61.08 Fat Grafting into Younger Recipients Positively Correlates with Volume Retention

Posted on January 18, 2018

C. P. Blackshear1, N. N. Chung1, D. M. Irizarry1, E. A. Brett1, J. M. Flacco1, A. Momeni1, G. K. Lee1, D. H. Nguyen1, M. T. Longaker1,2, D. C. Wan1  1Hagey Laboratory For Pediatric Regenerative Medicine,Department Of Surgery, Plastic And Reconstructive Surgery Division, Stanford University School Of Medicine,Stanford, CA, USA 2Institute For Stem Cell Biology And Regenerative Medicine,Stanford University,Stanford, CA, USA

Introduction:  Soft tissue deficits associated with various craniofacial anomalies can be addressed by fat grafting, although outcomes remain unpredictable. Furthermore, consensus does not exist for timing of these procedures. While some advocate approaching soft tissue reconstruction after the underlying skeletal foundation has been corrected, other studies have suggested earlier grafting may exploit a younger recipient niche that is more conducive for fat graft survival. As there is a dearth of research investigating effects of recipient age on fat graft volume retention, this study compared the effectiveness of fat grafting in younger versus older animals through a longitudinal, in vivo analysis.

Methods:  Human lipoaspirate from three healthy female donors was grafted subcutaneously over the calvarium of immunocompromised mice. Volume retention over 8 weeks was evaluated using micro-computed tomography in three experimental ages – 3-weeks old, 6-months old, and 1-year old. Histology was performed on explanted grafts to evaluate graft health and vascularity.

Results: Greatest retention of fat graft volume was noted in the youngest group compared to both older groups (*p < 0.05) at 6 and 8 weeks following grafting. Histological and immunohistochemical analyses suggested that improved retention in younger groups was associated with more robust vascularization.

Conclusion: Our study provides evidence that grafting fat into a younger recipient site correlates with improved volume retention over time, suggesting that beginning soft tissue reconstruction with fat grafting in patients at an earlier age may be preferable to late correction.

 

61.09 T Cells Potentiate Wound Repair By Regulating Extracellular Matrix Inflammation and Fibrosis

Posted on January 18, 2018

E. H. Steen1, X. Wang1, S. Balaji1, H. Li1, M. Fahrenholtz1, M. Butte4, P. Bollyky3, S. Keswani1,2  1Baylor College Of Medicine,Department Of Surgery,Houston, TEXAS, USA 2Texas Children’s Hospital,Department Of Pediatric Surgery,Houston, TEXAS, USA 3Stanford University,Department Of Medicine, Division Of Infectious Diseases And Geographic Medicine,Palo Alto, CALIFORNIA, USA 4University Of California, Los Angeles,Department Of Pediatrics, Division Of Allergy And Immunology,Los Angeles, CALIFORNIA, USA

Introduction: Lymphocytes, specifically T cells, have been shown to have a salutary effect in tissue repair by producing an anti-inflammatory milieu, which can result in decreased fibrosis. Further, it is established that tension impairs wound repair and increases fibrosis. How exactly the mechanical environment of a wound is sensed and transduced and how it affects the behavior of immune cells has not been fully elucidated. We hypothesize that select populations of lymphocytes are mechanosensitive and influence fibrosis and the dermal response to tissue injury.

Methods:  In a loss-of-function experiment, excisional stented wounds were made in mice devoid of T and B cells (SCID) and compared to wild type (WT) mice for degree of inflammation (CD45+ lymphocytes and F4/80+ macrophages) and fibrosis (trichrome, α -SMA staining). Adoptive transfer experiments were performed on SCID mice by adding the following lymphocyte populations from WT mice: 1) total lymphocytes (TL); 2) CD4+CD25-T cells; 3) CD4+CD25+T cells. Cutaneous wounds were then created in the syngeneic mice. Wound samples were harvested and analyzed as above. To understand the role of mechanosensitivity, healthy human donor lymphocytes were embedded in low, medium and high density hydrogels to mimic extracellular matrix (ECM) stiffening as would occur in fibrosis; subsequent gene expression was assessed by PCR array. This experiment was repeated with Pyk2 inhibitor to block lymphocyte mechanosensation. Data presented as means; p-values by ANOVA and t-test.

Results: SCID mice demonstrate increased dermal fibrosis (increased trichrome staining on days 14 and 30; α -SMA expressed on IHC on day 7) and an exaggerated inflammatory infiltrate compared to WT mice (CD45: SCID 34.5% vs WT 21%; F4/80: SCID 29.4% vs WT 3.2%; p>0.05). Adoptive transfer of all three T cell types was successful and reduces inflammatory infiltrates as compared to control SCID mice (neutrophils: TL 12.9%; C4+CD25-T cells 16.5%; CD4+CD25+T cells 1%; macrophages: TL 5.8%; CD4+CD25-T cells 25%; CD4+CD25+T cells 22.2%). Fibrosis decreases in the TL and CD4+CD25+ subpopulations (decreased trichrome staining on day 7). On PCR array of 84 genes, expression of CXCL5, TLR7, C3, and CCR2 in human lymphocytes changes in response to increasing ECM stiffness (CXLC5 323-fold, TLR7 7.4-fold, C3 20-fold, and CCR2 4.5-fold higher in high vs low density gels). The addition of Pyk2 inhibitor to the system returned gene expression to control values.

Conclusion: Our data suggests that lymphocytes are mechanosensitive and respond to increasing tension by enhancing expression of inflammatory cytokines and chemokines, in part via a Pyk2-dependent mechanism. It also suggests that certain T cell subsets have a significant role in governing the dermal fibrotic phenotype. Investigating the molecular cues between mechanical forces and lymphocytes in dermal scarring may lead to the identification of anti-fibrotic agents for the skin and beyond.

61.05 Differential Expression of Fatty Acid Synthase in Patients with Carotid Artery Stenosis

Posted on January 18, 2018

G. S. De Silva1, M. S. Darwech1, K. S. Desai1, N. S. Harroun1, C. F. Semenkovich3, M. A. Zayed1,2  1Washington University In St. Louis,Division Of Vascular Surgery,Saint Louis, MO, USA 2Veterans Affairs St. Louis Health Care System,Division Of Vascular Surgery,Saint Louis, MO, USA 3Washington University In St. Louis,Division Of Endocrinology, Lipid, And Metabolism,Saint Louis, MO, USA

Introduction:
Ischemic stroke is the third most common cause of death in the United States, and diabetes (DM) is one of the strongest risk factors. In the setting of DM, de novo tissue lipogenesis is significantly altered, leading to differential expression of lipid species in various tissue beds. Central to this process is Fatty Acid Synthase (FAS), which regulates synthesis of long chain saturated fatty acids in various tissues. We recently observed higher levels of circulating serum FAS (sFAS) in patients with advanced vascular disease.  We therefore hypothesized that sFAS expression and activity is altered between DM and non-DM patients

Methods:
For this study, we enrolled DM, non-DM, and control patients (no arterial occlusive disease) in an IRB-approved vascular biobank and database between October 2014 and February 2016. In consenting patients, fasting serum samples were collected and evaluated for sFAS content and activity using ELISA-based assays. Differences in sFAS content and activity across patient cohorts were summarized as mean ± SEM, and significance was determined using Kruskal-Wallis analysis, Mann-Whitney U test, and Spearman correlation analysis.  All p-values  0.05 were considered to be significant

