identified a subpopulation of GFP+/Lineage-/CD45- cells, putative BM-MPCs, defined by the expression of pro-vasculogenic genes, including Ang, Cd248, and Sca1, that was increased in wounds treated with ASC-seeded hydrogels (45.8% of GFP+/Lin- cells) compared to injected ASCs and PBS controls (32.7% and 22.8% of GFP+/Lin- cells), respectively. FACS quantification of GFP+/Lin-/CD45-/Sca1+ cells within the wounds demonstrated an increase of 38% in recruited BM-MPCs when wounds were treated with ASC-seeded hydrogels compared to ASC injection.

Conclusion:
ASC-seeded hydrogels upregulate BM-MPC functionality, and enhance BM-MPC recruitment, to effect greater neovascularization and accelerate wound healing.
 

26.07 Nanocarrier-decorated Mesenchymal Stem Cells for Therapeutic Wound Healing and Angiogenesis

Posted on December 22, 2014

Z. Liu1, B. Wang1, D. M. Castilla1, Y. Li1, R. Tian1, L. Kovalski1, O. C. Velazquez1  1University Of Miami,Surgery/Sylvester Comprehensive Cancer Center,Miami, FL, USA

Introduction: Mesenchymal stem cells (MSC) carry high therapeutic potential for tissue regeneration. However, targeted delivery of therapeutic MSC to injured tissue remains a challenge. We are aimed to develop a safe and targeted cell delivery method for therapeutic administration of MSC to injured tissues to achieve enhanced wound healing and neovascularization. Herein, we present a novel cell delivery platform by coating the surface of MSC with nanocarriers composed of nanoparticle–adhesion molecule complex (NAMC). These nanocarriers guide the coated MSC to their destination via molecular recognition and association with their counterpart adhesion molecules, which are highly or selectively expressed on the injured tissue, and execute their therapeutic roles.

Methods: Murine bone marrow-derived MSC, which were obtained from ROSA 26-LacZ+ mice and pre-labeled with Luciferase 2 (Luc2+) gene, were coated with nanocarriers (NAMC) or control nanoparticle-albumin complex (NAC). 1x 106 NAMC- versus NAC-decorated MSC were administrated through either systemic (i.v.) or local (wound tissue injection) approach to recipient mice on which 6-mm full thickness dorsal skin wounds were created (n=6/group).  An extra group of wounded mice were treated with saline as baseline control. Bio-distribution and selective wound tissue homing of administrated Luc2+ MSC were detected by IVIS (In Vitro Image System) at various time points. Increased homing of infused circulating NAMC-decorated MSC to wound tissues was validated by X-gal staining of LacZ+ MSC in wound tissues. Wound healing rate was calculated based upon daily digital photograph of wounds. Neovascularization was assessed by either Dil-perfusion and wound tissue confocal microscopy (for local administration) or immunohistochemistry analysis of wound tissue sections (for systemic administration). 

Results:Whole-body IVIS scan showed significantly increased Luc2+ NAMC-decorated MSC within wound tissues, particularly on day 8 (~10-fold increased bioluminescence signal, p=0.029). Intravenously infused NAMC-decorated MSC selectively homed to wound tissues, but not other organs. Increased homing of intravenously infused NAMC-decorated MSC to wound tissues was demonstrated by X-gal staining of wound tissues. Wound healing rate and neovascularization were significantly increased by both systemic (p≤0.01) and local (p<0.01) administration of NAMC-decorated MSC in comparison with NAC-decorated control MSC. 

Conclusion:We demonstrated the feasibility and efficacy of a novel form of nanocarrier for targeted delivery of therapeutic stem cells to wound tissues in mouse models for enhanced wound healing and neovascularization. This nanocarrier cell surface decoration method is suited for targeted delivery of virtually any type of therapeutic cells and holds promise to be developed as a clinically-relevant cell-based therapy for regenerative medicine and beyond.  

 

23.01 Immune-mediation Alters The Cytokine Profile Of Hormone-dependent And Hormone-independent Breast Cancer Cells In A 3d In Vitro Culture System

Posted on December 22, 2014

T. N. Augustine1, R. Duarte2, G. P. Candy2  1School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa 2School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Immune infiltration is a fundamental determinant of tumour progression and response to therapy, with interactions between immune, and tumour cells mediated by cytokines. In advanced breast cancer, a dominance of regulatory T (TREG) lymphocytes and the scarcity natural killer (NK) cells may reflect the primacy of adaptive immunity in the induction of tumour tolerance. We thus developed 3-dimensional (3D) models of the tumour microenvironment to determine induced cytokine profiles under immune-mediation.

