21.03 DNA released from PAD4-mediated NETosis enhances tumor growth in murine pancreatic cancer

Posted on January 25, 2017

J. Miller-Ocuin1, W. R. Doerfler1, X. Liang1, B. Boone1, A. Singhi1, M. Lotze1, H. Zeh1  1University Of Pittsburgh,Surgical Oncology/Surgery/Medicine,Pittsburgh, PA, USA

Introduction: Activated neutrophils release intracellular material in a process known as neutrophil extracellular trap (NET) formation. NETs result from histone citrullination, chromatin decondensation, and ultimately DNA release from the cell. Peptidyl arginine deiminase 4 (PAD4) is an enzyme required for NET formation. Increased NET formation is associated with cancer progression in preclinical models of murine pancreatic cancer. We hypothesized mice deficient in PAD4 would demonstrate decreased tumorigenesis.

Methods: Luciferase-transfected Panc02 cells were injected into the pancreas of WT controls or PAD4-/- mice. Tumorigenesis continued for 5 weeks in untreated experiments. DNAse (5mg/kg IP) or vehicle was injected daily for 3 weeks in treated experiments. Mice were imaged weekly using an in vivo imaging system (IVIS) for tumor growth; area of interest (ROI) is represented as photon/sec/cm2/Sr. At conclusion of experiments, mice were sacrificed for tumor weights, immunohistochemistry (IHC) and serum DNA quantification. Bone marrow chimeras using Pdx-Cre KrasG12D (KC) transgenic mice, which develop spontaneous pancreatic cancer, were generated with lethal irradiation followed by reconstitution with PAD4-/- or WT bone marrow. 21 days after treatment with cerulein, which induces pancreatitis to accelerate tumor growth, animals were sacrificed and organs harvested. Hematoxylin and eosin stained tumor sections were evaluated by a pancreatic pathologist.

Results: PAD4-/-tumor bearing mice showed decreased NETs on IHC and decreased serum DNA (406ng/ml vs 858ng/ml, p=0.03), a surrogate NET marker. Tumorigenesis was significantly decreased in PAD4-/- vs. WT mice (921mg vs. 326mg, p=0.001; ROI: 2.4×107 vs. 1.6×106, p=0.05). DNAse treatment of WT mice lead to significantly decreased tumor growth vs. sham treated controls (336mg vs. 206mg, p=0.05; ROI: 9.9×105 vs. 3.8×105, p=0.05) while there was no significant change in tumor growth in PAD4-/- animals treated with DNAse (195mg vs. 217mg, p=0.29; ROI: 8.5×105 vs. 6.9×105, p=0.43). There was a trend toward decreased high-grade precursors and invasive cancers in PAD4-/- bone marrow recipients as compared to WT recipients (p=0.29) with significantly diminished NET formation by isolated bone marrow supernatant DNA levels (197ng/mL vs. 796ng/mL, p=0.001).

Conclusion: Murine pancreatic tumors in PAD4-/-mice show decreased tumorigenesis and decreased NET formation. DNA released during NET formation leads to increased tumor growth, which is suppressed by DNAse administration. Future studies will focus on the mechanism through which NET DNA promotes tumor growth.

 

21.02 Gut Microbiome Promotes Pancreatic Oncogenesis by Inducing Innate and Adaptive Immune Suppression

Posted on January 25, 2017

M. Hundeyin1, S. Pushalkar1, D. Daley1, G. Werba1, N. Mohan1, S. Lall1, B. Wadowski1, B. Aykut1, E. Kurz1, U. Soni1, E. Morales-Vicente1, D. Saxena1, G. Miller1  1New York University School Of Medicine,Surgery,New York, NY, USA

Introduction:
Pancreatic ductal adenocarcinoma (PDA) is the 3rd most lethal cancer in the United States and accounts for 85% of all pancreatic malignancies. The gut microbiome has emerged as an important regulator in the balance between health and disease, including oncogenesis. However, the microbiome has not been directly linked to pancreatic oncogenesis. We postulated that hosts with PDA harbor an altered pancreatic and gut microbiome and that dysbiosis influences PDA progression. 

Methods:
To determine whether endoluminal gut bacteria can access the pancreas, we administered fluorescently-labeled Enterococcus faecalis to WT mice via oral gavage. To determine whether bacteria promote the progression of pancreatic oncogenesis, we employed the slowly progressive p48Cre;LSL-KrasG12D (KC) mouse model of PDA and rederived KC mice in a germ-free environment. To identify possible perturbations in the gut microbiome associated with the progressive pancreatic oncogenesis, we evaluated the composition of the gut microbial community of KC mice compared with age-matched WT littermate controls by performing 16S gene sequencing. Lastly to determine the mechanism of immunosuppression; we injected F21242 pancreatic tumor cell line orthotopically into the pancreas of WT mice after bacterial ablation and vehicle, isolated the pancreatic leukocytes and analyzed them by flow cytometry. 

Results:
In the pre-morbid state the intestinal microbiome is similar in mice bearing pancreas-specific oncogenic mutations and in controls; however, as mice age, PDA-bearing hosts develop a unique gut microbiome including expansion of Actinobacteria and Deferribacteres. We found that gut bacteria access the pancreas and the cancerous pancreas harbors a distinct microbiome in mice. Further, genotypically identical PDA-bearing mice that exhibit divergent disease phenotypes harbor stage-specific microbiomes suggesting that microbial structure is associated with disease aggressiveness. Germ-free or ablative antibiotic treated mice were protected against PDA whereas transfer of gut bacteria from PDA-bearing mice, but not from control mice, reversed the tumor-protection. Bacterial ablation was associated with innate and adaptive immunogenic reprogramming of the PDA tumor microenvironment including a marked reduction in myeloid-derived suppressor cells and immune-suppressive macrophages, increased Th1 differentiation of CD4+ T cells, and expansion and activation of cytotoxic CD8+ T cells. In addition, we show that gut bacterial ablation and PD-1 blockade offer synergistic efficacy.

Conclusion:
These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has marked potential as a biomarker and therapeutic target in PDA.
 

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

Posted on January 25, 2017

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

Introduction:

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

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

Methods:

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

Results:

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

Conclusion:

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

19.06 Release of HMGB1 by Platelets Promotes DVT Formation

Posted on January 25, 2017

M. R. Dyer1, Q. Chen1, A. Gutierrez1, B. Zuckerbraun1, M. Neal1  1University Of Pittsburgh,Surgery,Pittsburgh, PA, USA

Introduction:  Deep vein thrombosis (DVT) is a common and highly morbid complication following trauma. The damage-associated molecular pattern molecule, high-mobility group box-1 (HMGB1) has been shown to be a key mediator of the inflammatory response following trauma. In pro-inflammatory states neutrophils can be stimulated to release their nuclear content including DNA, histones, and granule contents to form what is referred to as neutrophil extracellular traps (NETs). NETs have been shown to be pro-thrombotic and are implicated in the development of DVT. Recently platelet-derived HMGB1 was found to be a key mediator of microvascular thrombosis and formation of NETs in the lung following trauma. We hypothesized that HMGB1 released from activated platelets would promote DVT formation via NET production.

