25.07 Histone Deacetylase 8 Inhibitor is Protective in Murine Model of Renal Ischemia Reperfusion Injury

Posted on January 31, 2019

L. N. Krumeich1, S. Concors1, P. Hernandez1, D. D. Aufhauser1, Z. Wang1, G. Ge1, W. Hancock2, M. H. Levine1  1Hospital Of The University Of Pennsylvania,Surgery,Philadelphia, PA, USA 2Children’s Hospital Of Philadelphia,Pathology,Philadelphia, PA, USA

Introduction:  Renal ischemia-reperfusion injury (IRI) is a major source of morbidity after renal transplantation, contributing to decreased graft survival. Histone deacetylases (HDACs) regulate diverse cellular processes. We have previously shown that Class I HDACs, specifically HDAC2 deletion, has a protective effect on renal IRI. Examining HDAC8, a Class I HDAC, is of interest in renal IRI as it has potential clinical translatability. 

Methods:  Whole-body tamoxifen-inducible HDAC -8 knockout (HDAC8 KO) mice, tamoxifen-treated wild type (WT) mice, and mice treated with HDAC8 inhibitor or DMSO control were used in this experiment. All animals were female, B6 strain mice. Mice were subjected to 28 minutes of warm renal IRI through unilateral clamping of the renal pedicle and contralateral nephrectomy under strict temperature control.  Creatinine and BUN were examined at 24, 48, 72, and 96 hours post IRI. 

Results: HDAC8 KO (Figure 1A & 1B) and HDAC8 inhibitor-treated (Figure 1C & 1D) mice developed significantly less renal injury after renal IRI than controls, with significantly decreased post-operative BUN and Cr (p<0.0001 for all). 

Conclusion: HDAC8 knockout and pharmacologic inhibition appear to be protective in a standard model of renal IRI. The benefit of HDAC8 pharmacologic inhibition represents a particularly important finding, as this is the first drug with this degree of observed benefit. Further clinical correlation and mechanistic understanding are needed for this candidate molecule. 

25.06 Immunologic Profiling of Rejection Risk in HIV-Positive Solid Organ Transplant Recipients

Posted on January 31, 2019

S. N. Chu2, S. Wisel1, B. Shaw1, K. Lee1, M. Mintz1, C. Ward1, E. Chuu1, K. Melli1, K. Sugisaki1, P. Stock1, Q. Tang1  1University Of California – San Francisco,Department Of Surgery,San Francisco, CA, USA 2University Of California – San Francisco,School Of Medicine,San Francisco, CA, USA

Introduction:  HIV(+) solid­ organ transplant recipients are predisposed to a three times higher rate of rejection episodes when compared to HIV(­-) recipients, but immunological correlates of rejection in this population have not previously been identified. Here we describe our investigation of immunologic phenotype and gene expression profiling to identify functional differences between Rejectors (Rej) and Non­-Rejectors (NR).

Methods:  Donor and recipient peripheral blood mononuclear cells (PBMCs) were collected prior to transplant. Rej were selected based on biopsy­-proven acute cellular rejection. Kidney transplant recipients were stratified by Rej (n=28) versus NR (n=56), as compared to matched HIV(­-) kidney transplant recipients, HIV(+) non­transplant controls and HIV(­-), ESRD(­-) healthy control subjects (n=25 per group). These patients were profiled using flow cytometric panels to characterize cellular subsets, activation status, and Treg phenotype. Groups were compared for variance using the Kruskal­-Wallis test, with pairwise comparison performed between groups by Dunn’s post­-test. For gene expression analysis, pre­-transplant HIV(+) liver recipient PBMCs from Rej (n=6) and NR (n=6) were co­-cultured in mixed lymphocyte reaction (MLR) in vitro with either CD40L-­stimulated donor or 3rd party B cells. Donor B cells were removed by immunodepletion and recipient cells were analyzed using a custom NanoString panel. Raw counts were normalized and p­-values were adjusted using the Benjamini­-Hochberg procedure.