Three-dimensional culture models were established by co-culturing CD4+CD25+ TREG lymphocytes and NK cells with hormone-dependent MCF-7 or hormone-independent MDA-MB-231 cell lines in growth factor reduced-Matrigel. Cytokine production was measured using a multiplex cytokine assay. Multivariate analyses were used to determine significant differences in cytokine production, and to explore associations between cytokines.

The results show that cytokine secretions in a 3D simulated breast tumour microenvironment are associated with the hormone-dependency of tumours. The effects of NK cell-mediation induced a significant increase in CCL2, CCL4 and CXCL8 secretion in the MCF-7 culture model. In the MDA-MB-231 culture model NK cells alone induced a significant increase in IL-12 and CCL2 inferring the dominance of TREG cell-mediation of cytokine secretion. Cluster analysis and principal components analysis indicate that IL-6 plays a significant role in the induction of a chemokine cascade in both culture models, with IL-1β implicated in the induction of proinflammatory environment in the MDA-MB-231 culture model.

The cytokine data suggests that hormone-dependent MCF-7 cells, as a weakly metastatic cell line, are capable of subverting cytokine secretion of NK cells and TREG lymphocytes to enhance their invasive potential. In contrast, hormone-independent MDA-MB-231 cells, a more aggressive phenotype, subvert TREG cell-mediated cytokine secretion for the maintenance of a proinflammatory microenvironment for tumour progression. Further, the data suggests a role for IL-6 and IL-1β in promoting such immune evasion and tissue invasion.

23.02 NIR Photoimmunotherapy of Pancreatic Cancer Significantly Decreases Recurrence after Resection

Posted on December 22, 2014

A. A. Maawy1, Y. Hiroshima4, Y. Zhang3, M. Garcia-Guzman5, L. Makings5, R. Heim5, G. A. Luiken2, R. M. Hoffman1,3, M. Bouvet1  1University Of California – San Diego,Surgery,San Diego, CA, USA 2OncoFluor, Inc.,San Diego, CA, USA 3AntiCancer, Inc.,San Diego, CA, USA 4Yokohama City University,Surgery,Yokohama City, , Japan 5Aspyrian Therapeutics,San Diego, CA, USA

Introduction:   Photoimmunotherapy (PIT) is a selective treatment modality in the treatment of cancer. Tumor selectivity and targeting is based on the use of a monoclonal antibody  specific to cancer epitopes conjugated to a near infra red (NIR)phthalocyanine dye (IR700). While surgical resection of pancreatic cancer offers the only real chance at a cure, recurrence rates and overall mortality is still high. PIT, if efficacious, could serve as a useful adjunct in the surgical treatment of pancreatic cancer that would help eliminate invisible microscopic disease on the tumor bed.  In this study, we chose to use anti-carcinoembryonic antigen (CEA) as the monoclonal antibody because it highly expressed in pancreatic cancer.

Methods:   Athymic nude mice were orthotopically implanted with the GFP expressing human pancreatic cancer cell line BxPC3. After engraftment, the mice were divided into two groups: Bright light surgery (BLS) + anti-CEA-IR700 + 690nm laser (PIT) and BLS only.  The anti-CEA-IR700 conjugate (100 μg) was administered to the treatment group via tail vein injection 24 hours prior to therapy. After 24 hours, tumors were surgically exposed and treated with the designated phototherapy intraoperatively at an intensity of 150 mW/cm2 for 30 minutes and serially imaged non-invasively for 8 weeks using the OV-100 small animal imager.

Results:  Over the course of 8 weeks there was a significant difference in tumor size between the PIT-BLS (2.14 mm2, 95% CI [6.34, -2.06] and BLS group (115.2 mm2, 95% CI [141.6, 88.8]) with p<0.001. There was also a significant difference in tumor weight between the PIT-BLS (6.65 mg, 95% CI [19.65, -6.35] and BLS (1100 mg, 95% CI [1406, 794] at 8 weeks with p<0.001. In the PIT-BLS group, there was no tumor detectable in 86% of the mice vs a 100% recurrence rate in the BLS group (p=0.04).