Methods:  We created transgenic mice lacking HMGB1 specifically on platelets (HMGB1 PF4), and subjected them to IVC ligation to induce DVT formation, with comparison to control (HMGB1 Flox). Recombinant HMGB1 or RNase free DNase was administered via tail vein injection 30 minutes prior to IVC ligation in some experiments. Clots were harvested at 24 and 72 hours following IVC ligation, quantified by length and weight, and analyzed for NET expression by immunofluorescence (IF). Blood was obtained by cardiac puncture to determine HMGB1 circulating levels.

Results: HMGB1 levels are increased following DVT formation compared to sham (32 vs. 10.8 ng/ml) and platelets are the predominant source of HMGB1 during DVT (HMGB1 Flox 44.3 vs. HMGB1 PF4 16.3 ng/ml, p<0.05). Recombinant HMGB1 increased clot burden at 24 hours compared to control (25.3 vs 11.6 mg, p<0.05). HMGB1 PF4 mice have decreased thrombus formation at 24 hours (HMGB1 Flox 12.9 vs. HMGB1 PF4 7.4 mg, p<0.05) and 72 hours (HMGB1 Flox 32.6  vs. HMGB1 PF4 19.5 mg, p<0.05). IF staining of harvested clots for citrullinated histone H3, a marker of NETs, demonstrated significantly less NETs in HMGB1 PF4 mice (HMGB1 Flox 2291 vs. HMGB1 PF4 126, p<0.05), indicating a role for platelet HMGB1 in NET formation. Treatment with DNase, which degrades NETs, eliminated the difference in clot burden between HMGB1 PF4 and HMGB1 Flox mice (9 vs. 11 mg, p=0.18).

Conclusion: HMGB1 is increased following DVT and platelets are the significant source of circulating HMGB1. Platelet released HMGB1 increases thrombus burden via formation of NETs in a murine model of DVT. These data provide a potential critical link between inflammation and post-trauma thrombotic complications.

 

19.05 Protease Tailored Flourogenic Substrates Outperform CEA in Identifying Mucinous Pancreatic Cysts

Posted on January 25, 2017

D. A. Dominguez1, S. Ivry3, S. Hatcher3, E. Gilbert2, S. Kumar2, W. Park6, M. Schmidt7, R. Brand4, A. O’Donoghue5, K. Kirkwood2, C. Craik3  1UCSF East Bay,Department Of General Surgery,Oakland, CA, USA 2UCSF,Department Of General Surgery,San Francisco, CA, USA 3UCSF,Department Of Pharmaceutical Chemistry,San Francisco, CA, USA 4University Of Pittsburgh,Department Of Gastroenterology,Pittsburgh, PA, USA 5UCSD,Department Of Pharmacy And Pharmaceutical Sciences,San Diego, CA, USA 6Stanford University,Department Of Gastroenterology,Palo Alto, CA, USA 7Indiana University,Department Of Surgery,Indianapolis, IN, USA

Introduction:  Risk stratification of pancreatic cystic lesions remains an area of great clinical uncertainty.  Measurement of cyst fluid CEA is used to predict which cysts are mucinous, and therefore may have malignant potential, but its accuracy limits its usefulness.  Due to this lack of definitive pre-operative characterization, some patients will die from undiagnosed cancers, while others undergo unnecessary pancreatic resections with significant morbidity.  Dysregulation of protease expression and activity has been previously reported in mucinous pancreatic cyst fluid.  We tested the hypothesis that differences in proteolytic activity between mucinous (MUC) and non-mucinous (NON-MUC) cysts could be used to improve our ability to identify pre-malignant pancreatic tumors.

Methods:  We first analyzed cyst fluid from a cohort of human pancreatic neoplasms (MUC, n=16; NON-MUC, n=7) using multiplex substrate-profiling by mass spectrometry (MSP-MS), which is an unbiased, comprehensive technology for analyzing patterns of proteolytic activity. We found that aspartyl protease activity was unique to MUC pancreatic cysts. Next, using shotgun proteomic analysis, we identified 2 proteases (Prot1, Prot2), that were selectively expressed in MUC cyst fluid.  Specific cleavage profiles were used to design a selective fluorescent substrate for each protease. Substrates were then used to assess the activity of each protease in a larger patient cohort (MUC, n=71; NON-MUC, n=39); ROC curves were generated for each substrate.  Performance was compared with CEA values (CLIA lab) using >= 192 ng/mL as a cut off for MUC cysts. 

Results:  Receiver operator characteristic (ROC) curves for Prot1 and Prot2 exhibited an area under the curve (AUC) of 0.98 and 0.82, respectively. Prot1 demonstrated a sensitivity of 93% and specificity of 100%.  Prot2 had a sensitivity of 70% and a specificity of 92%.  Testing required < 0.2 mL cyst fluid.  CEA had an AUC of 0.86, and a sensitivity and specificity of 65% and 94%, respectively.

Conclusion:  MSP-MS is a powerful technology to examine dysregulated proteolysis in complex biological fluids.  The resultant fluorogenic substrates outperformed CEA, the highest of which had a sensitivity of 93% and a specificity of 100%, for the detection of MUC pancreatic cysts.  Our fluorescent assay has the potential to be a rapid, inexpensive, and highly accurate predictor of MUC pancreatic cysts. 

 

19.04 DNA released from PAD4-mediated NETosis enhances tumor growth in murine pancreatic cancer

Posted on January 25, 2017

J. Miller-Ocuin1, W. R. Doerfler1, X. Liang1, B. Boone1, A. Singhi1, M. Lotze1, H. Zeh1  1University Of Pittsburgh,Surgical Oncology/Surgery/Medicine,Pittsburgh, PA, USA

Introduction: Activated neutrophils release intracellular material in a process known as neutrophil extracellular trap (NET) formation. NETs result from histone citrullination, chromatin decondensation, and ultimately DNA release from the cell. Peptidyl arginine deiminase 4 (PAD4) is an enzyme required for NET formation. Increased NET formation is associated with cancer progression in preclinical models of murine pancreatic cancer. We hypothesized mice deficient in PAD4 would demonstrate decreased tumorigenesis.