Results: HIV(+) Rej were found to have markers of increased pre­-transplant immune activation as compared to NR, with a bias toward activation of the innate immune system. They exhibited a significantly altered monocyte phenotype, including decreased HLA­-DR expression on CD14+CD16+ intermediate monocytes. Moreover, Rej have increased B cell activation by HLA­-DR expression and less activated Tregs by decreased percentage of CD39+ Tregs. The frequency of Tregs did not differ between the two groups. After alloantigen stimulation, Rej showed increased gene expression of T­-cell activation markers, CD28 and ICOS. Interestingly, NR displayed upregulation of regulatory ligands in the leukocyte immunoglobulin­like receptor (LILR) family, including LILRB1, LILRA1, LILRB4, as well as a higher proportion of PD1+ CD8+ T cells compared to Rej. Differential gene expression was preserved irrespective of stimulus by either donor or 3rd party.

Conclusion: Overall, our results suggest that increased rates of rejection in HIV(+) kidney and liver transplants correlate with pre­-transplant, recipient­-specific immune dysfunction. Concordance in gene expression profile following stimulation with donor or 3rd party suggests that differential gene expression is an intrinsic, recipient­-driven propensity to immune activation in Rej and immune regulation in NR.

25.05 BLyS Deficient Rats Inhibit Alloantibody Production and B Lymphocyte Proliferation in Rodent Model

Posted on January 31, 2019

N. M. Bath1, B. M. Verhoven1, N. A. Wilson1, R. R. Redfield1  1University Of Wisconsin,Transplant,Madison, WI, USA

Introduction: APRIL (A proliferation inducing ligand) and BLyS (B Lymphocyte Stimulator) are two critical survival factors for B lymphocyte maturation and plasma cells, the main source of alloantibody. We generated rats deficient in APRIL and BLyS to characterize the effects of targeting these cytokines in our established rodent model of antibody mediated rejection (AMR) in kidney transplant.  Here we report our initial phenotyping and response to alloantigen in these novel rodents.

Methods: Using CRISPR/Cas9 we engineered APRIL-/- and BLyS-/- Lewis rats. Collected tissues were analyzed using flow cytometry, ELISPOT, and immunohistochemistry. APRIL-/- and BLyS-/- rats were sensitized with Brown Norway (BN) blood (complete MHC mismatch). Flow cross match and a 3 day mixed lymphocyte reaction (MLR) was performed with wild type (WT), APRIL-/-, and BLyS-/- rats to assess DSA (Donor Specific Antibody) production and cell proliferation, respectively.

Results: When challenged with alloantigen, sensitized BLyS-/- had significant decreases in DSA when compared to WT and APRIL-/-. MLR demonstrated a significant decrease in BLyS-/- cell proliferation when challenged by BN splenocytes compared to APRIL-/- and WT (p<0.02). Additionally, BLyS-/- significantly depleted antibody secreting cell production of IgM and IgG in all tissues compared to WT and APRIL-/- (p<0.04). BLyS-/- demonstrated a significant reduction of splenic marginal zone B lymphocytes detected by anti-PAX5 compared to both WT and APRIL-/- (p<0.0001).

Conclusion: BLyS-/- produced fewer alloantibodies and demonstrated a significant reduction in cell proliferation when challenged with alloantigen. Antibody secreting B lymphocytes and splenic germinal centers are also depleted in BLyS-/-, which translates into a reduction of alloantibody production. Future studies will characterize rodents deficient in both APRIL and BLyS and apply this to kidney transplant model as a method to prevent AMR.

25.04 The Human Antibody and Cellular Response to MHC Compatible Swine Cell

Posted on January 31, 2019

J. M. Ladowski1, G. Martens1, L. Reyes1, M. Tector1, A. J. Tector1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction:  Xenotransplantation is a solution to the growing need for life-saving transplantable organs. Recent advances in genetic engineering allow for rapid manipulation of the swine genome. We hypothesize that genetically engineered cells possessing recipient-matched class I major histocompatibility complex (MHC), on a swine MHC deficient background, would reduce both antibody- and cellular-mediated rejection.