Conclusion:  PIT causes significant tumor cell death with high specificity in the treatment of pancreatic cancer as a surgical adjuvant in orthotopic nude mouse models. Animals treated with BLS-PIT appear to be cured from an aggressive metastatic pancreatic cancer.  PIT holds promise in the treatment of this highly lethal cancer and may serve as a useful adjunct to surgery in the eradication of otherwise undetectable microscopic disease.  The results of the present report suggest that BLS-PIT be evaluated clinically in the future.

 

23.03 Defining Immunological Aspects of Regional Chemotherapy Using Immunocompetent Murine Melanoma Model

Posted on December 22, 2014

M. Tsutsui1, Z. Sun1, P. Speicher1, P. Dolber1,2,3, J. Dannull1, S. Nair1, D. Tyler1,2  1Duke University Medical Center,Surgery,Durham, NC, USA 2Durham VA Medical Center,Durham, NC, USA 3Duke University Medical Center,Pathology,Durham, NC, USA

Introduction:

The effectiveness of regional therapeutics may be related to the degree of local and systemic anti-tumor immune response generated. We utilized an immunocompetent mouse model of advanced extremity melanoma to define the immune aspects of regional chemotherapy infusion.

Methods:

B16F10.9OVA melanoma cells were inoculated subcutaneously in one or both hindlimbs of C57BL/6 mice. After tumor diameter reached 5 mm, isolated limb infusion (ILI) was performed using melphalan or vehicle in one hindlimb with or without anti-CTLA-4.  Mice experiencing a complete response were rechallenged with a second tumor inoculation.

Results:

Tumor doubling and quadrupling time were significantly longer using melphalan ILI with systemic anti-CTLA-4 than without (P = 0.005, Hazard Ratio: HR 1.96 and P = 0.02, HR 1.72, respectively); no anti-CTLA-4 effect was observed on tumors that received vehicle ILI. Of the mice rechallenged with a second tumor inoculation, inhibition of tumor growth at the second inoculation site was only observed in mice treated with melphalan ILI and systemic anti-CTLA-4. In animals carrying tumors on both hindlimbs, tumor doubling and quadrupling times on the non-ILI-treated side were significantly longer in animals whose contralateral hindlimb was treated with melphalan ILI and systemic anti-CTLA-4 than with melphalan ILI alone (P = 0.011, HR 5.05 and P = 0.004, HR 9.1, respectively). Systemic tumor specific immune responses were detectable in the regional draining lymph nodes only in animals treated with both melphalan ILI and systemic anti-CTLA-4.

Conclusion:

Optimizing immunologic aspects of regional melanoma therapy may significantly complement systemic immunotherapy strategies.

23.04 Melanoma-Associated Fibroblasts Are a Promising Therapeutic Target

Posted on December 22, 2014

Z. Liu1, H. Shao1, M. G. Moller1, O. C. Velazquez1  1University Of Miami,Surgery/Sylvester Comprehensive Cancer Center,Miami, FL, USA

Introduction:  Cancer-associated fibroblasts (CAF) play critical roles in promoting primary tumor development, growth and progression by stimulating tumor cell proliferation, survival, migration, angiogenesis as well as providing a niche supporting the metastatic colonization of disseminated tumor cells in distant organs. Moreover, CAF appear to be relevant to the development of drug resistance and tumor recurrence. Hence, CAF are a promising therapeutic target. We have previously demonstrated that activation of the Notch1 signaling pathway confers normal human dermal fibroblasts a suppressive phenotype to melanoma growth. Here, we further investigated whether manipulation of Notch signaling in melanoma-associated fibroblasts (MAF) isolated from human melanoma patients alter their regulatory phenotype by which affects melanoma growth. 

Methods:  MAF were isolated from human primary and metastatic melanoma lesions and characterized. Notch pathway activity in MAF versus normal human dermal fibroblasts was examined using Notch pathway RT2-PCRArray and immunoblotting. Enforced activation of Notch pathway was achieved by transducing MAF with lentiviral vector encoding active form of Notch1 (NIC). The effect of MAF engineered to carry high Notch activity on melanoma growth was tested by in vitro co-culture and in vivo co-engrafting animal model. 

Results: Isolated MAF are a-smooth muscle actin (a-SMA) and fibroblast activation protein (FAP) positive. MAF exhibited a relatively lower Notch activity compared to normal human dermal fibroblasts. Enforced activation of Notch1 pathway downgraded cellular activities of MAF. MAF engineered to carry high Notch activity significantly inhibited melanoma cell growth in vitro and retarded xenografted human melanoma cell growth on mouse skin. 