Methods: Luciferase-transfected Panc02 cells were injected into the pancreas of WT controls or PAD4-/- mice. Tumorigenesis continued for 5 weeks in untreated experiments. DNAse (5mg/kg IP) or vehicle was injected daily for 3 weeks in treated experiments. Mice were imaged weekly using an in vivo imaging system (IVIS) for tumor growth; area of interest (ROI) is represented as photon/sec/cm2/Sr. At conclusion of experiments, mice were sacrificed for tumor weights, immunohistochemistry (IHC) and serum DNA quantification. Bone marrow chimeras using Pdx-Cre KrasG12D (KC) transgenic mice, which develop spontaneous pancreatic cancer, were generated with lethal irradiation followed by reconstitution with PAD4-/- or WT bone marrow. 21 days after treatment with cerulein, which induces pancreatitis to accelerate tumor growth, animals were sacrificed and organs harvested. Hematoxylin and eosin stained tumor sections were evaluated by a pancreatic pathologist.

Results: PAD4-/-tumor bearing mice showed decreased NETs on IHC and decreased serum DNA (406ng/ml vs 858ng/ml, p=0.03), a surrogate NET marker. Tumorigenesis was significantly decreased in PAD4-/- vs. WT mice (921mg vs. 326mg, p=0.001; ROI: 2.4×107 vs. 1.6×106, p=0.05). DNAse treatment of WT mice lead to significantly decreased tumor growth vs. sham treated controls (336mg vs. 206mg, p=0.05; ROI: 9.9×105 vs. 3.8×105, p=0.05) while there was no significant change in tumor growth in PAD4-/- animals treated with DNAse (195mg vs. 217mg, p=0.29; ROI: 8.5×105 vs. 6.9×105, p=0.43). There was a trend toward decreased high-grade precursors and invasive cancers in PAD4-/- bone marrow recipients as compared to WT recipients (p=0.29) with significantly diminished NET formation by isolated bone marrow supernatant DNA levels (197ng/mL vs. 796ng/mL, p=0.001).

Conclusion: Murine pancreatic tumors in PAD4-/-mice show decreased tumorigenesis and decreased NET formation. DNA released during NET formation leads to increased tumor growth, which is suppressed by DNAse administration. Future studies will focus on the mechanism through which NET DNA promotes tumor growth.

 

19.03 Gut Microbiome Promotes Pancreatic Oncogenesis by Inducing Innate and Adaptive Immune Suppression

Posted on January 25, 2017

M. Hundeyin1, S. Pushalkar1, D. Daley1, G. Werba1, N. Mohan1, S. Lall1, B. Wadowski1, B. Aykut1, E. Kurz1, U. Soni1, E. Morales-Vicente1, D. Saxena1, G. Miller1  1New York University School Of Medicine,Surgery,New York, NY, USA

Introduction:
Pancreatic ductal adenocarcinoma (PDA) is the 3rd most lethal cancer in the United States and accounts for 85% of all pancreatic malignancies. The gut microbiome has emerged as an important regulator in the balance between health and disease, including oncogenesis. However, the microbiome has not been directly linked to pancreatic oncogenesis. We postulated that hosts with PDA harbor an altered pancreatic and gut microbiome and that dysbiosis influences PDA progression. 

Methods:
To determine whether endoluminal gut bacteria can access the pancreas, we administered fluorescently-labeled Enterococcus faecalis to WT mice via oral gavage. To determine whether bacteria promote the progression of pancreatic oncogenesis, we employed the slowly progressive p48Cre;LSL-KrasG12D (KC) mouse model of PDA and rederived KC mice in a germ-free environment. To identify possible perturbations in the gut microbiome associated with the progressive pancreatic oncogenesis, we evaluated the composition of the gut microbial community of KC mice compared with age-matched WT littermate controls by performing 16S gene sequencing. Lastly to determine the mechanism of immunosuppression; we injected F21242 pancreatic tumor cell line orthotopically into the pancreas of WT mice after bacterial ablation and vehicle, isolated the pancreatic leukocytes and analyzed them by flow cytometry. 

Results:
In the pre-morbid state the intestinal microbiome is similar in mice bearing pancreas-specific oncogenic mutations and in controls; however, as mice age, PDA-bearing hosts develop a unique gut microbiome including expansion of Actinobacteria and Deferribacteres. We found that gut bacteria access the pancreas and the cancerous pancreas harbors a distinct microbiome in mice. Further, genotypically identical PDA-bearing mice that exhibit divergent disease phenotypes harbor stage-specific microbiomes suggesting that microbial structure is associated with disease aggressiveness. Germ-free or ablative antibiotic treated mice were protected against PDA whereas transfer of gut bacteria from PDA-bearing mice, but not from control mice, reversed the tumor-protection. Bacterial ablation was associated with innate and adaptive immunogenic reprogramming of the PDA tumor microenvironment including a marked reduction in myeloid-derived suppressor cells and immune-suppressive macrophages, increased Th1 differentiation of CD4+ T cells, and expansion and activation of cytotoxic CD8+ T cells. In addition, we show that gut bacterial ablation and PD-1 blockade offer synergistic efficacy.

Conclusion:
These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of PDA and that the microbiome has marked potential as a biomarker and therapeutic target in PDA.
 

19.02 Personalized Medicine in Cleft Lip and Palate: Functional Genomics Analysis of IRF6 Gene Variants

Posted on January 25, 2017

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

Introduction:

Cleft lip and/or palates (CL/P) are among the most common congenital malformations. Mutations in the transcription factor gene IRF6 represent the most significant genetic contributors to orofacial clefts. Hundreds of novel sequence variations in IRF6 have been reported. However, the functional significances of the myriad of IRF6 mutations remain difficult to ascertain as in silico prediction programs have poor predictive power and often provide conflicting results. To address this gap in knowledge, we hypothesized that the protein function of IRF6 gene variants could be rapidly assessed by their ability to rescue the irf6-/- mutant phenotype in animal models.

Methods:

CRISPR genome editing was used to generate irf6-/- zebrafish. Previously uncharacterized IRF6 variants were identified and categorized by computational predictions from Polyphen2 and SIFT. Variants were introduced to wildtype irf6 cDNA by site-directed mutagenesis; mRNA was produced by in vitro transcription and microinjected into irf6-/- embryos to assess for phenotypic and functional rescue.

Results:

All offspring of irf6-/- females displayed an embryonic rupture phenotype caused by defects in the embryonic periderm, a simple squamous epithelium surrounding the embryo that models the human oral epithelium. The periderm rupture model is validated for use in the analysis of human IRF6 function, as injection of human IRF6 mRNA into mutant zebrafish embryos can fully rescue the mutant phenotype. Human IRF6 gene variant mRNAs were then injected in a rapid and accurate rescue assay to assess the effects of human IRF6 mutations on protein function.

The IRF6 gene variant rescue results revealed that neither location of the mutations within the IRF6 protein nor computational programs were accurate predictors of whether observed human IRF6 mutations actually resulted in defective proteins. Several variants predicted to be benign failed to rescue, while others predicted to be deleterious by both computational programs were able to rescue not only periderm rupture, but also normal craniofacial development.