Results:
26 patients (13 DM, 13 Non-DM) undergoing carotid endarterectomy (CEA), as well as 13 control patients were prospectively enrolled and retrospectively reviewed.  There were no significant differences between demographics of enrolled patients except the use of metformin in the DM cohort.  sFAS activity was significantly higher in DM compared to non-DM patients (7.1 ± 0.85 vs 4.5 ± 0.74 pmoles of NADPH consumed/mg of protein; p=0.05). Both DM and non-DM patients had higher sFAS activity compared to control patients (2.7 ± 0.72; p =0.01). Although we did not observe a difference between FAS content between DM and non-DM patients (6.0 ± 1.4 vs 6.2 ± 1.2 ng/mL, p=0.99), patients with carotid artery stenosis had higher sFAS levels relative to control patients (6.1 ± 0.93 vs 1.5 ± 0.23 ng/mL; p<0.001) 

Conclusion:
Our study confirms that sFAS circulates in the serum of DM and non-DM patients with high-grade carotid artery occlusive disease. sFAS activity appears to be higher in the serum of diabetic patients.  These findings suggest that sFAS and its downstream effectors may serve as important biomarkers for disease progression in patients with DM and arterial occlusive disease.
 

61.06 Adjuvant Therapy Improves Survival In a Novel Immunocompetent Model of Pancreatic Cancer Resection.

Posted on January 18, 2018

B. Giri1, V. Sethi1, B. Garg1, A. Farrantella1, S. Kurtom1, S. Modi1, Z. Malchiodi1, L. Hellmund1, S. Ramakrishnan1, S. Banerjee1, A. Saluja1, S. Lavania1, V. Dudeja1  1University Of Miami,Department Of Surgery,Miami, FL, USA

Introduction:  Most patients with localized pancreatic ductal cancer (PDAC), even after tumor resection and systemic chemotherapy, succumb to disease recurrence. Current animal models for PDAC do not recapitulate the patterns of disease recurrence following resection of primary tumor. The aim of the current study was to study recurrence and evaluate therapeutic strategies in an immunocompetent, clinically relevant model of PDAC resection. 

Methods:  Cancer cells derived from tumors arising in KPC (KrasLSL.G12D/+; p53R172H/+; PdxCretg/+) mice were co-injected with stromal cells (Pancreatic stellate cells from C57/B6 wild type mice) into the pancreas of immunocompetent mice to simulate the stroma rich tumor seen in human PDAC. Tumors were allowed to grow, resected 10-20 days later (after excluding metastatic disease) and animals were serially followed for tumor recurrence. Therapy with Gemcitabine (200mg/kg/week) and Paclitaxel (25 mg/kg/week) was used as a model to study the effect of adjuvant therapy in animals who underwent tumor resection. 

Results: Similar to that seen in human disease, tumors formed after co-injection of KPC cells and stromal cells had a dense desmoplastic reaction. Resection of primary tumors at 20 days after implantation revealed uniform tumor volumes and microscopic examination of resected tumors showed negative margins indicating R0 resection. Resembling human PDAC, most animals developed intra-abdominal recurrence on follow up. Furthermore, when tumors were resected at 20 days, these animals showed improved overall survival (Median survival: 54.7) compared to mice that did not undergo resection (Median survival: 47). There was an even greater improvement in overall survival in mice which underwent adjuvant chemotherapy (Median survival: 71.5) demonstrating usefulness of this model in studying clinically relevant scenarios.  

Conclusion: We describe a novel immunocompetent animal model that recapitulates human disease in morphology and recurrence patterns. We show that it can be used to evaluate therapy in clinical scenarios and may help characterize factors responsible for disease recurrence. 
 

61.07 A Retrievable RESCUE Stent for Hemorrhage Control in a Porcine Model of Caval Injury

Posted on January 18, 2018

C. C. Go1, J. M. Kuhn3, Y. Chen2, Y. Chun2, B. Tillman1,3  1University Of Pittsburgh Medical Center,Division Of Vascular Surgery,Pittsburgh, PA, USA 2Swanson School Of Engineering,Department Of Industrial Engineering,Pittsburgh, PA, USA 3University Of Pittsburgh,McGowan Institute Of Regenerative Medicine,Pittsburgh, PA, USA

Introduction: Traumatic and iatrogenic injuries of the inferior vena cava (IVC) present a difficult and life-threatening problem. Our group has previously demonstrated the feasibility of a retrievable RESCUE stent for angiographic control of hemorrhage in a terminal model. The goals of this present study were to evaluate hemorrhage control, demonstrate continued venous return angiographically, and characterize hemodynamics of the animal before and after stent deployment using a survival porcine model of caval injury.

Methods: A retrievable RESCUE stent was constructed with a previously described “petal and stem” design with a nitinol scaffold and then covered with a sleeve of polytetrafluoroethylene. Three anesthetized pigs underwent standardized 22-French injury of the IVC. Animals were passively hemorrhaged for 1200 mL followed by stent deployment from a percutaneous femoral access and imaged with venography. After one hour, the RESCUE stent was retrieved, the injury repaired, and resuscitation with cellsaver blood and crystalloid was completed. Animals were followed for 48 hours postoperatively.

Results: Upon injury, 1200 mL of blood loss occurred in under 5 minutes. Deployment of the RESCUE stent took less than 2 minutes under fluoroscopy and provided instantaneous hemostasis, while venography confirmed normal venous return to the heart (Figure 1). The average mean arterial pressure of the three animals was 47 mm Hg at baseline, fell to 32 mmHg after injury, and returned to 44 mmHg after stent placement and resuscitation. Central venous pressures followed a similar trend: 10 mmHg prior to injury, 8 mmHg post-injury, and 10 mmHg post-stent/resuscitation. The stent was retrieved easily with sheath advancement at the time of permanent vascular repair. All animals survived the 48-hour postoperative period with stable hemoglobin levels and absence of organ failure as determined by serum chemistries.

Conclusion: A RESCUE stent provided rapidly-deployed hemorrhage control in a porcine model of caval injury. Its use reversed hemodynamic instability and allowed for eventual permanent repair over an hour after injury with successful postoperative recovery. This approach may be useful for “damage control” when exposure is limited due to massive caval bleeding or when vascular expertise is not immediately available. Further investigation may reveal whether the continued blood flow offered by the RESCUE stent translates to improved outcomes as compared to balloon occlusion or IVC crossclamping.

61.04 Metabalomic Signatures and Nitric Oxide Pathway Perturbations of End Stage Renal Disease Patients

Posted on January 18, 2018

K. M. Staton1, J. Rozowsky1, E. Quinlivan2, C. Kuppler1, S. Gray1, T. Garrett2, S. Berceli1  1University Of Florida,Vascular Surgery,Gainesville, FL, USA 2University Of Florida,Experimental Pathology,Gainesville, FL, USA

Introduction:

The systemic deleterious effects of End Stage Renal Disease (ESRD) are numerous and well established, however, advances in our understanding of the fundamental biology within the local small-molecule environment has been limited.  This study utilizes high-throughput techniques to quantify global and targeted metabolomics of ESRD in comparison to healthy counterparts.  We hypothesize that ESRD patients exhibit critical deviations in their metabolome, particularly within those elements surrounding nitric oxide (NO) production.  These changes may provide a mechanistic explanation of previously established functional and structural vascular abnormalities associated with ESRD.