Conclusion: 

Notch signaling pathway appears to be a ‘molecular switch’ in determining the function of tumor stromal fibroblasts. Notch signaling activity is lower in MAF. Increasing Notch signaling activity in MAF confers them a tumor-suppressing phenotype towards melanoma growth. Our study demonstrated that Notch signaling functions as a critical molecular determinant in governing the tumor-regulating role of tumor stromal fibroblasts and provided a novel approach to target tumor microenvironment by manipulation of Notch signaling in stromal fibroblasts.

 

 

23.05 Tumor Stroma and Melanoma Progression Depends on Host Endothelial Cell Derived SDF-1

Posted on December 22, 2014

Z. N. Maan1, M. S. Hu1, A. J. Whittam1, L. H. Fischer1, D. Duscher1, G. G. Walmsley1, G. W. Krampitz1, J. Barrera1, D. Atashroo1, M. Rodrigues1, A. Whitmore1, M. Findlay1, P. H. Lorenz1, M. T. Longaker1, G. C. Gurtner1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction: The surrounding vascularized stroma of malignant cells provides a microenvironment critical for tumor development and subsequent progression of cancer. Understanding the signaling mechanisms governing the microenvironment and its interplay with tumor cells has the potential to inform more efficient tools for cancer management. Stromal-derived factor-1 (SDF-1) has been implicated in regulating stem cell microenvironments and has also been shown to influence cancer biology, though its specific role remains unclear. Utilizing newly developed murine models, we investigated the role of host endothelial derived SDF-1 in mediating endothelial-fibroblast interactions during tumor stroma formation and cancer progression.

Methods: Murine B16 melanoma cells were seeded onto a pullulan-collagen hydrogel, which were subcutaneously implanted in SDF-1 endothelial (eKO) and fibroblast knockout (fKO), CXCR4 fibroblast knockout (frKO), and floxed control mice. The mice were photographed and weighed and had their tumor size measured at regular intervals. After 28 days, tumors were harvested, weighed and processed for histology. The effects of SDF-1 on fibroblast proliferation, migration, survival and angiogenic profile were assessed in vitro.

Results: eKO mice demonstrated significantly reduced tumor burden compared to fKO, frKO, and control mice, in terms of height (*p < 0.05), weight (*p < 0.05) and volume (*p < 0.05). SDF-1 increased fibroblast proliferation (*p<0.001), migration (*p<0.01), and survival (* p < 0.05) in vitro. Co-culture demonstrated that decreased endothelial production of SDF-1 significantly reduced fibroblast expression of VEGF (*p < 0.05) and FGF-2 (*p < 0.05) in vitro.

Conclusion: Endothelial cell SDF-1 (eSDF-1) plays a pivotal role in cancer biology, regulating the expression of cytokines responsible for neovascularization and modulating fibroblast behavior and survival capacity, thereby modulating tumor stroma formation and tumor progression.

 

23.06 Neutrophil Extracellular Traps Promote Tumor Progression after Liver Ischemia Reperfusion

Posted on December 22, 2014

S. Tohme1, H. Huang1, A. Al-Khafaji1, A. Tsung1  1University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA

Introduction: Previous studies have shown that after liver ischemia-reperfusion (I/R), the growth of hepatic metastatic tumors increase; however, the mechanisms remain unclear. We have previously shown that during I/R neutrophils release Neutrophil Extracellular Traps (NETs), extracellular fibers composed of DNA and neutrophil proteins, which heighten the inflammatory response and subsequent liver injury. Some emerging data suggests that NETs may play a role in tumor progression, but the role of NETs in I/R-mediated acceleration of tumor growth is unknown. We hypothesize that NETs formed in response to liver I/R promote tumor growth and progression.

Methods: In-vitro, neutrophils were harvested from mice bone marrow. Neutrophils were treated with PMA, a well-known stimulator of NET formation, for 4hrs. Media was collected and co-cultured with mc38 cancer cells. MTT assays, Western blots, invasion and migration assays were used for analysis. In-vivo, colorectal liver metastases were induced in C57BL/6 mice by spleen injection of mc38 cells. Animals were then subjected to partial liver I/R vs. sham surgery followed by splenectomy with or without daily DNase I injections, a known inhibitor of NETs. The livers were harvested 3 weeks later for analysis.