Methods:  Two CRISPR gRNA plasmids were designed to remove the entire coding sequence of the swine class I MHC and co-transfected with a third plasmid containing a promoterless Hygromycin resistance gene surrounded by PhiC31 recombinase sequences, followed with a promoterless human class I MHC cDNA. The response of MHC-matched individuals to the human MHC expressing swine cell was evaluated in a flow cytometry crossmatch (FCXM), 24-hour IFN-y ELISPOT assay, and a mixed lymphocyte reaction (MLR) to measure an immediate, recall, and delayed response.  

Results: A cell line expressing human class I MHC was successfully generated using the described approach. Individuals with no preformed to the class I MHC chosen in this experimental model demonstrated significantly less IgG antibody binding to the human MHC positive swine cell compared to the MHC deficient parent line (one-way ANOVA, p < 0.0001). The ELISPOT revealed significantly more IFN-y release for both MHC-matched and non-MHC-matched individuals in response to the human MHC positive swine cell line (paired two-tailed Wilcoxon test, p = 0.0078 and 0.0156 respectively). A human MHC expressing swine cell elicited less, but not significant, proliferation in the MLR assay compared to the swine MHC expressing cell for MHC-matched but significantly less for non-MHC-matched individuals (Figure 1 unpaired, two-tailed Wilcoxon test p = 0.1250 and 0.0312 respectively).

Conclusion: The development of the CRISPR/Cas9 system allows for complex genetic engineering strategies to be achieved rapidly. This study demonstrates that expression of human MHC on a MHC devoid swine cell can reduce the humoral and cellular response for MHC-matched individuals, but may result in a higher recall response as measured by IFN-y production.

Figure 1:  Results of the proliferation in the CFSE-based MLR of HLA-A2 positive (Figure 1A) and HLA-A2 negative (Figure 1B) PBMC responders to the HLA-A2+ AEC (Lane 1) and SLA class I positive AEC (Lane 2). An unpaired, two-tailed Wilcoxon test for the four HLA-A2 positive responders found no statistical significance between the HLA-A2 positive AEC vs the SLA class I positive AEC (p = 0.1250). For the six HLA-A2 negative responders, statistical difference was found between the HLA-A2 positive AEC vs the SLA class I positive AEC (*, p = 0.0312).

25.03 Uncoupling adaptive immune responses exacerbates hepatic ischemia-reperfusion injury

Posted on January 31, 2019

M. A. Zimmerman1, J. Kim1, A. Martin1, L. Fojut1, J. Yee1, J. C. Hong1  1Medical College Of Wisconsin,Transplant Surgery,Milwaukee, WI, USA

Introduction:  Liver ischemia reperfusion injury (IRI) is a major cause of severe allograft dysfunction following orthotopic liver transplant. Innate immune responses contribute to hepatocellular injury following reperfusion. We hypothesize that the cross talk between neutrophils and natural killer cells (NK) plays an important role in tissue damage in hepatic IRI

Methods:  A murine model of partial liver ischemia was initially employed using a hanging-weight system in 2 groups: C57Bl/6 and SCIDbeige (lack functional B and T lymphocytes, defective NK cell function). In addition, IRI was performed in a third group of SCIDbeige following neutrophil depletion (ND). All animals underwent 45 minutes of ischemia and 4 or 24 hours of reperfusion. Biochemical and histologic injury was assessed. Cytokine profiles were analyzed by reverse-transcription polymerase chain reaction (RT-PCR). 

Results: Immunodeficient animals exhibit similar tissue injury compared to C57Bl6 (Figure 1A and B) at both 4 and 24 hours of reperfusion. ND in the setting of altered NK function exacerbates IRI compared to both C57Bl/6 of non-ND SCIDbeige (p<0.05). Injury in the non-ND SCIDbeige animals is associated with elevated interferon-g (IFNg), interleukin-10 (IL-10), and interleukin-6 (IL-6) expression (Figure 1C) (p<0.05).

Conclusion: Severe combined immunodeficiency does not protect the liver from IRI. Defective NK cell function is associated with elevated IFNg, IL-10, and IL-6 expression. Finally, neutrophil depletion, in the setting of altered NK function, exacerbates tissue injury as early as 4 hours following reperfusion.