 

Methods:

Human plasma samples were obtained from 5 patients with normal renal function and 5 patients with ESRD currently on hemodialysis.  Global metabolomic analysis was performed by LC-MS, and metabolites identified by their retention time and exact mass analysis using an in-house library.  Targeted metabolite analysis was performed by stable isotope dilution LC-MS/MS on 31 NO-associated metabolites of specific interest. Descriptive statistical analysis was performed to compare the respective metabolomes of ESRD to those with normal renal function.  

 

Results:

 

Targeted Metabolomics: Patients with ESRD in comparison to control patients had significant differences p-value <0.05 in 6 of the targeted 31 (19%) metabolites.  Of those metabolites 5 of 6 (83%) were increased significantly in the ESRD population, with 1 of 6 (17%) significantly decreased (FIGURE).  Global Metabolomics: 3788 metabolite signatures were detected, of which 1099 (29%) were mapped to specific compounds.  523 of the identified 1099 metabolites (47.6%) were found to be different (p<0.05) in the ESRD population, with 212 (41%) significantly increased vs 311 (59%) significantly decreased.

Conclusion:

With the loss of glomerular filtration we see significant alterations within the small-molecule environment of those with ESRD.  Targeted metabolomics provide insight into how these differences alter key regulators of vascular and cardiovascular homeostasis.  Our data would suggest patients with ESRD have decreased production of NO.  This is supported by our findings of ESRD patients exhibiting significant increases in both competitive NO inhibitors (SDMA, Methyl Arginine, and ADMA) and end products of alternative NO-related pathways (Citrulline, and Ornithine).  Expanding our understanding of the small-molecule environment and its effects will provide important insights into the etiology of altered vascular pathophysiology observed in the ESRD population and offer potential novel targets of intervention to re-establish normal homeostasis within the cardiovascular system. 
 

61.03 Distinct Lipid Mediator Profile Associated With Arteriovenous Fistula Maturation

Posted on January 18, 2018

J. Rozowsky1, K. Staton1, K. O’Malley1, M. S. Conte2, M. R. Spite3, S. Berceli1  1University Of Florida,Vascular Surgery,Gainesville, FL, USA 2University Of California – San Francisco,Vascular Surgery,San Francisco, CA, USA 3Brigham And Women’s Hospital,Anaesthesia,Boston, MA, USA

Introduction:

Active resolution of acute inflammation occurs at the interface between innate and adaptive immunity. Once thought to be a passive process, endogenous lipid mediators (resolvins and protectins) have been shown to be key promotors during this resolution phase. These mediators have been traditionally thought to protect organs from collateral damage and stimulate the clearance of inflammatory debris, more recently they have been shown to be important in monocyte recruitment and activation.  The link between cell-mediated inflammation and vascular pathophysiology has been firmly established and mounting evidence suggests an important role for these cells in flow-mediated vascular adaptation.  Undefined, however, is the importance of inflammation, and its subsequent resolution, in modulating adaptation in the extreme flow environment that characterizes a dialysis access arteriovenous fistula (AVF). We hypothesize active inflammation is critical to AVF maturation, and elevated levels of circulating, pro-resolving mediators act to dampen inflammation and impede AVF maturation.

Methods:

In patients undergoing single-stage AVF (n=20),  plasma was collected at two time points (pre-op and 6 weeks post-op) and analyzed for 22 different lipid mediators using liquid chromatography/mass spectrometry. AVFs were categorized as successful (n=10) if they were used for dialysis in four subsequent visits or failed (n=10) if they did not meet this benchmark. Because of the non-normal distribution, a two-factor Scheirer-Ray-Hare test, was used to identify lipids that are differentially expressed between the outcome groups or over time.

Results:

Six of 22 lipids (14-HDHA, 13-HDHA, 7-HDHA, 4-HDHA, 5-HEPE, and 15-HETE), all precursors to the bioactive mediators, were significantly associated with AVF maturation outcome (p-value < 0.05; false discovery rate (FDR) < 0.1), with all 6 of these compounds elevated in AVFs that failed to mature. None of the lipids demonstrated significant time dependent variations or time*outcome interactions.

Conclusion:

AVF maturation failure was associated with elevated levels of select precursors, upstream of the bioactive pro-resolvin mediators. While it is tempting to speculate that this pattern of compounds results in early dampening of the inflammatory process and maturation failure, it is also plausible that the observed accumulation of precursors is secondary to a stalled production of pro-resolving mediators and an overall escalation of inflammatory processes.  Further investigation regarding the biologic activity of these precursor compounds, and their overall implication in modulating local inflammation, is needed.

 

61.02 Patient Derived Xenografts Capture Cancer Cachexia and Preserve the Tumor Immune Signaling Profile

Posted on January 18, 2018

M. H. Gerber1, S. Judge2, D. Delitto1, A. Delitto2, R. Nosacka2, S. Wallet3, A. Judge2, J. Trevino1  1University Of Florida,Department Of Surgery,Gainesville, FL, USA 2University Of Florida,Department Of Physical Therapy,Gainesville, FL, USA 3University Of Florida,Department Of Oral Biology,Gainesville, FL, USA

Introduction:  Cancer cachexia is a debilitating syndrome associated with pancreatic adenocarcinoma that is not well understood. Patients with pancreatic adenocarcinoma present with a wide range of cachexia that is not necessarily related to tumor burden. Current preclinical models for studying pancreatic cancer cachexia involve subcutaneously injecting highly passaged cell lines into the flanks of mice. Cellular injections lack the tumor associated stroma component and its associated local and systemic immune signals. We hypothesize that using true patient derived xenografts (PDX), we can mimic cancer cachexia and immune signaling phenotypes in a murine model.

Methods:  Human pancreatic cancer PDX were created by implanting fresh surgical specimens of pancreatic cancer directly into the flanks of mice. At the time of surgery, a separate tumor sample and muscle biopsy were obtained from the patients. The tumor, spleen, and muscle tissues were collected from mice at endpoint. Expression of genes associated with cachexia were analyzed in both the human and mouse muscles. The patient tumor sample as well as the mouse tumors and spleens were lysed and analyzed using Luminex technology to detect multiple cytokines and chemokines of both human and mouse origin.

Results: Two patients with varying degrees of cachexia had PDX murine models created by implanting each graft subcutaneously into the flanks 5 mice labeled G1 and G2 with 5 mice receiving sham surgery. The two patients had varying degrees of fold changes in cachexia related genes including FoxO1, Socs3, STAT3, Atrogin-1, and MuRF1 that were significantly different compared to controls. The PDX mice mirrored the patient variability in muscle atrophy gene expression fold changes. Both G1 and G2 PDX mice had significant muscle atrophy compared to controls in the tibialis anterior muscles (G1: P < 0.001; G2: P < 0.001) and the gastrocnemius muscles (G1: P < 0.001; G2: P < 0.001).  The G1 and G2 PDX tumor and spleen lysates had significant differences in expression of multiple human and mouse cytokines/chemokines including KC, IL-8, IL-6, IP-10, IL-1beta, VEGF, GRO, FGF-2, GM-CSF, TGF-alpha, MDC, and IFN-alpha. The relative cytokine and chemokine ratios in PDX G1 and G2 tumor lysates were similar in their respective patient tumor lysates. 