Results: In vitro, there was a significant increase in proliferation of mc38 cells co-cultured with stimulated neutrophil media compared to untreated mc38 cells by MTT assay. This surge in proliferation was significantly decreased when DNaseI was added. Similarly, there a was significant increase in the invasion and migration of mc38 cells treated with stimulated media compared to control or addition of DNaseI. By using western blot analysis, the addition of neutrophil media resulted in the activation and phosphorylation of STAT3, a protumorigenic transcription factor, and p38, p65 and JNK, mitogen activator protein kinases implicated in tumor growth and development under stressful conditions. The activation of these proteins was significantly decreased with the addition of DNaseI. In vivo, Mice in the I/R group had grossly significantly more tumor growth and higher liver/body weight when compared to mice in the sham group. Addition of DNaseI to mice receiving I/R resulted in a significant decrease in tumor growth. Histologically, the tumors from the I/R + DNaseI group has less proliferation by Ki67 staining compared to the I/R group. There was a similar decrease in the activation of the MAP kinases from the tumor tissue obtained from the I/R + DNase1 mice compared to the I/R group. There was no difference in tumor growth between mice in the sham groups with or without DNaseI.

Conclusions: Liver I/R is a strong stimulus for metastatic tumor growth. NETs formed during I/R contributes to tumor growth by activating protumorigenic signaling pathways. DNaseI may represent a novel therapy for targeting NET-mediated tumor growth during liver I/R.  

23.07 Rhodiola Crenulata Inhibits Wnt/β-Catenin Signaling in the Treatment of Glioblastoma Multiforme

Posted on December 22, 2014

M. C. Mora1,2, K. E. Wong1,2, M. V. Tirabassi3, R. B. Arenas1,2, S. Schneider2  1Baystate Medical Center,Surgery,Springfield, MA, USA 2Pioneer Valley Life Science Institute,Springfield, MA, USA 3Baystate Children’s Hospital,Surgery,Springfield, MA, USA

Introduction:
Extracts from Rhodiola crenulata, a Tibetan plant, have anti-neoplastic effects on a variety of cancers. The purpose of this study is to determine if Rhodiola crenulata extract exhibits anti-neoplastic properties on Glioblastoma Multiforme (GBM) in-vitro.

Methods:
Human U-87MG GBM cell line was pretreated with 200ug/ml of RC or vehicle control for 24, 48, 72, and 96 hours.  Cell proliferation was then measured using a MTS calorimetric assay. Clonogenicity assay was used to further evaluate cell proliferation in which 100 cells were treated with vehicle control, 100ug/ml of RC, radiation, or radiation and RC together and were observed for colony growth.  Neurosphere formation was evaluated by phase contrast microscopy comparing treatment with 100ug/ml RC to vehicle control.  For the remainder of experiments cells were pretreated with 200ug/ml RC for 72 hours. qRT-PCR was performed to evaluate the expression of proliferation and differentiation genes. Expressions of β-catenin and Glial fibrillary acidic protein (GFAP), a protein marker of differentiation, were measured with immunocytochemistry (ICC). To assess transcriptional activity of the Wnt/β-Catenin axis, a luciferase-β-Catenin-reporter assay was conducted. After transfection, cells were treated with 200μg/ml of RC or vehicle control and a dual luciferase reporter assay was performed to quantify luciferase activity up to 72 hours following RC treatment.

Results:
MTS assay revealed a 65% decrease in proliferation with RC therapy starting at 48 hours (p=0.01) and a 70% reduction observed at 96 hours (p=0.0006).  Colony formation was reduced by more than half in cells treated with RC (p=0.03) and colonies were further reduced if treated with radiation and RC together (p=0.009). Neurosphere formation was eliminated in all wells after treatment with RC (Figure 1A-B).  C-myc and cyclin-D1, genes of proliferation associated with the Wnt/ β-catenin pathway, had a 60-fold and 55-fold reduction in expression respectively (p<0.05) after RC treatment. ID-1, oct-4, and snail, genes of differentiation were noted to have a 95-fold, 50-fold, and 40-fold reduction in expression respectively, (p<0.05) after treatment with RC.  ICC revealed that RC induced GFAP expression (Figure1C-D) and decreased nuclear expression of β-catenin. Luciferase assay performed on luciferase-β-Catenin-reporter transfected cells revealed decreased Wnt promoter activity (p=0.0041) following treatment with RC.