Conclusion:

The zebrafish irf6-/- rescue assay rapidly addressed the functional significance of novel IRF6 mutations and overcame computational limitations for predicting variant pathogenicity. These validated biological results can help improve future computational predictions, and moreover, unlock the possibility for rapidly characterizing yet undiscovered IRF6 variants from orofacial cleft patients, illustrating a generalizable functional genomics paradigm of personalized medicine.

19.01 Arid1a Has Context-Dependent Oncogenic and Tumor Suppressive Roles in Liver Cancer

Posted on January 25, 2017

S. C. Wang1,2, X. Sun2, I. Nassour1,2, X. Luo2, J. Chuang2, L. Li2, T. Maples2, C. Celen2, L. H. Nguyen2,3, S. Zhang2, H. Zhu2  1University Of Texas Southwestern Medical Center,Division Of Surgical Oncology,Dallas, TX, USA 2University Of Texas Southwestern Medical Center,Children’s Research Institute,Dallas, TX, USA 3Howard Hughes Medical Institute,Chevy Chase, MD, USA

Introduction:

ARID1A, a component in the SWI/SNF chromatin-remodeling complex, is one of the most commonly mutated genes in cancer. Because a majority of the mutations are loss-of-function, ARID1A is widely assumed to be a tumor suppressor. However, the functional effect of ARID1A loss is unclear. Here, we used a genetically engineered mouse model to dissect the effects of Arid1a loss in liver cancer.

 

Methods:

A liver-specific Tet-Off MYC overexpression liver cancer model was used. The tetracycline transactivator was driven by the liver activator protein promoter and MYC expression was under the control of the tetracycline response element (LAP-tTA; TRE-MYC, referred heretofore as LAP-MYC mice). MYC was induced at birth by removal of doxycycline. Liver specific Arid1a knockout (LKO) was achieved by crossing in Albumin-Cre; Arid1af/f . A lentiviral system was used to stably express shArid1a in the mouse hepatoma Hepa1c1c7 cell line. The Arid1a overexpression construct was delivered with adenovirus.

 

Results:

LAP-MYC LKO mice had significantly improved survival as compared to LAP-MYC mice (Figure A; HR: 0.45; 95% CI: 0.28 to 0.80, P < 0.01) suggesting that Arid1a had oncogenic properties. When we overexpressed Arid1a in LAP-MYC mice, we found increased tumor burden, confirming that Arid1a could be oncogenic (Figure B; P < 0.01). Next, we transplanted Hepa1c1c7 cell lines that stably expressed shArid1a in the subcutaneous tissue of immunocompromised mice. The tumors grew at the same rate as cells that had normal Arid1a expression. These data suggest that Arid1a was required for normal initiation of liver cancer but did not constrain the growth of established cancer cells.

 

RNA-seq of LAP-MYC and LAP-MYC LKO tumors demonstrated pro-invasion and metastasis gene programs were upregulated in the LAP-MYC LKO tumors, signifying that in this context, Arid1a had tumor suppressive effects. To test the functional metastatic effects of Arid1a loss, we delivered the shArid1a expressing Hepa1c1c7 cells systemically via tail vein injections and found that the Arid1a knockdown cells resulted in more lung metastases than control cells (Figure C; P < 0.001). This showed that in established cancers Arid1a was tumor suppressive and its loss conferred increased metastatic potential.

 

Conclusions:

Arid1a in liver cancer has both oncogenic and tumor suppressive properties dependent on temporal context. It potentiates tumor initiation and constrains metastases. Other contexts, such as tissue type and dosage, should also be explored to fully characterize the role that Arid1a plays in cancer.

04.20 Effects of CD47mAb Treatment on VEGF Signaling in the Huh-7 Hepatocellular Carcinoma Cell Line

Posted on January 25, 2017

D. Chirumbole1, M. Xu1, X. Wang1, G. A. Upadhya1, Y. Lin1, W. C. Chapman1  1Washington University,Transplant Section, General Surgery,St. Louis, MO, USA

Introduction:
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and studies suggest that CD47mAb immunotherapy could be an effective therapeutic option. CD47 ligation has also been shown to inhibit vascular endothelial growth factor (VEGF) signaling in various cell types. However, few studies have considered the effects of CD47mAb treatment on VEGF signaling and tumor progression in HCC. The purpose of this study is to investigate the effects of CD47mAb treatment on VEGF signaling in the Huh-7 HCC cell line.  

Methods:
The Huh-7 HCC cell line was treated with Control IgG, CD47mAb1 (VX1000R), or CD47mAb2 (VX1004R) at a concentration of 10 µg/mL for 24 hours. Cells were then collected for study. ELISA, qRT-PCR, Western blot, and immunofluorescent staining analyses were performed to investigate the expression of VEGFs, VEGFRs, epidermal growth factor receptor (EGFR), and downstream markers related to cell growth and proliferation at the mRNA or protein level. 

Results:
VEGFR-1 and -2 expression was significantly reduced in VX1004R-treated cells compared to control in terms of relative density to β-actin (VEGFR-1: 0.181±0.105 vs. 0.618±0.064, p=0.004; VEGFR-2: 0.127±0.071 vs. 0.259±0.038, p=0.048). Phosphorylated ERK1/2 and total p38 MAPK were also significantly reduced after VX1004R treatment (phospho-ERK1/2: 0.582±0.020 vs. 0.736±0.088, p=0.042; p38 MAPK: 0.857±0.077 vs. 1.120±0.014, p=0.004), as was the expression of proliferating cell nuclear antigen (PCNA) (0.457±0.044 vs. 0.546±0.003, p=0.047). On the other hand, there was no significant change in phosphorylated p38 MAPK expression after treatment. VX1000R-treated cells showed a trend toward decreased expression of most markers, but there were no significant differences at p<0.05. Expression of vegf-a mRNA was significantly reduced in both CD47mAb-treated groups (VX1004R: fold change=0.593±0.061, p=0.002; VX1000R: fold change=0.775±0.046, p=0.010). Expression of vegf-b mRNA was reduced in VX1004R-treated cells only (fold change=0.693±0.083, p=0.010). There was a non-significant trend toward decreased EGFR expression in both CD47mAb-treated groups. 

Conclusion:
The results of this study suggest that CD47mAb treatment may inhibit VEGF signaling, downstream kinase activation, and cell proliferation in the Huh-7 cell line and therefore could inhibit tumor progression. VX1004R appears to be a more effective treatment than VX1000R. Further study is needed to investigate the effects of CD47mAb treatment on VEGF signaling in other HCC cell lines and in an in vivo system.   
 