 

25.02 Preconditioning By Portal Vein Embolization An Hepato-Protective Strategy For Extended Hepatic Resections

Posted on January 31, 2019

M. I. Orue-Echebarria1, P. Lozano1, J. López-Baena1, E. Vara2, L. Olmedilla3, C. J. Lisbona3, J. Vaquero4, J. García Sabrido1, J. Asencio1  1General University Hospital Gregorio Marañón,Department Of General And Digestive Surgery,Madrid, MADRID, Spain 2Complutense University,Department Biochemistry And Molecular Biology,Madrid, Spain 3General University Hospital Gregorio Marañon,Department Of Anesthesia,Madrid, Spain 4General University Hospital Gregorio Marañon,Laboratory In Hepatology,Madrid, Spain

Background: Portal vein embolization is usually performed weeks before extended hepatic resections to increase the future liver remnant and prevent posthepatectomy liver failure. Portal vein embolization performed closer to the operation also could be protective, but worsening of portal hyper-perfusion is a major concern.

Methods: A subtotal (90%) hepatectomy was performed in swine undergoing or not undergoing (control) a portal vein embolization 24 hours earlier (n = 10/group). Blood tests, hepatic and systemic hemodynamics, hepatic function (plasma disappearance rate of indocyanine green), liver histology, and volumetry (computed tomographic scanning) were assessed before and after the hepatectomy. Hepatocyte proliferating cell nuclear antigen expression and hepatic gene expression also were evaluated.

Result: Swine in the control and portal vein embolization groups maintained stable systemic hemodynamics and developed similar increases of portal blood flow (302 ± 72% vs 486 ± 92%, P = .13). Portal pressure drastically increased in Controls (from 9.4 ± 1.3 mm Hg to 20.9 ± 1.4 mm Hg, P < .001), while being markedly attenuated in the portal vein embolization group (from 11.4 ± 1.5 mm Hg to 16.1 ± 1.3 mm Hg, P = .061). The procedure also improved the preservation of the hepatic artery blood flow, liver function, and periportal edema. These effects occurred in the absence of hepatocyte proliferation or hepatic growth and were associated with the induction of the vasoprotective gene Klf2.

Conclusion: Regenerative preconditioning by portal vein embolization represents a potential hepato-protective strategy for major hepatic resections. 

25.01 Combination Radiotherapy with NF-κB Inhibitor Enhances Antitumor Effect of Gallbladder Cancer

Posted on January 31, 2019

N. Takada1,2, H. Sugano1,2, Y. Shirai1, N. Saito1, R. Hamura1,2, T. Taniai1,2, H. Shiba1, K. Eto1, T. Uwagawa1,3, T. Ohashi2, K. Yanaga1  1The Jikei University School of Medicine,Department Of Surgery,Minato-ku, Tokyo-to, JP, Japan 2The Jikei University School of Medicine,Division Of Gene Therapy, Research Center For Medical Science,Minato-ku, Tokyo-to, JP, Japan 3The Jikei University School of Medicine,Division Of Clinical Oncology And Hematology, Department Of Internal Medicine,Minato-ku, Tokyo-to, JP, Japan

Introduction: For gallbladder cancer (GBC), surgical resection is the only curative treatment, but it is difficult to detect early and the prognosis of advanced GBC remains very poor. Radiotherapy alone or in combination with chemotherapy has been reported as a neoadjuvant therapy for patients with locally advanced unresectable GBC, but its effect is limited. Nuclear factor kappa B (NF-κB) is activated in various cancer cells and involved in tumor growth, invasion and metastasis. In addition, NF-κB is activated by chemotherapy and radiotherapy. This radiation-induced NF-κB activation plays an important role in the regulation of cell apoptosis, inflammation and oncogenesis including invasion and angiogenesis, which result in treatment tolerance. We previously reported that nafamostat mesilate (NM), a synthetic serine protease inhibitor commonly used for pancreatitis and disseminated intravascular coagulation (DIC) in Japan inhibited NF-κB activation and induced antitumor effects for pancreatic cancer. We hypothesized that NM may inhibit radiation-induced NF-κB activation and enhance the antitumor effect of radiotherapy for GBC cells.