Conclusion:
Rhodiola crenulata extract effectively suppresses proliferation, stimulates differentiation, and eliminates tumorsphere formation of GBM cells in-vitro. The effects observed are likely secondary to the inhibition of the Wnt/β-catenin signaling pathway. 
 

23.08 Patient-Derived Pancreatic Cancer Xenografts Reflect Patient Tumor Biology and Predict Outcome

Posted on December 22, 2014

R. Marayati1, C. J. Tignanelli2, J. Yeh1,2,3  1Lineberger Comprehensive Cancer Center,Chapel Hill, NC, USA 2University Of North Carolina At Chapel Hill,Department Of Surgery,Chapel Hill, NC, USA 3University Of North Carolina At Chapel Hill,Department Of Pharmacology,Chapel Hill, NC, USA

Introduction: Pancreatic cancer is a lethal malignancy with an extremely poor prognosis and lack of effective therapy. Patient-derived xenograft (PDX) models have been used as a preclinical platform to investigate the underlying biology of pancreatic cancer and to evaluate new biomarkers and anti-cancer therapies. We sought to assess whether PDX models reliably reflect the tumor biology and course of disease in patients with pancreatic cancer.

Methods: Surgically resected pancreatic ductal adenocarcinomas were obtained from 56 de-identified patients after IRB approval. Tumors were engrafted either orthotopically or subcutaneously into immunocompromised mice and passaged over time. PDX tumors were considered successful if they reached a volume of at least 200 mm3 and were passaged at least twice. The time to 200 mm3 was defined by the number of weeks it took for a tumor to reach a volume of 200 mm3 in the initial passage. At each passage, pancreatic ductal adenocarcinoma histology was confirmed by hematoxylin and eosin staining and KRAS mutation status was determined by pyrosequencing. Overall survival (OS) and recurrence-free survival (RFS) were calculated using the Kaplan-Meier method and compared using the log-rank test. Pearson’s chi-square and Fisher's exact tests were used to compare categorical variables (IBM SPSS Statistics v20).

Results: Out of 56 engrafted patient tumors, 37 (66%) met our criteria for success. The median follow-up of our patient cohort was 34 months. Patients with successfully engrafted tumors had a significantly shorter median OS (12 months vs. 21 months, p=0.039). Successful engraftment did not correlate with known pathological variables such as differentiation, lymph node involvement, stage of disease, or tumor margin status. Furthermore, successful engraftment was independently predictive of OS in a multivariate Cox regression model that included lymph node involvement, stage, and tumor margin status, with a hazard ratio of 0.497 (95% CI [0.248, 0.997], p=0.049). Of the 37 successfully engrafted tumors, 79% had KRAS mutations. Patients whose tumors took longer than 20 weeks to reach a volume of 200 mm3 had a significantly longer median RFS (18 months) than those patients whose tumors took less than 20 weeks (9 months, p=0.013). In a multivariate Cox regression model that included KRAS mutation status, lymph node involvement, stage, and tumor margin status, time to 200 mm3 was the single independent predictor of RFS with a hazard ratio of 3.429 (95% CI [1.075, 10.936], p=0.037).

Conclusions: Our results show that PDX models accurately recapitulate the growth pattern and inherent tumor biology of patients with pancreatic cancer, in that tumors that grow faster are associated with earlier recurrences. In addition, patients with successfully engrafted tumors have a significantly poorer overall survival, suggesting that these tumors represent a more aggressive subset for which novel therapies are needed.

23.09 PI3K and Pan-ErbB Inhibition Overcomes ErbB Cross-Talk in Pancreatic Ductal Adenocarcinoma

Posted on December 22, 2014

C. J. Tignanelli1, J. Stratford2,3, R. A. Moffitt2, J. Yeh1,2,3  1University Of North Carolina At Chapel Hill,Department Of Surgery,Chapel Hill, NC, USA 2University Of North Carolina At Chapel Hill,Lineberger Comprehensive Cancer Center,Chapel Hill, NC, USA 3University Of North Carolina At Chapel Hill,Department Of Pharmacology,Chapel Hill, NC, USA

Introduction:
 

     Resistance to single kinase inhibition is one of the main challenges in the treatment of cancer patients. We have previously shown that treatment with BKM120 (a pan-class 1 PI3K inhibitor, currently in Phase I/II clinical trials) resulted in tumor growth inhibition (p = 0.017) but not regression in a pancreatic ductal adenocarcinoma (PDAC) patient derived xenograft (PDX) mouse model, suggesting that tumors may be adapting to PI3K inhibition. When evaluating possible mechanisms of resistance we identified ErbB1, ErbB2 and ErbB3 activation in response to BKM120 treatment in both cell lines and PDX tumors.  The ErbB family is a well-established therapeutic target in multiple cancers. Intense cross-talk is known to occur between ErbB isoforms perhaps contributing to the limited effectiveness of single ErbB inhibition seen in the clinic in the case of erlotinib in PDAC. We hypothesized that pan-ErbB inhibition would be required in combination with BKM120 for optimal tumor response.