Conclusion: Patient derived xenografts in pancreatic ductal adenocarcinoma capture cancer cachexia and preserve the cytokine/chemokine patient heterogeneity found in the local and systemic immune environments. The preservation of individual patient tumors using this PDX model will help us better understand the driving forces behind cancer cachexia and may lead towards personalized therapies.

60.08 Cationic Polymer Inhibits Pancreatic Cancer Invasion in vitro and Metastasis in vivo

Posted on January 18, 2018

I. A. Naqvi1, R. Gunaratne1, J. McDade1, D. Rouse1, J. Lee1, B. Sullenger1, R. White2  1Duke University Medical Center,Surgery,Durham, NC, USA 2University Of California – San Diego,Surgery,San Diego, CA, USA

Introduction:  Pancreatic cancer (PC) has the poorest prognosis of all major cancers with a 5-year survival of less than 5%. Cell-free DNA (cfDNA) has been well-validated as a biomarker that correlates with tumor burden. More recently, a role for cfDNA in tumor progression has also been identified. The latter occurs through activation of toll-like receptors (TLRs) in both tumor cells and the host environment, which can upregulate pro-metastatic signaling pathways. In addition, tumor-derived extracellular vesicles (EVs) such as microparticles and exosomes have also been implicated in promoting metastasis by activating pro-invasive pathways in tumor cells and pre-conditioning secondary sites for metastatic establishment. Our laboratory has previously shown that cationic polymers can neutralize cfDNA and abrogate inflammation in disease models of autoimmunity and infection. We investigated the ability of the cationic polymer, PAMAM-G3, to bind and inhibit cfDNA and EVs and thereby inhibit tumor invasion in vitro and metastasis in vivo. 

Methods:  Transwell-Matrigel invasion assays were performed using multiple PC cell lines. Cells were plated in the upper chamber with no serum +/- cfDNA, EVs, or PAMAM-G3. The bottom chamber was plated with complete media as a chemo-attractant. Cells treated with cfDNA, EVs, or PAMAM-G3 were also analyzed for activation of pro-inflammatory pathways (NFkB, MAPK, etc.). The in vivo model was performed in C57B6 mice that were injected with bioluminescent murine PC cells into their spleens and followed for liver metastases with bioluminescent imaging. Experimental mice were treated biweekly with intraperitoneal PAMAM-G3 (20 mg/kg) or saline starting 48 hours after pancreatic tumor cell implantation.

Results: PAMAM-G3 significantly inhibited in vitro invasion of pancreatic cancer cell lines in response to cancer patient serum-derived cfDNA and EVs. Moreover, PAMAM-G3 (20 mg/kg) treatment led to a significant reduction in liver metastasis without affecting primary tumor growth in vivo. Serum derived from saline treated mice induced higher levels of invasion than serum from polymer treated mice and exogenous polymer blocked this effect (Figure). (**** = p<0.0001, *** = p<0.001, ** = p<0.01, NS = not significant by t-test)

Conclusion: Cationic polymers such as PAMAM-G3 may represent a novel class of therapeutics to combat pancreatic cancer metastasis. These polymers can bind and neutralize cfDNA and tumor-derived EVs, thereby blocking activation of pro-inflammatory pathways within tumor cells, and reducing tumor invasion and metastasis. Cationic polymers will be tested in other pre-clinical models of cancer and in dosing studies for potential progression to clinical translation.

60.09 Innate Inflammation Regulation in Non-Human Primate Model of Brain Death and Kidney Transplantation

Posted on January 18, 2018

S. K. Odorico1, L. Zitur1, T. Zens1, P. Chlebeck1, J. Danobeitia1, A. D’Alessandro1, L. Fernandez1  1University Of Wisconsin,Department Of Surgery, Division Of Transplantation,Madison, WI, USA

Introduction: Brain dead (BD) kidney grafts in transplantation (TX) fail at higher rates than living donors; innate inflammation is well documented during BD but little is known about coagulation, fibrinolysis and contact regulation, which occurs in conjunction with the innate immune response.

Methods: Our group investigated the dynamics of these upstream inflammatory cascades through a 20-hour (20H) non-human primate (NHP) model of BD and kidney TX that mimics a clinical setting with three treatment groups: vehicle-treated BD (VTBD), sham surgery control and naïve. Analytes of the three pathways were measured via ELISA. Sham (N=4) and VTBD (N=7) protein measurements were compared within and between groups and correlated with outcomes. Gene expression microarrays of kidney biopsies were completed for sham (N=8), VTBD (N=8) and naïve (N=6) groups following the 20H BD period.

Results: CONTACT: An inflammatory state resulted from inducing BD. Factor XII (FXII) and bradykinin (BK), two pro-inflammatory analytes, were up-regulated at both 12H and 20H in VTBD (FXII: p=0.0842 and 0.0419; BK: p=0.0642 and 0.0903) compared to baseline (BL). BK increased 117.2% from BL at 20H in VTBD. Neither prekallikrein nor plasma kallikrein was significantly affected, and this cascade lacked significant differentiation between VTBD and sham. COAGULATION: Thrombin (p=0.0103), fibrinogen (p=0.0134) and vWF (p=0.0005) were all significantly upregulated following the 20H BD period compared to BL in VTBD. However, only thrombin (p=0.0753) increased in VTBD compared to sham. In VTBD, increased fibrinogen was correlated with increased day four serum creatinine (R2=692, p=0.0381). FIBRINOLYSIS: We observed a statistically significant increase in plasminogen (p=0.0002), tissue plasminogen activator (tPA) (p=0.0153) and tPA activity (p=0.0148) following the 20H BD period in VTBD compared to BL. Only plasminogen increased significantly in VTBD compared to sham (12H: p=0.0165, 20H: p=0.0038). MICROARRAY: VTBD genetic profiles lacked contact, coagulation and fibrinolysis activation compared to naïve animals. Interestingly, VTBD had a 14-fold macrophage activation (p<0.05) compared to naïve. Activation of other immune cell types did not occur. Additionally, platelet degranulation (10-fold enrichment, p<0.001) and associated-gene activation (13 genes, p<0.05) occurred in VTBD. Lastly, a down-regulation in metabolic processes in VTBD compared to naïve occurred. However, no clear clustering of genes between sham, naïve and VTBD was observed.

Conclusion: Protein and genetic analysis of contact, coagulation and fibrinolytic pathways portrayed a moderate inflammatory state building throughout the 20H BD period with minor differences between VTBD and sham. In addition, our results indicate an activation of macrophages and platelets, dictating further investigation into the potential roles these innate inflammatory cells play during BD in kidney TX.