Methods: In vitro, we assessed NF-κB activity, cell viability, induction of caspase cascade and quantification of apoptosis of human GBC cells (NOZ) in the following four groups: 1) radiation (5Gy) alone, 2) NM alone, 3) combination (radiation?5Gy?and NM), or 4) vehicle as control. In combination group, the cells were incubated with NM for 3 hours before radiation therapy. In vivo, xenograft GBC mice model were established by injecting subcutaneously with 5×106 NOZ cells into the right flank. At one week after injection, the animals were treated with intraperitoneal (i.p.) injection of NM (30 mg/kg) 5 times a week as NM group, i.p. injection of the equal amount of distilled water 5 times a week and radiation (5Gy) once as radiation group, or i.p. injection of NM (30 mg/kg) 5 times a week and radiation (5Gy) once as combination group for 2 weeks. For control group, the equal amount of distilled water was injected (i.p.) 5 times a week.

Results:In vitro, as compared with radiation group, combination group had lower NF-κB activity (2026.12 ± 84.87 ng/mg protein vs. 1315.12 ± 88.98 ng/mg protein, p<0.01), and reduced cell viability (62.99 ± 4.02% vs. 31.07 ± 6.47%, p<0.01). The levels of apoptotic proteins (Cleaved caspase-3, -8, -9 and cleaved PARP) in combination group were greater than those in other groups. The proportion of apoptotic cells in combination group was significantly higher than that in radiation group (53.80 ± 2.83% vs. 15.14 ± 1.70%, p<0.01). In vivo, tumor growth in combination group was significant slower than that in radiation group (9.50 ± 3.27mm vs. 6.65 ± 1.77mm, p<0.05).

Conclusion:NM suppressed radiation-induced NF-κB activation and enhanced the antitumor effect of gallbladder cancer.

24.10 Dietary Aryl Hydrocarbon Receptor Ligands Shape the Composition and Function of the Microbiota

Posted on January 31, 2019

K. M. Brawner1, W. Van Der Pol2, L. Duck3, V. Yeramilli1, L. Smythies3, C. Morrow4, C. Elson3, C. Martin1  1University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Center For Clinical And Translational Science,Birmingham, Alabama, USA 3University Of Alabama at Birmingham,Medicine,Birmingham, Alabama, USA 4University Of Alabama at Birmingham,Cell, Developmental, And Integrative Biology,Birmingham, Alabama, USA

Introduction: Proper immune responses to gut microbes are mediated through host-cell receptor signaling. The aryl hydrocarbon receptor (Ahr) is engaged by microbial-derived tryptophan (Trp) derivatives to contribute to the development of a functional intestinal barrier.  We have shown that removal of dietary sources of Ahr ligands results in decreased fecal immunoglobulin (Ig) A levels. The effect of dietary Ahr ligand manipulation on intestinal microbiome composition and function is largely unknown. 

Methods: We randomly assigned mouse littermates to a conventional diet or an Ahr ligand-free diet.  DNA from fecal samples was isolated for 16S rRNA gene sequencing or fecal samples were cultured anaerobically. Faecalibaculum rodentium was grown anaerobically in broth with or without carbohydrates and supplemented or not with Trp.  Ahr ligands in F. rodentium –conditioned supernatants were measured by luciferase assay.   An in vitro model of IgA transcytosis was used to determine if fecal microbiota from Ahr ligand-free diet mice and F. rodentium-conditioned supernatants are capable of degrading IgA.  For gene expression analysis, RNA was harvested from primary epithelial cells used in IgA transcytosis assays.

Results: We observed a global alteration of fecal microbiota upon dietary Ahr ligand deprivation.  Mice on the Ahr ligand-free diet harbored a microbiota that was less diverse compared to mice on the conventional diet. The most common DNA sequence from the combined dataset of both groups of mice belonged to the genus Allobaculum. This genus was enriched in the feces of mice on the Ahr ligand-free diet.  The Allobaculum sequences in our dataset were most closely related to F. rodentium. Compared with non-conditioned broth, F. rodentium-conditioned supernatant contained lower amounts of Ahr ligands.  Cultured fecal bacteria from mice on the Ahr ligand-free diet, but not the conventional diet, were able to degrade IgA.  Finally, F. rodentium-conditioned supernatant was also capable of IgA degradation without affecting IgA transcytosis.