04.19 Differential Sepsis Survival Between Genetically Identical Mice from Different Vendors

Posted on January 25, 2017

K. Fay1, N. Klingensmith1, J. Lyons1, C. Chen1, C. Coopersmith2, M. L. Ford3  1Emory University School Of Medicine,Department Of Surgery,Atlanta, GA, USA 2Emory University,Critical Care Center,Atlanta, GA, USA 3Emory University,Transplant Center,Atlanta, GA, USA

Introduction:  Previous studies have shown that mice with identical genetic backgrounds obtained from different vendors possess differential immuno-phenotypic characteristics based on environmental factors including the composition of the microbiome. However, immune function in the setting of murine septic peritonitis has been previously assumed to be consistent within mice of the same strain. Here, we aimed to determine whether vendor-specific environmental factors impact survival and immune function following CLP.

Methods:  6 week old B6 mice from Charles River (CR) and Jackson Lab (JAX) underwent cecal ligation and puncture (CLP) one to two days after arrival to prevent acclimation to an environment different from their original vendor. To assess survival, each group underwent CLP and was followed for seven days. To assess immunophenotype, mice were sacrificed at 24 hours after CLP and underwent splenic harvest. Cells were then processed and stained for phenotypic and functional markers to be analyzed via flow cytometry. Data are presented as mean±SEM. 

Results: CR B6 mice exhibited worsened lymphopenia in the setting of sepsis compared to JAX B6 mice, and a significantly decreased frequency of CD4+ and CD8+ T cells (36.9±0.8 vs 44.1±2.0, p<0.05 and 25.0±1.8 vs 29.5±0.8, p<0.01). When function of these T cells was examined via cytokine analysis, CR B6 mice possessed significantly reduced frequencies of IL-2- secreting T cells as compared to JAX B6 mice, and this was true for both the CD4+ and CD8+ T cell compartments (8.7±0.4 vs 15.7±2.1, <p0.05 and 5.8±0.3 vs 12.2±1.5, p<0.01, respectively). This immune profile of worsened lymphopenia and decreased frequencies of IL-2-secreting CD4+ T cells observed in CR septic mice has been previously associated with worsened mortality. However, CR B6 mice exhibited profoundly superior survival following CLP as compared to JAX B6 mice (90% vs. 0%, p<0.0001). Further evaluation for possible mechanisms underlying this finding revealed that CR septic mice possessed significantly increased frequencies of IFNγ+ CD4+ cells compared to JAX septic mice (2.4±0.1 vs 1.5±0.2, p<0.01). This was not associated with a difference in the frequencies of dendritic cells between the two groups (21.1±1.6 vs. 25.9±3.7). However, CR septic mice did demonstrate significantly higher frequencies of effector memory CD4+ T cells compared to JAX septic mice (10.6±0.3 vs 7.3±0.4, p<0.01). 

Conclusions: Having the same genetic background does not result in an identical immunophenotype following the induction of sepsis in mice obtained from different vendors. Interestingly, despite having some immunologic features previously associated with increased mortality, Charles River B6 mice exhibited markedly better survival compared to Jackson Lab B6 mice.  We conclude that the presence of increased IFNγ-secreting CD4+ and effector memory cells could underlie the improved survival observed in Charles River B6 mice. Previous work has demonstrated an effect of the microbiome on antigen specific T cell proliferation and function, which may play a role in the differences with see between Charles River and Jackson Lab mice. Evaluation of differences between the microbiota of the two groups will be an avenue of future exploration. 

 

04.18 Sexual Dimorphism in Inflammation and Obesity: Disparate Effects on PMN Activation

Posted on January 25, 2017

M. E. Diebel1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Marian And Michael Ilitch Department Of Surgery,Detroit, MI, USA

Introduction: There is conflicting data regarding the impact of obesity on the outcomes of trauma and critically ill patients.  Sexual dimorphism has been demonstrated in both trauma and obese patient populations, with a protective effect in females related to the estrogen (E2) hormone.  The effects of gender and sex hormones on the obese trauma patient are unknown.  It has been shown that E2 has anti-inflammatory effects in a number of clinical and experimental settings.   We postulated that there would be sex hormone related differences in polymorphonuclear (PMN) activation when co cultured with adipose tissue under conditions that simulate physiologic stress.  This was studied in vivo.
 

Methods: PMNs from normal human volunteers were incubated with a co culture of adipocytes and macrophages (“adipose tissue”, AT) under normal laboratory or stress conditions consisting of an anoxia-reoxygenation challenge and Epinephrine (10-3 µM ).  In subgroups, E2 or testosterone (DHT) was added at physiologic concentrations.  PMN adhesion (CD11b, CD62) and degranulation (CD35) markers were determined by flow cytometry.

Results: See table. (mean ± SD; N = 4 for each group)

Conclusions: Our data show that there is sexual dimorphism in PMN activation following exposure to AT under stress conditions. This suggests that the female sex hormone E2 may abrogate PMN induced tissue injury in the obese patient following shock and other stress conditions.

 

04.17 Surgical patients with infection have suppressed monocyte function and increased IRAK-M expression

Posted on January 25, 2017

N. J. Galbraith1, S. P. Walker1, S. A. Gardner1, J. V. Carter1, S. Manek1, S. Galandiuk1, H. C. Polk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:
Some patients who suffer major infection or trauma respond with an exaggerated compensatory anti-inflammatory response. The purpose of this study was to study monocyte function of patients with surgical infection, and determine the expression of negative regulators of toll-like receptor signaling. . 

Methods:

Monocytes were isolated following phlebotomy after written informed consent in colon and rectal surgery patients diagnosed with deep surgical site infection. Samples were studied for baseline parameters of monocyte function, including HLA-DR, and immune response was studied by ex-vivo lipopolysaccharide (LPS) stimulation (100 ng/mL) for 4h. At 4 h, levels of HLA-DR and cytokine responses determined by flow cytometry (FACS) and ELISA, respectively. Monocyte gene expression of negative regulators including suppressor of cytokine signaling 3 (SOCS3),  interleukin-1 receptor-associated kinase M (IRAK-M), Src homology region 2 domain-containing phosphatase-1 (SHP-1), and A20 (TNFAIP3, Tumor Necrosis Factor, Alpha-Induced Protein 3) were measured by qRT-PCR. Follow-up samples from the same patients were obtained after resolution of the infection where possible. Healthy controls were studied for comparison. 

Results:

Sixteen patients with deep surgical site infection were studied, the majority of which were diagnosed with intra-abdominal abscesses and who were hemodynamically stable at the time of sampling. Ten healthy controls were included for comparison. Baseline monocyte HLA-DR expression was similar between patients and healthy controls, but patients with deep surgical site infection had a lower HLA-DR expression in response to LPS (p<0.05). Infected patients had a suppressed TNF-α production when compared against healthy volunteers (p<0.05). In contrast, infected patients had higher levels of circuiting plasma IL-10 levels (p<0.05).

Of the negative regulators studied, IRAK-M was the most consistently up-regulated in infected patients as compared to healthy controls (p<0.05). Interestingly, the expression of IRAK-M partially returned to the levels of healthy controls following clinical recovery from infection. 