Methods:

      PDAC cell lines were treated with 475 nM of BKM120 and either 25 nM of siErbB1, siErbB2, siErbB3, or 40nM dacomitinib (a pan-ErbB inhibitor currently in Phase III clinical trials). Target inhibition was confirmed by immunoblotting.  Cellular viability was measured using a cellular growth assay after 72 hours of treatment.

Results:
 

     We found that the combination of BKM120 and dacomitinib inhibited proliferation in 10 of 10 PDAC cell lines (p < 0.01) and was more effective than BKM120 alone. Furthermore, BKM120 and dacomitinib showed impressive synergy across all cell lines with a mean combination index of 0.24 (0.00245 – 0.49). We next evaluated whether inhibition of any single ErbB family member would be sufficient for synergy with BKM120. We observed significantly greater growth inhibition after treatment with BKM120 and dacomitinib (68%) in the HPAC cell line compared with BKM120 + siErbB1 (48%, p < 0.001), BKM120 + siErbB2 (51%, p < 0.001), or BKM120 + siErbB3 (55%, p = 0.002), suggesting that single ErbB inhibition is not as effective as pan-ErbB inhibition.

Conclusion:

     Our results suggest that inhibition of any single ErbB will not be sufficient to overcome the adaptive response of tumors to PI3K inhibition. Instead, combined treatment with a pan-ErbB and PI3K inhibitor will be necessary.  Combination studies in PDX models are ongoing. Pan-ErbB and PI3K inhibition in PDAC may be more effective than either single agent alone and should be considered in clinical trials.

23.10 Identification of Glioblastoma Stem-Like Cell-Binding Human Antibodies by Yeast Biopanning

Posted on December 22, 2014

J. S. Kuo1, M. Zorniak1, E. V. Shusta2, J. S. Kuo1  1University Of Wisconsin,Neurological Surgery,Madison, WI, USA 2University Of Wisconsin,Chemical And Biological Engineering,Madison, WI, USA

Introduction:  Glioblastoma multiforme (GBM) is a poorly treated human brain cancer with few tools available for detection and isolation of their therapeutic-resistant, glioblastoma cancer stem-like cell (GSC) populations. A non-immune, human single-chain antibody (scFv) yeast display library was mined for human GSC-specific antibodies via biopanning.

Methods:  Standard molecular biology techniques were employed, along with novel yeast biopanning strategy involving positive and negative selections (Wang et al, Nature Methods. 2007 Feb;4(2):143-5) followed by screening identified clones against patient-derived GSC and control normal neural stem cell lines. Briefly, GSC-binding scFv candidates were isolated after combining nine rounds of enrichment via positive screening with several rounds of negative screening against normal human astrocytes, neural stem cells, and serum-cultured GBM tumor. 

Results: Clonal scFv assessment by restriction enzyme fingerprinting revealed 62 unique scFv clones. Each yeast-displayed scFv clone was characterized for qualitative binding selectivity against 12 distinct human lines of normal, GSC, and patient-matched GBM cells. Clone scFv-9.7, in particular, demonstrated substantial binding specificity for five GSC lines representing three different classes of tumor invasiveness and various neural progenitor lineages. GSC-specificity was further verified using secreted and purified scFv-9.7, which detected highly infiltrative GSCs from tumor xenografts via flow cytometry, and successfully targeted and fluorescently visualized tumor xenografts in vivo when conjugated with a near-infrared dye. 

Conclusion: In summary, rapid screening via yeast antibody library biopanning identified human GSC-specific antibodies for potential development into immunotargeted diagnostics and therapeutics in brain cancer. 