60.10 Interstitial pressure affects efficiency of lymphangiogenesis in 3D culture

Posted on January 18, 2018

W. Jiao1, G. Lee1, S. Park1, B. Shaw1, C. Sung1, B. Han1, Y. Hong1, A. K. Wong1  1University Of Southern California,Plastic Surgery,Los Angeles, CA, USA

Introduction:  Lymphedema affects over 140 million individuals worldwide and can be caused by surgical injury or resection of lymph nodes.  Patients with lymphedema are at risk for infection, impaired extremity function, and in rare cases malignant transformation.  Pathogenesis begins with dysfunctional lymphatic drainage which then leads to fluid stasis, inflammation, fibrosis, and progressive stiffness of the soft tissue.    We hypothesize that a progressive increase in interstitial pressure has an adverse effect on lymphatic endothelial cell function, thus further contributing to disease progression.  To test this hypothesis, we cultured lymphatic endothelial cells (LEC) in 3-D scaffolds of different stiffness conditions to determine the effect of interstitial pressure on LEC proliferation and tube formation.  

Methods:  Human primary LECs were isolated from neonatal foreskin under an IRB approved protocol and cultured in Endothelial Cell Basal Medium (EBM, Lonza) with 15% FBS. LECs (1×104) were encapsulated in a range of percentages of biomimetic collagen hydrogels with physiologically relevant pressure including: 5.25 % (3.37 pKa), 6.0 % (5.57 pKa), 7.5 (12.8 pKa),and  9.0 % (15.31 pKa).  Experiments were performed in quadruplicate.  3-D in vitro cultures were observed for their phenotypic behavior and analyzed at defined time points for three weeks.  Two-way ANOVA (Tukey’s multiple comparison test) was used for multi-group statistical analysis.

Results:  Lymphangiogenesis, lymphatic vessel-like branching, and formation of collateral lymphatic-like structures were observed at 3 days post cell-seeding in 3D hydrogel and increased in a time dependent fashion (Fig. 1). Immunofluorescence demonstrated that these branch-like structures were positively stained for podoplanin, an LECs specific marker.  The optimal percentage of gel concentration which favored the formation of these structures was 6.0% gel. Using group comparisons, 6.0% and 7.5% groups were statistically different from 5.25% and 9.0 % groups (p = 0.0198).  There was no statistical difference between 6.0% and 7.5 % groups, and 5.25% and 9.0 % groups.  

Conclusion:  The physiologic response to lymphatic injury and lymph fluid obstruction involves lymphangiogenesis and formation of collateral vessels to provide alternative drainage pathways.   As lymphedema progresses towards a fibrotic stage, interstitial pressure increases.  In this study, we used a 3D in vitro model to demonstrate that lymphangiogenesis occurs optimally at 6.0-7.5% gel (5.57 to 12.8 kPa), which is in the normal physiologic range.  At lower (3.37 kPa) or higher (15.31kPa) pressures, lymphangiogenesis is less efficient.  This data may be used to guide medical/pro-lymphangiogenic and surgical approaches to the management of lymphedema.

 

 

60.07 Lipidomic Profiling Identifies Phospholipase A2 as a Novel Effective Therapy for HNSCC In Vivo

Posted on January 18, 2018

C. Subramanian1, T. M. Rajendiran2, T. Soni3, M. S. Cohen1  1University Of Michigan,General Surgery,Ann Arbor, MI, USA 2University Of Michigan,Pathology And Michigan Regional Metabolomics Resource Core,Ann Arbor, MI, USA 3University Of Michigan,Michigan Regional Metabolomics Resource Core,Ann Arbor, MI, USA

Introduction: Of the 60,000 HNSCC cases diagnosed annually in the US, 60% present with advanced stage III/IV disease requiring multimodality therapy yet five-year survival rates have not significantly changed in over three decades with a median survival of less than a year. It is therefore imperative to find better therapies for patients with advanced disease and one of the best routes is through identification of novel yet tumor-specific biomarkers from which the drugs can be designed. Since lipids play an important role in tumor development and progression of cancer and can be very specifically overproduced in several tumors, we hypothesized that lipid metabolic profiling of HNSCC is a unique untapped mechanism to identify novel therapeutic targets for HNSCC.

Methods: Mass spectrometry (LC/MS), based shotgun untargeted global lipidomic profiling was used to explore the lipid signatures which included 63 HNSCC patient tumors. Validated HNSCC cell lines UMSCC1,12, 22B and MDA1986 were also grown in 2D culture for tumor xenograft implantation for comparison. Phospholipase A2 activity was measured using the cPLA2 assay kit from Cayman as per the manufacturer’s protocol. UMSCC 22B xenografts were developed and the mice were treated i.p. with10 mg/ kg of cPLA2 inhibitor arachidonyl trifluoromethyl ketone (ATK) or vehicle for 15 days and the tumor volumes were measured.

Results: Unsupervised principal component analysis of the lipid signature showed a very distinct and clear separation between the benign and cancer tissues. A total of 576 lipids were identified of which 322 were significantly different between benign and cancer tissues (p<0.05). Evaluation of the top 40 significantly expressed lipids showed up regulation of free fatty acids (24:1, 24:2,24:3 and 22;3), phospholipids such as lysophosphotidyl choline (LPC) in cancer tissues compared to benign tissues whereas glycero lipids such as triglycerides (TG) were down regulated in cancer tissues. Since upregulation of LPC is catalyzed by PLA2, its activity was measured both in tumor samples as well as in HNSCC cell lines. Tumor samples showed 3.0 fold higher expression levels of cPLA2 compared to benign samples (p<0.01) whereas cell lines showed only 2.5 fold upregulation compared to control fibroblast cell lines (p<0.01). Treatment of HNSCC xenogaft with ATK resulted in significant (>90%) reduction in tumor volume compared to vehicle treatment groups (p<0.01). Animal weight and organs all showed no signs of toxicity with treatment.

Conclusion: Lipid profiling of HNSCC patient tumors identified up regulation of FFA and LPC indicating PLA2 as a potential therapeutic target for HNSCC. Blocking of cPLA2 activity using ATK resulted in significant growth reduction of HNSCC xenografts without any measured toxicity. This is a very exciting, novel, and highly translatable treatment option for HNSCC with high clinical potential that warrants further preclinical validation.

 

60.04 Synergistic Apoptosis Following Endoplasmic Reticulum Stress Aggravation in Mucinous Colon Cancer

Posted on January 18, 2018

B. D. Honick1, A. K. Dilly1, S. Hong1, Y. J. Lee1,2, H. J. Zeh1, D. L. Bartlett1, H. A. Choudry1  2University Of Pittsburg,Department Of Pharmacology & Chemical Biology,Pittsburgh, PA, USA 1University Of Pittsburg,Department Of Surgery,Pittsburgh, PA, USA

Introduction: Cancer cells up-regulate endoplasmic reticulum (ER) stress response related molecular signaling pathways, known as unfolded protein response (UPR), in order to accommodate high protein turnover associated with rapidly proliferating cells. We hypothesized that mucinous colon cancers would have higher basal ER stress owing to high mucin 2 (MUC2) protein production, and that ER stress aggravation in these tumors would overwhelm cytoprotective capacity of UPR and induce apoptosis.  

Methods:  In vitro studies were conducted using explant tissue from mucinous and non-mucinous colon cancers, normal colon, LS174T cells (MUC2-secreting human colon cancer cells), and stably transfected LS174T cells expressing dominant negative TCF4 (differentiated goblet-like cells controlled by Tet-on system that express higher MUC2 levels than wild-type LS174T cells). ER stress aggravators included ER calcium pump inhibitor celecoxib and fatty acid synthase inhibitor orlistat.