Conclusion:

In this study, patients with deep surgical site infection have lower levels of HLA-DR and TNF-α following LPS stimulation, suggestive of impaired cytokine responses and antigen presenting function. IRAK-M, a negative regulator of TLR signaling, was up-regulated in patients during infection, with a partial return to normal levels following clinical recovery. IRAK-M may play a role as a biomarker in identifying “high risk” patients.

04.16 The Innate Immune System Suppresses the Progression of Metastatic Pancreatic Cancer

Posted on January 25, 2017

A. D. Michaels1, S. J. Adair2, M. G. Mullen1, S. Nagdas2, J. B. Persily1, N. Jha1, J. T. Parsons2, T. W. Bauer1  1University Of Virginia,Surgery,Charlottesville, VA, USA 2University Of Virginia,Microbiology, Immunology, And Cancer Biology,Charlottesville, VA, USA

Introduction:  Despite a margin-negative (R0) resection, most patients with localized pancreatic adenocarcinoma (PDAC) eventually die from metastatic disease and therefore likely harbor occult metastases at the time of resection. The ability to inhibit growth of occult metastases could greatly improve survival of patients with PDAC. Prior studies suggest a role of the innate immune system in suppressing the progression of PDAC hepatic micrometasases. To test this hypothesis, we utilized a murine model of micrometastatic PDAC with patient-derived xenografts (PDX).

Methods:  Patient-derived PDAC cells expressing luciferase were injected into the spleens of two strains of mice resulting in liver metastases, followed by splenectomy to remove the primary tumor. The mouse strains used were athymic nude mice which lack cell-mediated immunity but have functioning innate immunity and NOD scid gamma (NSG) mice which lack both cell-mediated immunity as well as functional macrophages, dendritic cells, and NK cells. Metastatic tumor burden was evaluated by serial bioluminescent imaging. When disease burden threatened life, mice were sacrificed and necropsied.  Gene expression profiling (GEP) was performed on matched hepatic and peritoneal metastases from NSG mice. 

Results: The spleens of 12 nude and 12 NSG mice were injected with PDX 449 cells. There was faster and more extensive clearance of tumor cells in nude mice. By post-injection day 42, the majority of the NSG mice had extensive abdominal disease whereas only three nude mice had mild-to-moderate tumor burden. Figure 1 displays representative bioluminescent imaging at days 2 and 42. At necropsy, gross liver metastases were present in 5/12 (42%) NSG mice and 100% had peritoneal metastases. By bioluminescent imaging through 78 days, 4/12 (33%) nude mice had peritoneal metastasis and there was no evidence of any hepatic metastasis. For both metastatic sites, the NSGs had significantly more disease (liver, p=0.04; peritoneum, p=0.001). GEP of metastases from three NSG mice demonstrated significant differences between tumors from the different sites. Hierarchical clustering by innate immunity-related genes revealed that hepatic tumors cluster separately from peritoneal tumors with the most substantial differences in the TLR, VEGFA, and MAPK signaling pathways.

Conclusion: The innate immune system significantly inhibits progression of occult metastatic PDAC cells in the liver and peritoneum. Immunotherapy approaches which augment the innate immune system could greatly improve survival in patients with PDAC.

 

04.15 Cancer-Associated Plastic IL17+Foxp3+ and ex-Th17 Foxp3+ Cells are Immunosuppressive

Posted on January 25, 2017

S. E. Berkey1, D. L. Bartlett1, N. Obermajer1  1University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA

Introduction: Regulatory T (Treg) cells are integral in maintaining immune homeostasis and are associated with the inflammatory immunosuppression observed in cancer. Expansion of Treg cells within the tumor is a known barrier to successful cancer immunotherapy. In previous experiments, we have demonstrated that Th17 cells are a novel source of tumor-induced forkhead box P3 (Foxp3+) cells in addition to the natural (n)Treg and induced (i)Treg cells, which develop from naïve precursors. During tumor growth, Th17 cells progressively transdifferentiate into IL17+Foxp3+ and ex-Th17 IL17negFoxp3+ T cells. Here, we aimed to characterize the immunosuppressive ability of the ex-Th17 IL17negFoxp3+ T cells.

 

Methods: Using novel Th17eYFP-Foxp3mRFP reporter mice, CD4+ cells were isolated from splenocytes. The cells were cultured under Th17-driving conditions (IL6, IL23, and TGF-β) for 3-4 days, followed by Treg– driving conditions (TGF-β) for an additional 3 days, and then sorted into Th17-Treg subsets (eYFP+IL17+Foxp3neg, eYFP+IL17+Foxp3+, eYFP+IL17negFoxp3neg, eYFP+IL17negFoxp3+, eYFPnegIL17negFoxp3neg and eYFPnegIL17negFoxp3+ (inducible (i)Treg cells)). CD4+ cells were then isolated from CD45.1 mice and stained with the proliferation dye, carboxyfluorescein succinimidyl ester (CFSE). These CD4+ cells were activated with anti-CD3e Ab in the presence of CD4neg fraction and Th17-Treg subsets (2:1 ratio of CD4+ target cells: Th17-Treg subset). The cells were incubated for 72 hours and then analyzed by flow cytometry. The percentage of proliferating cells (CFSEneg, gated on live/CD45.1+CD4+ cells) was calculated as a fraction of proliferating cells compared to control condition where CD4+ cells were activated with anti-CD3e Ab in the absence of Th17-Treg subsets.

 

Results: We demonstrate that IL17+Foxp3+ and ex-Th17 IL17negFoxp3+ T cells have greater suppressive effects on CD4+ T cell proliferation compared to Th17 cells and have similar suppressive effects as (i)Treg cells.

 

Conclusions: The conversion of Th17 into Foxp3+ T cells is an alternative route of IL17+Foxp3+ and IL17negFoxp3+ T cell development in tumors. Due to their immunosuppressive properties, transdifferentiated Treg cells represent a novel target and warrant new therapeutic approaches aimed at eliminating Foxp3+ T cells in tumor immunotherapy.

04.14 Exploring Immunologic Aspects of Regional Therapy in Melanoma

Posted on January 25, 2017

J. A. Perone1, T. Tamasa1,2, M. Tsutsui2, P. Dolber1, I. V. Pinchuk1, D. S. Tyler1,2, K. Olino1  2Durham Veterans Affairs Medical Center,Durham, NC, USA 1University Of Texas Medical Branch,Surgery,Galveston, TX, USA

Introduction:

In- transit melanoma is an aggressive, rare disease subtype presenting with multiple subcutaneous lesions. Treatment options include resection, intralesional therapy, and regional chemotherapy with isolated limb infusion (ILI).  Despite short-term efficacy of ILI, disease recurrence and development of metastatic disease are common.  New research has focused on the ability of chemotherapy, particularly doxorubicin to elicit immunogenic cell death. We hypothesized that both intra-lesional (ITI) and regional treatment with doxorubicin would lead to not only treatment responses due to its cytolytic activity, but would also result in immunogenic cell death and the activation of innate immune responses. 