Conclusion: Here we show that removal of dietary Ahr ligands alters the composition and function of intestinal microbiota.  The ability of F. rodentium-conditioned supernatants to degrade Ahr ligands suggests Allobaculum thrives in an environment in which Ahr is inactive.  Our IgA transcytosis experiments indicate a cause and effect relationship between altered microbiota and reduced fecal IgA on the Ahr ligand-free diet.  We also show that F. rodentium is capable of IgA degradation, recapitulating our results from cultures of whole fecal samples.  To our knowledge, this is the first time a specific commensal species has been implicated in Ahr ligand and IgA degradation.        

 

24.09 Intra-tumor Heterogeneity is Associated with Immune Response and Improved Survival in Colon Cancer

Posted on January 31, 2019

A. L. Butash1, T. Kawaguchi2, L. Yan3, Q. Qi3, X. Peng3, M. Asaoka1, G. Mann1, E. Otsuji2, K. Takabe1  1Roswell Park Cancer Institute,Department Of Surgery,Buffalo, NY, USA 2Kyoto Prefectural University of Medicine,Department of Surgery,Kyoto, KYOTO, Japan 3Roswell Park Cancer Institute,Department Of Bioinformatics And Biostatistics,Buffalo, NY, USA

Introduction:
Tumor heterogeneity arises from differences among cancer cells that are inherited during cell division.  It implies the coexistence of sub-populations of cancer cells that differ in their genetic, phenotypic, or behavioral characteristics. Several studies have documented a role for intra-tumor heterogeneity (ITH) in driving tumor progression and treatment resistance in colon cancer. However, ITH, especially related to intra-tumor immune microenvironment, is not well examined in colon cancers.  In this study, we aim to examine the association of ITH and immune gene signatures and its clinical relevance in colon cancers.

Methods:
Integrated genomic and clinical data was collected from colon cancer patients in The Cancer Genome Atlas (TCGA). ITH was estimated by calculating Mutant Allele Tumor Heterogeneity (MATH) using computational analyses. Intra-tumor immune signatures and tumor infiltrating immune cell composition were calculated using integrated transcriptomics and CIBERSORT or TIMER, respectively. Clinical relevance was assessed by Kaplan-Meier survival curve. 

Results:
Colorectal tumors with high ITH were found to have fewer activated CD4 T-cells (p<0.0074) and CD8 T-cells (p<0.0084) when compared to tumors with low ITH. A similar trend was seen with decreased expression of immune checkpoint molecules; such as PD-1, PDL-1, CTLA4, and LAG3 (p<0.01, p<0.0067, p<0.023, p<0.005 respectively) in tumors with high ITH, reflecting the overall decreased presence of immune cells. Expression of cytolytic molecules; PRF1 (p<0.0046) and GZMA (p<0.00059), as well as the immune cytolytic activity (p<0.0023) were also lower in tumors with high ITH. We also retrieved a genomic classifier created of >200,000 microsatellite loci to identify microsatellite instability (MSI), and found that MSI-high tumors showed lower ITH, suggesting that genomic instability could be associated with intra-tumor heterogeneity. MSI-high tumors were associated with low ITH (p<0.0001). Gene Set Enrichment Analysis (GSEA) demonstrated that enrichment of immune-response gene sets was observed in the lower ITH tumors. Low MATH was associated with improved overall survival (OS) while high MATH demonstrated worse survival (p<0.029), as well as disease-free survival (p<0.0093). In right sided colorectal tumors, low ITH was associated with improved survival (p<0.0023), but this trend was not observed with left sided colorectal tumors in this patient population.

Conclusion:
We found that low ITH was associated with enhanced intra-tumor immunogenicity or immune response and is prognostic of improved survival in colon cancer. Our study is in agreement with the notion that ITH is determined by tumor immune microenvironment including anti-tumor immunity, which is expected to have future implications for clinical application. 
 