Results: Higher levels of ER stress response proteins, including GRP78/BiP (immunoglobulin heavy chain-binding protein), ATF4 (activating transcription factor 4), CHOP (C/EBP homologous protein) assessed by real-time PCR and immunofluorescence, were detected in mucinous colon cancer explant tissue and MUC2 over-expressing LS174T-dnTCF4 cells as compared to non-mucinous cancer explant tissue, normal colon epithelium and wild-type LS174T cells. These data suggest a correlation between MUC2 protein production and basal ER stress levels. Treatment of LS174T cells with ER stress inducers celecoxib or orlistat alone decreased cell viability (assessed by MTS assay and phase-contrast microscopy) and induced apoptosis (measured by flow cytometry with annexin-V and propidium iodide staining) and ER stress response proteins (BiP, ATF4, CHOP evaluated by western blot) in a dose-dependent fashion, with doses > 75 µM (celecoxib) and > 200 µM (orlistat) required to achieve > 50% cell death. Combination treatment of LS174T cells with celecoxib (50 µM) and orlistat (100 µM) demonstrated synergistic loss of cell viability (> 50% cell death by MTS assay) and induction of apoptosis (flow cytometry) and ER stress response proteins (western blot). Western blot demonstrated synergistic induction of PUMA (p53 upregulated modulator of apoptosis), cleaved caspases 9 and 3, and cleaved PARP (poly ADP-ribose polymerase) as the downstream mechanism for apoptosis following combination therapy in LS174T cells.

Conclusion: Mucinous cancers have higher basal ER stress than non-mucinous cancers, perhaps due to the higher protein turnover associated with abundant MUC2 production. These tumors may be especially vulnerable to drugs that overwhelm the ER stress response through disrupting protein homeostasis. Ongoing studies will determine whether ER stress aggravation by this combination therapy triggers a switch from autophagy to apoptosis.

 

60.05 The contribution of Enterococcus-mediated plasmin(ogen) activation in anastomotic leak pathogenesis

Posted on January 18, 2018

R. A. Jacobson1,2, O. Zaborina1, J. C. Alverdy1  1The University Of Chicago,Surgery,Chicago, IL, USA 2Rush University/Cook County,Surgery,Chicago, IL, USA

Introduction:  Recent work in our lab has demonstrated that Enterococcus faecalis (E. faecalis) alone can cause anastomotic leak (AL) in rodents by activating tissue associated pro-matrix metalloprotease 9 (pro-MMP9) leading to pathologic degradation of submucosal collagen in the anastomotic wound. However the fibrinolytic protease plasminogen (PLG) 1) is concentrated and cleaved to active plasmin at sites of tissue injury 2) activates pro-MMP9 and 3) directly cleaves collagen, therefore its role in AL pathogenesis needs to be clarified. Here we hypothesized that PLG is also activated by E. faecalis and acts synergistically with MMP9 leading to further enhancement of collagen degradation. Therefore, the aim of this study was to define the relative contribution of PLG in the molecular pathogenesis of AL via its effect on collagen degradation.

 

Methods:  A collagenolytic E. faecalis strain (E44), previously identified to play a putative role in AL was used in these experiments. Plasmin(ogen) activation was assessed with a fluorogenic substrate specific to plasmin. Fluorescein-tagged gelatin, type 1 collagen and type 4 collagen assays were employed to assess E. faecalis-mediated collagenolylsis in the presence of PLG, the known plasminogen activator urokinase (uPA), and pro-MMP9. All purified enzymes were purified recombinant human forms Tranexamic acid (TXA) was used to inhibit plasmin(ogen) activation. 

Results:

Plasmin(ogen) activation: E. faecalis alone activated PLG in a concentration-dependent fashion (reaction velocity increased by 0.32 RFU/s per E. faecalis colony forming unit, R2 = 0.99 ). The effect was amplified in the presence of uPA (1.57 RFU/s per CFU, R2 = 0.96). This process was inhibited by TXA in a concentration-dependent fashion. 

 

Collagenolysis/gelatinolysis: E. faecalis-mediated cleavage of type 4 collagen was unchanged by the addition of pro-MMP9, but was significantly increased in the presence of PLG with and without its activator uPA. In the presence of PLG and uPA, addition of pro-MMP9 appeared to synergistically increase collagenolytic activity (Panel A). TXA diminished overall gelatinolytic activity in a dose dependent manner (1.16 AU 10mM TXA vs 3.50 AU E.faecalis + PLG/uPA/proMMP9, p<0.05, panel B). Results were redemonstrated in gelatin and type 1 collagen cleavage assays.

Conclusion: Our data suggest that the molecular pathogenesis of anastomotic leak may involve synergistic activation of plasmin(ogen) and pro-MMP9 in a manner that drives collagenolytic activity to supraphysiologic levels. That this process can be suppressed by the use of TXA, offers a novel therapeutic role to apply this FDA approved agent to high risk anastomotic surgery. 

 

60.06 Topical application of a Dual PI3K/mTOR Inhibitor for the Prevention of Anal Carcinogenesis In Vivo

Posted on January 18, 2018

B. L. Rademacher1, L. M. Meske1, K. A. Matkowskyj2, E. D. LaCount1, E. H. Carchman1  1University Of Wisconsin,Department Of Surgery, Division Of General Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Pathology And Laboratory Medicine,Madison, WI, USA

Introduction:  Patients with anogenital human papilloma virus (HPV) infection are at high risk of developing squamous cell dysplasia that can progress to squamous cell carcinoma of the anal canal (SCCA). We have previously shown that dual PI3K/mTOR inhibition results in decreased dysplasia and SCCA with systemic drug administration in our HPV mouse model of anal carcinogenesis. Here we sought to investigate the effect of local, topical application of a dual PI3K/mTOR inhibitor, BEZ235, on tumor free survival, histopathologic changes and autophagy.

Methods:  K14E6/E7 mice were given no treatment (Control), topical BEZ235 (BEZ), the carcinogen DMBA (DMBA), or both DMBA and BEZ for a total of 20 weeks. Mice were assessed weekly for tumor development. At 20 weeks they were euthanized and their anal samples examined for histopathologic changes at the anal transition zone (ATZ). Slide sections of the ATZ were assessed for mTOR and PI3K activity by staining for pS6 and pAKT expression (immunohistochemistry), respectively, and evidence of autophagic function via LC3β and p62 expression (immunofluorescence). Tumor free survival analysis was conducted used Kaplan Meier statistics, and all comparisons of mean differences in histopathologic score or protein signal were conducted using a one-way ANOVA.

Results: Regarding tumor free survival, mice receiving DMBA alone survived, on average, 16.9 weeks prior to tumor onset, whereas mice receiving both DMBA and BEZ survived, on average, 19.3 weeks (P<0.000001). Histopathological analysis revealed a significant decrease in mean score comparing DMBA with DMBA plus BEZ (P<0.000001). Comparing DMBA versus DMBA plus BEZ, IF revealed efficacy of topically applied dual PI3K/mTOR inhibitor, via significant decreases in both pS6 and pAKT (P<0.001 for both comparisons). Compared to Control mice, both BEZ and DMBA plus BEZ treated mice had significantly higher LC3β expression, signifying autophagic induction (P<0.005 for both comparisons), whereas DMBA, BEZ, and DMBA plus BEZ treated mice had significantly lower p62 expression, signifying increased autophagic function (P<0.0005 for all comparisons).