 

Methods:

B16 F10.9-OVA melanoma was injected intra-dermally into C57Bl/6 female mice to create a solitary leg tumor. Mice were randomized into 4 treatment groups: saline ITI, saline ILI and Doxorubicin ITI or ILI.  Mice were followed for tumor growth, survival and local toxicities. Immunologic evaluation was performed by flow cytometry and a 34-cytokine bioplex assay of the treated tumors.

 

Results:

Mice treated with doxorubicin both with ILI or ITI had delayed tumor growth (p<0.0001, one-way ANOVA) and longer survival (p<0.0001, log-rank test) when compared to saline controls. Bioplex analysis 7 days following treatment with chemotherapy showed an increase in G-CSF production within the doxorubicin treated tumors while VEGF production decreased (Figure 1).

 

Conclusion:

ITI and ILI with doxorubicin leads to a local immune response as well as direct cytotoxic effects in an orthotopic model of murine melanoma.  This suggests that an innate immune response is present likely due to both infiltration of neutrophils and macrophages.  The decrease in VEGF production is also important as this may also improve the ability within the local tumor microenvironment to overcome immunosuppressive elements.

04.13 The prognostic Impact of HLA Class I (A, B, C), HLA-E and HLA-G expression in Pancreatic Cancer

Posted on January 25, 2017

S. W. De Geus1, H. A. Prevoo1, N. G. Dekker-Ensink1, B. A. Bonsing1, H. Morreau2, A. L. Vahrmeijer1, P. J. Van Den Elsen3, C. J. Van De Velde1, P. J. Kuppen1  1Leiden University Medical Center,Surgery,Leiden, 2333 ZA, Netherlands 2Leiden University Medical Center,Pathology,Leiden, 2333 ZA, Netherlands 3Leiden University Medical Center,Immunohematology,Leiden, 2333 ZA, Netherlands

Introduction:  Pancreatic cancer has a notoriously dismal prognosis, which is to a large degree caused by its high metastatic potential. Even after complete surgical resection of the primary tumor, distal recurrence rates range from 46% to 89%. Tumor evasion and suppression of the host immune surveillance system has demonstrated to affect clinical outcome in various malignancies. However, the role of immune system in pancreatic cancer remains mainly unclear. The purpose of this study was to investigate the clinical impact of classical human leukocyte antigen (HLA) class I (A, B, C), and non-classical HLA-G and HLA-E tumor expression in pancreatic cancer patients.

Methods:  Immunohistochemical stainings were performed for HLA class I (A, B, C), HLA-G and HLA-E tumor expression in pancreatic adenocarcinoma patients (n = 137) who underwent primary surgery between 2003 and 2013. Survival analysis was performed using the multivariate Cox proportional hazard models for overall (OS) and relapse-free (RFS) survival. 

Results: HLA class I, HLA-G and HLA-E expression was observed in respectively 50%, 17% and 96% of the pancreatic adenocarcinoma patients. On multivariate analysis, primary tumor stage pT2-4 (HR, 2.188; 95% CI, 1.029-4.653; p=0.042), lymph node stage pN1 (HR, 1.327; 95% CI, 0.809-2.176; p=0.262), and HLA-G expression (HR, 1.743; 95% CI, 1.010-3.009; p=0.046) were associated with unfavorable OS. HLA-G expression resulted in median OS of 10.0 months (95% CI, 3.3 – 16.7 months), whereas absence of HLA-G expression resulted in median OS of 18.0 months (95% CI, 13.9 – 22.1 months). In addition, primary tumor stage pT2-4 (HR, 1.745; 95% CI, 0.851-3.577; p=0.128), lymph node stage pN1 (HR, 1.281; 95% CI, 0.785-2.090; p=0.321), and HLA-G expression (HR, 1.820; 95% Cl, 1.053-3.146; p=0.032) were also independently predictive for adverse RFS. Median RFS was 7.0 months (95% CI, 3.0 – 11.0 months) for patients demonstrating positive HLA-G expression compared to 12.0 months (95% CI, 8.8 – 15.3 months) for patients with absent HLA-G expression. HLA class I and HLA-E expression showed no correlated with OS or RFS in pancreatic adenocarcinoma patients.    

Conclusion: This study demonstrated that HLA-G expression was significantly associated with adverse OS and RFS in pancreatic adenocarcinoma patients. Consequently, immunohistochemistry for these markers may aid identification of patients with a poor prognosis, who likely benefit less from an upfront surgical strategy. In addition, these results provide further evidence for the immunogenic character of pancreatic cancer and subsequent potential for therapeutic strategies targeting the immune system.

 

04.12 Improved Survival in Neonatal Sepsis Following Alum Pretreatment is Independent of Neutrophils

Posted on January 25, 2017

S. L. Raymond1, J. C. Rincon1, A. Cuenca1, D. C. Nacionales1, L. L. Moldawer1, S. D. Larson1  1University Of Florida,Department Of Surgery,Gainesville, FL, USA

Introduction: Sepsis is one of the leading causes of death among neonates with over 1 million deaths annually. Development of neonatal sepsis results in part from a failure of host innate immunity. Alum has long been used as a vaccine adjunct and is currently FDA approved for clinical use. However, the exact mechanisms by which alum enhances immunity are poorly understood. The purpose of this study is to delineate the mechanisms by which alum enhances neonatal protective immunity and improves survival in sepsis.

Methods: To determine the impact of adaptive immunity, we studied Rag1-knockout mice which do not develop functional B or T cells. 6 day old neonatal wild type C57BL/6J (WT) and Rag1-knockout mice received pretreatment with Imject alum®, 100  μg s.c. and 24 hours later underwent i.p. administration of cecal slurry (CS) (LD40-60) to induce polymicrobial intra-abdominal sepsis. Survival was assessed for 7 days. To determine the contribution of innate immunity, 5 day old neonatal WT mice were depleted of neutrophils or Gr1+ cells (neutrophils, monocytes, dendritic cells) by administration of either anti-Ly6G antibody (1 mg/kg i.p.) or anti-Gr1 antibody (60 μg i.p.), respectively. At day 6, Imject alum®, and on day 7, cecal slurry were administered. Survival was assessed for 7 days. 

Results: Alum pretreatment significantly improved survival following polymicrobial sepsis in both neonatal WT and Rag1-knockout mice compared to untreated controls (p<0.05). Administration of anti-Ly6G resulted in a >95% reduction of circulating neutrophils. Alum pretreatment improved survival in anti-Ly6G neutrophil-depleted neonatal mice following sepsis (p<0.05) (Fig. 1). Conversely, following Gr1+ cell depletion, mortality was increased compared to non-depleted mice (p<0.05). Additionally, alum treatment following Gr1+ cell depletion failed to improve survival in sepsis.   