24.08 Effects of Plant Based Enteral Nutrition Formula on Colonic Gene Expression in a Murine Colitis Model

Posted on January 31, 2019

R. G. Ramos1, M. Rogers1,2, A. Yeh1,2, J. Tian1,2, B. Firek1,2, E. Novak1,2, K. Mollen1,2, M. Morowitz1,2  1University Of Pittsburg,Surgery,Pittsburgh, PA, USA 2Children’s Hospital Of Pittsburgh Of UPMC,Pediatric Surgery,Pittsburgh, PA, USA

Introduction:

The beneficial effects of a high fiber diet on epithelial integrity, immune reactivity, and bacterial diversity have been well documented in the medical literature. Previously we have shown that when compared to standard mouse chow or conventional enteral nutrition (CEN), plant based enteral nutrition (PBEN) formulas significantly decrease disease activity in murine colitis models. To define the mechanisms underlying this beneficial effect, we utilize RNA Seq to compare colonic gene expression in mice with colitis on different nutritional regimens.

Methods:

Four groups of four C57BL6 male mice each were fed regular mouse chow, PBEN (Liquid Hope), CEN1 (PediaSure), or CEN2 (Vital) for 7 days. On day 8, 2% dextran sodium sulfate (DSS) was started. On day 12, mice were sacrificed and colon was preserved for RNA-seq analysis. Colonic gene expression was characterized with RNA-seq and reference matched with KEGG. R software with vegan package was used for analysis. False discovery rate (FDR) was used to adjust for multiple hypothesis testing, logarithmic fold change (LFC) was used to measure effect size, statistical significance was defined as a p value < .05.

Results:

As we have seen previously, weight loss and disease activity scores were significantly lower in PBEN animals (see figure). RNA Seq of whole colon tissue from each dietary group demonstrated a statistically significant change in transcription of the following pathways in the PBEN colitis group: interleukin 17 (IL-17) and tumor necrosis factor α  (TNF-α ) signaling, vitamin A metabolism, nucleotide binding oligomerization domain domain-like receptors (NOD), and inflammatory bowel disease (IBD) associated pathways. A twofold change in expression levels was identified in the following pathways with LFC, upregulated genes include: Gasdermin-C4 (Gsdmc4), antigen peptide transporter 1 (Tap1), aldehyde dehydrogenase family 1 subfamily A2 (Ald1a2), UDP-Glucuronosyltransferase-2B7 (UGT2B7), members of major histocompatibility complex II (MHC), signal transducer and activator of transcription 1 (STAT1), capase 1 (Casp1), caspase 4 (Casp4), and mitofusin 2 (Mfn2). Downregulated genes include: TNF ligand superfamily member 11 and 14 (RANKL and LIGHT), IL-17 receptor A (IL17RA), interleukin 1 beta (IL-1β ) and interleukin 6 (IL-6).

Conclusion:

The downregulated TNF-α  signaling (RANKL and LIGHT) and IL-17 signaling (IL17RA, IL-1β  and IL-6) pathways, along with the upregulated vitamin A metabolism (Ald1a2 and UGT2B7), IBD (MHC II and STAT1) and NOD (Casp1, Casp4, and Mfn2) pathways offer a biologically plausible explanation of the molecular mechanisms underlying PBEN’s beneficial effects on epithelial integrity and inflammation.

24.07 TAK228 Enhances Antitumor Activity of Eribulin in Triple Negative Breast Cancer

Posted on January 31, 2019

N. Owusu-Brackett1, K. W. Evans1, A. Akcakanat1, E. Yuca1, E. Ileana Dumbrava1, F. Janku1, F. Meric-Bernstam1  1University Of Texas MD Anderson Cancer Center,Houston, TX, USA

Introduction: Phosphatase and tensin homologue (PTEN) negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. Triple negative breast cancers (TNBC) are often PTEN-deficient, making mTOR a compelling target. We evaluated the efficacy of catalytic mTOR inhibitor TAK228 alone and in combination with eribulin in triple negative breast cancers (TNBC) with and without PTEN loss.