Conclusion: Consistent with systemic delivery of a dual PI3K/mTOR inhibitor, topical application of BEZ235 shows prolonged tumor free survival. Furthermore, this finding is confirmed via targeted inhibition of the PI3K/mTOR pathway resulting in activation of autophagy and decreased carcinogenesis.

 

60.03 Rituximab Decreases Lymphoproliferative Tumors and Increases Success of Patient-Derived Xenografts

Posted on January 18, 2018

J. L. Leiting1, M. C. Hernandez1, L. Yang2, J. R. Bergquist1, M. J. Truty1  1Mayo Clinic,Department Of Surgery,Rochester, MN, USA 2Mayo Clinic,Center For Individualized Medicine,Rochester, MN, USA

Introduction:
Patient-derived xenografts (PDX) provide clinically relevant translational models that accurately recapitulate individual patient tumor histopathologic and molecular phenotypes. Maintaining high engraftment rates is critical and loss of PDX models are due to engraftment failure or development of lymphoproliferative tumors (LTs). Previous PDX work has suggested the etiology of LTs are due to tumor associated lymphocytes and/or Epstein Barr Virus activation in an immunodeficient environment. Here, we report our efforts to decrease rates of LTs from a high volume PDX program in order to improve PDX engraftment efficiency. To address the issue of LTs, we hypothesized that routine injection of rituximab (an anti-CD20 antibody) at time of tumor implantation would maximize engraftment by reducing the rate of LTs.

Methods:
With IRB and IACUC approval, surgically resected primary patient tumors were implanted into the flanks of NOD SCID mice according to our protocol. We assessed the effect of routine rituximab injection in pre- and posttreatment groups. Implanted mice were monitored weekly for time to tumor formation (TTF) and all derived PDX models were verified by a GI cancer pathologist.  Chi squared and Fisher’s Exact test were used for statistical comparison.

Results:
A total of 697 generations of PDX have been implanted with 290 individual patient tumors. These include 145 pancreatic cancers (60 treatment naïve, 85 neoadjuvant), 66 cholangiocarcinomas (CCA), 38 hepatocellular carcinomas (HCC), and 41 miscellaneous GI tumors. Of these, 308 generations received rituximab compared to 389 that did not. Overall rates of LTs were 11.3% (8.6% in primary PDX engraftments and 13.3% in subsequent generations). LT rates varied by tumor type with HCCs having the highest overall rate (19.7%), followed by naïve pancreatic cancers (11.1%) and CCAs (10.1%). Rituximab treatment decreased LT rates from 13.1% to 9% (p<0.001) in the entire cohort and as a result increased overall successful PDX engraftment. This was consistent in both primary PDX engraftment (10.5% vs 3%, p<0.001) as well as subsequent generations (16.2% vs 10%, p<0.001.) Rituximab had the most significant decrease in LTs in CCAs (13.1% vs. 5.1%, p=0.0019) and HCCs (26% vs. 13%, p<0.001).

Conclusions:
Engraftment failure due to LT formation is detrimental and leads to excessive costs and inefficiencies. Rates of LTs appear to decrease and overall engraftment rates increase with routine rituximab treatment at the time of implantation for both primary and secondary passages. Historically difficult to engraft tumors like CCAs and HCCs appear to benefit the most by rituximab treatment. Further study evaluating the etiology of LTs in PDX is warranted. 

60.02 Rectal Cancer Patient Derived Xenograft Model to Advance Personalized Therapy

Posted on January 18, 2018

S. A. Becker1, Y. Zhu1, C. Wang4, A. W. Cross2, E. Curl2, K. E. Armeson5, K. E. Hurst1, D. N. Lewin2, G. Warren6, B. J. Hoffman4, E. G. Hill5, V. J. Findlay2, E. R. Camp1  1Medical University Of South Carolina,Department Of Surgery,Charleston, SC, USA 2Medical University Of South Carolina,Department Of Pathology,Charleston, SC, USA 3Medical University Of South Carolina,Charleston, SC, USA 4Medical University Of South Carolina,Department Of Medicine,Charleston, SC, USA 5Medical University Of South Carolina,Department Of Public Health Sciences,Charleston, SC, USA 6Medical University Of South Carolina,Department Of Radiation Oncology,Charleston, SC, USA

Introduction:
Neoadjuvant 5-fluorouracil chemoradiation (5FU/RT) has established benefits in locally advanced rectal cancer (LARC) patients; however, therapeutic resistance is observed in ~70% of the cases. To advance care, predictors of 5FU/RT response still need to be identified and novel personalized approaches are desperately needed. Our multi-disciplinary research team hypothesized that a unique “bedside-to-bench” LARC patient-derived xenograft (PDX) model established from pre-neoadjuvant 5FU/RT LARC endoscopic biopsy samples could serve as a therapy predictive platform used for testing future personalization strategies.

Methods:
Mouse xenografts were created from pre-neoadjuvant 5FU/RT LARC endoscopic biopsy samples (IRB approved protocol). Dissociated cancer cells were mixed with Matrigel (1:1 ratio) and subcutaneously (SQ) injected in NSG mice to yield the passage 0 (P0) tumors. Dissociated tumor cells were passaged in an expanded number of mice to create subsequent passages for investigation. P2-3 xenografts were used for histologic characterization and for in vivo therapy experiments. Therapy groups include 1) DMSO control; or 2) 5FU (40 mg/kg ip X2) + RT (1Gy daily for 5 days) 3) Cetuximab (1mg/dose ip twice weekly for 3 weeks) +5FU/RT. Xenograft response, as measured by tumor weight and serial volume measurements, was compared to the corresponding human LARC.

Results:
After optimization, we achieved an outgrowth (human tumor to P0) rate of 73% (n= 19/26) and engraftment (P0 to P1) of 85%. Clinical factors did not predict outgrowth but tumors with distant metastasis demonstrated increased xenograft doubling times. Xenograft histology was conserved in 88% of cases compared with the human LARC tumor. Xenograft 5FU/RT response based on tumor volume correlated with clinical tumor regression grading response to neoadjuvant (5FU/RT) (Figure 1). KRAS wild type tumors demonstrated response to Cetuximab in addition to 5FU/RT (p<0.05).

Conclusion:
A novel LARC patient-derived xenograft (PDX) platform mirror images the biology, architecture, and therapy heterogeneity observed in LARC patients suggesting our model represents an effective platform to develop future personalization therapeutic strategies.
 

60.01 A Tumor Infiltrating Nanoparticle Delivery Platform for In Vivo KRAS Knockdown

Posted on January 18, 2018

B. A. Krasnick1, M. S. Strand1, N. Sankpal1, Y. Bi1, P. Goedegebuure1, S. Wickline2, H. Pan2, R. C. Fields1  1Washington University,Surgery,St. Louis, MO, USA 2University Of South Florida College Of Medicine,Cardiology,Tampa, FL, USA

Introduction: For patients with metastatic colorectal cancer (CRC), 5 year survival is ~10%. Unlike conventional chemotherapy, where treatment is not targeted and often inefficient, nanoparticles (NPs) have the potential to allow precise delivery of cargo (such as siRNA or other small molecule inhibitors) directly to sites of disease. The cargo being delivered can theoretically target multiple different pathways simultaneously, including those not “druggable” by other means (such as KRAS).  Here, we describe our peptide based, endosomolytic NP system designed to deliver siRNA against KRAS to several model CRC tumor systems.
 