 

24.01 Demonstration of the Effects of Portal Vein Ligation on Glucose Metabolism Using In Vivo Multi-modal PET/MRI Measurements in Healthy Rat Liver

Posted on December 22, 2014

A. Fülöp1, A. Budai1, D. Korsós1, V. Hegedűs1, L. Harsányi1, I. Horváth2, N. Kovács3, D. Máthé3, K. Szigeti2, A. Szijártó1  11st Department of Surgery, Semmelweis University, Budapest, Hungary 2Department of Biophysics and Radiation Biology; Semmelweis University; Budapest; Hungary 3CROmed Translational Research Centers; Budapest; Hungary

Introduction:
Portal vein ligation (PVL) results in ipsilateral atrophy and hypertrophy of contralateral liver segments. It is unknown how PVL affects metabolic patterns of hepatic tissues. The aim of this study is to evaluate the effect of PVL on glucose metabolism, using multi-modal PET/MRI imaging in healthy rat liver. 

Methods:
Male Wistar rats (n=30) underwent PVL. 2-deoxy-2-(18F)fluoro-D-glucose (FDG) PET/MRI imaging and morphological/histological examination were performed before; 1-, 2-, 3-, 7-days after PVL. Dynamic PET data were collected and the standardized uptake values (SUV) for ligated and non-ligated liver lobes were calculated in relation to cardiac left ventricle (SUVVOI/SUVCLV) and mean liver SUV (SUVVOI/SUVLiver). 

Result:
PVL induced atrophy of ligated lobes, while non-ligated liver tissue showed compensatory hypertrophy. Dynamic PET scan revealed altered FDG kinetics in both ligated and non-ligated liver lobes. SUVVOI/SUVCLV significantly increased in both groups of lobes, with a maximal value at 2nd postoperative day and returned near to the baseline 7 days after the ligation. After PVL, ligated liver lobes showed significantly higher tracer uptake compared to the non-ligated lobes (significantly higher SUVVOI/SUVLiver values were observed at postoperative day 1, 2 and 3). The homogenous tracer biodistribution observed before PVL reappeared by 7th postoperative day. 

Conclusion:
Our study demonstrated an altered glucose metabolism in both ligated and non-ligated liver lobes. The observed alterations in FDG uptake dynamics should be taken into account during the assessment of PET data until the PVL induced atrophic and regenerative processes are completed. 

24.02 Photodynamic Therapy of Human Lung Cancer Xenografts in Mice

Posted on December 22, 2014

C. Nwogu1, P. Pera1, K. Attwood1, W. Bshara1, R. Pandey1  1Roswell Park Cancer Institute,Buffalo, NY, USA

Introduction: Photodynamic therapy may be effective for treatment of peripheral tumors in patients unable to tolerate surgery. We hypothesized that a novel photosensitizer, PS1, would be more effective than the standard agent, Porfimer sodium (Photofrin® or PFII), in treating human lung cancer xenografts in mice.

Methods: Patient-derived NSCLC xenografts were established subcutaneously in 20 SCID mice. There were two treatment and two control groups. Two groups of 5 mice were injected with PS1 or PFII. 24 hours later, the subcutaneous tumors in these mice were treated with laser light at a wavelength of 630nm for PFII and 665nm for PS1. 4 mice were treated with laser light with no photosensitizer and 6 mice received no treatment at all. The mice were observed for 60 days. Bonferroni adjusted methodology was used to compare the tumor growth rates between treatment groups. The tumor growth endpoint, time-to-1000mm3, was evaluated using standard Kaplan-Meier methods and compared between groups using the log-rank test. All analyses were conducted in SAS v9.3 (Cary, NC). One representative tumor in each group was cut and stained with H&E and Caspase3 to evaluate necrosis and apoptosis.

Results: Tumor re-growth pattern in the mice is illustrated in figure 1. The median time-to-1000mm3 was 12, 12, 26 and 52 days for the control, light only, PF II and PS1 groups (p<0.001). H&E analysis revealed <1%, 0%, 67% and 80% necrosis, respectively from representative samples in the same four groups. Caspase3 positivity in these groups was 2%, <1%, 17% and 39% respectively.

Conclusion:

The mice treated with PS1 exhibited a longer time for tumor regrowth, showed more tumor necrosis and apoptosis compared to the other treatment groups. Thus, the novel photosensitizer, PS1, was demonstrated to be more effective than Porfimer sodium in treating human lung cancer xenografts in a preclinical pilot study and deserves further study.

 

Figure 1: Effect of photodynamic therapy on human NSCLC implanted in SCID mice

 

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