Conclusion: Pretreatment of neonatal mice with the commonly used pediatric vaccine adjuvant alum improves survival in polymicrobial intra-abdominal sepsis. We demonstrate, for the first time, that alum improves survival independent of functional B or T cells and in the absence of neutrophils. Our data suggests that the protective effect of alum is likely dependent on non-granulocyte Gr1+ myeloid cells, mainly monocytes and dendritic cells. These novel findings suggest a therapeutic potential of administering FDA approved alum-based adjuvants to neonates to enhance innate immune function and prevent neonatal sepsis.
 

04.11 Psychological Stress During Pregnancy Alters Immune Function in Maternal and Neonatal Mice

Posted on January 25, 2017

S. Deas1, M. Melendez-Ferro1, V. Yeramilli4, C. Culbreath2, R. Lorenz5, C. Martin1,3  1University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 2Inova Fairfax Hospital,Surgery,Falls Church, VA, USA 3Children’s Of Alabama,Surgery,Birmingham, ALABAMA, USA 4University Of Alabama at Birmingham,Microbiology,Birmingham, Alabama, USA 5University Of Alabama at Birmingham,Pathology,Birmingham, Alabama, USA

Introduction:  Psychological and environmental stress have been linked to several health conditions. During pregnancy, stress can negatively affect both the mother and child. Potential psychological stressors include a challenging home environment or life event, substance abuse, or chronic health problems. The impact of maternal psychological stress on the neonatal immune system remains unknown. A key element of immune development in neonates is the transfer of protective antibodies from the mother to the newborn. Specifically, the presence of immunoglobulin A (IgA) and early bacterial colonization are key processes in the development of passive immunity in neonates. IgA protects the mucosal surface of the gut by binding to pathogenic bacteria and preventing colonization; in the early stages of development, it is passed from mother to child in breast milk. The gut microbiome further protects mucosa, and it is primarily transmitted from the vaginal microbiome during passage through the birth canal and feeding. We hypothesize that prenatal psychological stress depresses immune function in the mother, thereby impairing transmission of passive immunity to neonates. 

Methods:  8-week-old C57BL/6 littermates underwent timed breeding. The females of three mating pairs were subjected to daily psychological stress by using a well-established restraint model during days 7-14 of the gestational period. The control group experienced no stress. Maternal vaginal microbiome was sampled two days prior to delivery via vaginal lavage; samples were then cultured on Schaedler agar in aerobic conditions for 24 hours at 37°C. Enzyme-linked immunosorbent assay (ELISA) was used to measure fecal IgA levels in in 2-week-old pups.  

Results: Two-week-old mice from the stressed group had significantly less fecal IgA compared with the control group; stress group mean = 51658.51 ng/mL, standard error = 11982.92; control group mean = 200320.22 ng/mL, standard error = 57674.83 (P value = 0.0033). Microbial culture of vaginal microbiome from the mother revealed 98 colony forming units (CFUs) for the stressed group, compared to 187 CFUs for the control group (see figure).

Conclusion: Maternal gestational stress results in significantly less fecal IgA in offspring compared to controls. Additionally, stressed dams had lower vaginal bacterial colonization compared to controls, which could explain this finding. Future experiments will further elucidate this mechanism by analyzing serum IgA levels in the pups and stress hormones in dams and pups.  

 

04.10 Stat3 inhibition enhances myeloid derived suppressor cell apoptosis via Fas/FasL signaling

Posted on January 25, 2017

P. Guha1, J. C. Gardell1, M. Cunetta1, M. Lima1, J. Darpolor1, S. Ferree1,2, N. Espat1,2, S. C. Katz1,2  1Roger Williams Medical Center,Surgical Oncology,Providence, RI, USA 2Boston University,Boston, MA, USA

Introduction: Myeloid derived suppressor cells (MDSC) mediate intrahepatic immune suppression in liver metastasis (LM) patients. MDSCs are a heterogeneous group of immature myeloid cells that limit the efficacy of genetically engineered chimeric antigen receptor T cells (CAR-T).  We seek mechanistic targets to limit MDSC expansion or induce death to enhance CAR-T efficacy for LM.

Methods: Tumor burden of murine LM model treated with DMSO or STATTIC or BBI was measured using bioluminescence. Flow cytometry, western blot, qPCR and immunofluorescence was performed on MDSC isolated from tumors. LDH assay and CFSE was used to measure MDSC induced suppression of CAR-T.

Results:Previously, we have shown that the GM-CSF/Jak2/STAT3 axis drives liver MDSC (L-MDSC) expansion and suppressive function in murine LM model. We hypothesized that blocking STAT3 activation would cause decreased L-MDSC viability in the tumor microenvironment. STATTIC and BBI target STAT3 by inhibiting its activation, dimerization and nuclear translocation. Both STATTIC and BBI caused a reduction in tumor burden in our LM model with decrease in L-MDSC and pSTAT3 levels as compared to the vehicle (DMSO) control mice. When L-MDSC isolated from STATTIC or BBI treated tumor bearing mice were co-cultured with CAR-T cells, there was a significant decrease in immunosuppression as evidenced by enhanced tumor cell cytotoxicity (29+4% DMSO,54+4% STATTIC p=0.02, 52+7% BBI p=0.01) and CAR-T expansion (22+2% DMSO,36+3% STATTIC p=0.001, 38+4% BBI p=0.001). There was significant increase in apoptosis of L-MDSCs (table) isolated from STATTIC or BBI treated tumor bearing mice as compared to DMSO control. Investigation of apoptosis pathway signaling molecules showed that there was an up-regulation of pro-apoptotic Bax (STATTIC-3.6 fold and BBI-2.7 fold over DMSO, p<0.05) and down-regulation of anti-apoptotic Bcl2 (STATTIC-0.8 fold and BBI-0.5 fold over DMSO, p<0.05) protein in L-MDSCs. This was also associated with significant decrease in proliferation markers such as phosphorylated Erk (pErk) (STATTIC-0.3 fold and BBI-0.5 fold over DMSO, p<0.05), phosphorylated Akt (pAkt) (STATTIC-0.3 fold and BBI-0.4 fold over DMSO, p<0.05). Interestingly, there was an increase in phosphorylated p38 (pp38) MAPK activity (STATTIC-2.7 fold and BBI-3.2 fold over DMSO, p<0.05), Fas (table) and TNFα (STATTIC-3.4 fold and BBI-2.8 fold over DMSO, p<0.05) expression in MDSCs. 

Conclusion: Our findings indicate that STAT3 inhibition caused an increase in L-MDSC apoptosis through up-regulation of Fas/Fas-L with down-stream pro-apoptotic signaling via p38MAPK. Thus, blockade of STAT3 in tumor bearing mice may enable us to rescue the anti-tumor immunity for LM through induction of L-MDSC death.

 

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