Methods: Quasar 705 (Q705) tagged siRNA NPs (Fluorescent NP) were given via IV injection to mice, and in vivo uptake was assessed. To assess Fluorescent NP uptake we utilized confocal microscopy, in vivo and ex vivo imaging systems, as well as multi-color flow cytometry. CT26 murine CRC cells, which harbor a KRAS mutation are used throughout. For patient derived xenografts (PDXs), a unique KRAS mutant CRC cell line, 322, was derived. Subcutaneous (SQ) CT26 ± GFP/Luciferase labelled murine CRC tumors were created by injecting tumor cells into the right flank of BALB/C mice. For liver tumors, a hemi-splenectomy liver metastasis model was used, with CT26 GFP/luciferase labelled CRC cells injected into the BALB/C hemispleen. A KRAS specific siRNA was used create our KRAS siRNA NP (KRAS NP) for IV treatment, and KRAS RNA level was assessed via RT-PCR.

 

Results: Our Fluorescent NP localizes to SQ murine CRC tumors (Figure Part A, N=5 per group—Fluorescent NP or Control NP treated) with minimal non-tumor uptake, as well as 322 PDX CRC tumors (not shown). Our Fluorescent NP localizes specifically to GFP positive murine CRC tumor cells and not to non-tumor cells in a SQ tumor model (Figure Part B, N=3 per group—Fluorescent NP and Control NP treated). In our liver metastasis model, Fluorescent NP is delivered specifically to GFP+ tumor cells in the liver (Figure Part C). Mice harboring CT26 SQ tumors were then treated with KRAS NP, which leads to a 50% knockdown in tumor KRAS RNA expression as compared to Control NP treated mice (p<0.0001).

 

Conclusion: Our peptide based nanoparticle localizes to in vivo CRC cells in metastatic and heterotopic model systems. This system can be used to specifically target KRAS expression. We are currently exploring the therapeutic potential of this system in multiple pre-clinical systems as a novel platform for targeted delivery of precision therapeutics. 

 

59.10 Psychosocial Stress and Dysbiosis Alter Intestinal Immunity in C57BL/6

Posted on January 18, 2018

S. Deas1, J. Neilson1, K. Brawner1, C. Morrow2, C. Martin1  1University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Department Of Cell, Developmental, And Integrative Biology,Birmingham, Alabama, USA

Introduction:  It is widely accepted that stress can negatively impact intestinal immune function. IgA is an established regulator of intestinal homeostasis and has been demonstrated to regulate bacterial translocation. The precise mechanism of how chronic psychological stress and bacterial dysrugulation affect the immune system and intestinal homeostasis is not known, but may be due to cortisol release by the hypothalamic-pituitary-adrenal (HPA) axis. We hypothesize that chronic stress and a state of dysbiosis impair intestinal immune function, perhaps mediated by dysregulation of the gut-brain immune axis.

Methods:  8-week-old C57BL/6 littermates were assigned to one of three experimental groups over a 7-day period. Group 1 was subjected to daily psychological stress via a well-established restraint model for one hour daily. Group 2 was given 1mg/mL vancomycin and 0.1mg/mL gentamicin in their drinking water to induce dysbiosis. Group 3 was subjected to both daily psychological stress and the antibiotic cocktail. The control group experienced no stress or antibiotic treatment. After the 7-day period, mesenteric lymph nodes (MLN) were harvested and cultured on Schaedler agar and incubated at 37°C for 3.5 days. Select bacterial colonies from our Group 3 MLNs were analyzed via 16S PCR for species identification. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure fecal IgA and serum corticosterone levels. 

Results: Microbial culture of MLN from the control group revealed minimal bacterial growth, with an average of 1.5 pinpoint colony forming units (CFUs) per plate. CFUs were difficult to quantify from other groups due to morphology. All experimental groups were characterized by irregular, raised, undulate growth. The 16S PCR analysis of isolates from Group 3 revealed that the predominant phylum involved in MLN translocation was Proteobacteria. Finally, the Group 2 had significantly less fecal IgA compared to the control group; P-value = 0.045*. The Group 3 had significantly less fecal IgA compared to Group 1; P-value = 0.0045# (Figure 1). There was no significant difference in serum corticosterone between the control and experimental groups.

Conclusion: 
The combination of chronic stress and dysbiosis severely impairs the intestinal immune response, demonstrated by decreased fecal IgA. Serum corticosterone levels were not significantly different between control and experimental groups, suggesting that dysregulation of the HPA axis may not be the mechanism explaining the observed dampening of intestinal immunity. Bacterial translocation, as evidenced by MLN culture, indicates that this may be due to direct barrier dysfunction. Future experiments will seek to further elucidate this mechanism.

59.09 Prrx1 Identifies the Fibroblast Sub-Population Responsible for Scarring in the Ventral Dermis

Posted on January 18, 2018

M. S. Hu1, T. Leavitt1, R. Ransom1, J. Garcia1, U. Litzenburger1, G. Walmsley1, C. Marshall1, L. Barnes1, A. Moore1, E. Zielins1, C. Chan1, D. Wan1, P. Lorenz1, H. Chang1, M. Longaker1  1Stanford University,Division Of Plastic Surgery, Department Of Surgery,Palo Alto, CA, USA

Introduction:
Scarring and fibrosis are an enormous public health concern, resulting in excessive morbidity and mortality, in addition to countless lost health care dollars. Currently, there are many treatments for cutaneous scarring available, but few have proven successful, and there is an estimated $12 billion annual market in the United States for such treatments. We previously identified a sub-population of fibroblasts responsible for the bulk of connective tissue deposition in the dorsal dermis during embryonic development, cutaneous wound healing, and melanoma stroma formation. Whether these findings translate to the ventral dermis has yet to be elucidated.

Methods:
Prrx1-derived fibroblasts were traced by crossing Prrx1Cre and ROSA26mTmG mice. Prrx1-derived fibroblasts were characterized using flow cytometry, histology, and ATAC-seq analysis at various stages of embryonic development.

 

Results:
Lineage tracing of fibroblasts within the ventral dermis revealed a sub-population, labeled by the embryonic expression of Prrx1, acting as the key contributor to connective tissue deposition during scar formation. This lineage increased as a proportion of total fibroblasts within the ventral dermis over the course of gestation, associated with the transition from scarless to scarring repair. Differential patterns of chromosomal accessibility based on ATAC-seq data further demonstrated the heterogeneic nature of fibroblasts within the ventral dermis.

Conclusion:
Analogous to findings in the dorsal dermis, fibroblasts of the ventral dermis show functional heterogeneity. Prrx1 identifies the fibroblast sub-population with fibrogenic potential in the ventral dermis. As in the dorsal dermis, selectively ablating this fibroblast sub-population may lead to decreased cutaneous scarring and a novel therapeutic to decrease scarring and fibrosis.

 

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