Methods: We tested TAK228 in combination with eribulin in a panel of TNBC cell lines with cell proliferation assays. Western blot analysis was performed to assess PTEN status and PI3K pathway inhibition. In vivo, antitumor efficacy of TAK228 as a single agent as well as in combination with eribulin was evaluated.

Results: Five of eight triple negative breast cell lines were sensitive to TAK228, independent of PIK3CA/PTEN status. Western blotting demonstrated inhibition of mTORC1/2 signaling as demonstrated by decreased phosho-AKT, phospho-S6 and phospho-4EBP1. In vitro, TAK228 was synergistic with eribulin in all eight TNBC cell lines. The combination of TAK228 and eribulin did not enhance apoptosis but increased G2/M growth arrest (p<0.001). In vivo, TAK228 led to modest growth inhibition in TNBC derived xenografts with no tumor regression observed. In two TNBC PDXs with PTEN loss, one with intrinsic eribulin sensitivity, another eribulin resistance, TAK228 in combination with eribulin did not enhance in vivo efficacy. In a third PTEN-negative TNBC model, eribulin alone achieved disease stabilization, but the combination of TAK228 and eribulin led to significantly smaller tumor volumes compared to eribulin alone (p<0.001).

Conclusion: TAK228 enhances the antitumor efficacy of eribulin in TNBC models in vitro, and enhanced in vivo activity in selected models. Further study is needed to determine the potential of this combination, and optimal patient selection strategies.

 

24.06 An innovative approach to protecting immune function in breast cancer through Hsp90 inhibition

Posted on January 31, 2019

T. Wang1, C. Subramanian1, M. Cohen1  1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction: Breast cancer is the second leading cause of cancer-related death in women in the US. To date, use of immunotherapy has been difficult due to decreased mutational burden in breast cancer compared to other cancers such as melanoma. Hsp90 has been an exciting target for the development of anti-tumor agents, as multiple cancer signaling pathways can be disrupted simultaneously through Hsp90 inhibition; however, current Hsp90 inhibitors have significant immunosuppressive effects by decreasing dendritic cell maturation, antigen uptake, and tumor recognition by T cells. Recently our group has developed novel Hsp90 inhibitors that target only the carboxy-teminus (KU758) or are selective to the beta-isoform of Hsp90 (KUNB31). We hypothesize that these novel approaches to Hsp90 inhibition will have decreased immunosuppressive effects (due to their improved selectivity for client proteins) compared to traditional N-terminal inhibitors (17-AAG) and would be more ideal drugs for combination with immunotherapy.

Methods: Validated MCF7 (ER+) and MDA-MB-231 (triple neg) breast cancer cells were treated with 17-AAG, KU758, and KUNB31 for 30 hr. Dendritic cells (DC) were isolated from murine bone marrow and plated on a 96-well plate. The supernatant from the treated cells was then transferred to DCs for 24 hours. IL-6, TNA-α, and TGF-β secretion was measured by ELISA assay. SPSS software was used for statistical analysis and all experiments were repeated in triplicate.

Results: IL-6 secretion from DCs was decreased by exposure to 17-AAG treated MCF7 cells compared to untreated cells by 45.9% (p=0.05). Similar results were observed in exposure to 17-AAG treated MDA-MB-231 cells (52.6% decrease, p=0.001). In contrast, IL-6 levels were not significantly changed with exposure of DCs to both cell types treated with KU758 or KUNB31, except slightly at high concentrations >10uM of KUNB31 (decreased by 15.2%, p=0.13). In DCs exposed to Hsp90 inhibitor-treated MCF7 cells, there was decreased TNF-α expression with all drugs, but the greatest effect was noted with 17-AAG (decreased by 39.6%, p=0.07). TNF-α expression was significantly decreased by 81.9% (p=0.01) from DCs exposed to 17-AAG treated MDA-MB-231 cells. In contrast, no significant decrease in either TNF-α expression or TGF-β secretion was seen with KU758 and KUNB31 treatment. See Figure 1.

Conclusion: Compared to N-terminal inhibitors, our novel approach to Hsp90 inhibition may be better targets for combination with immunotherapy agents due to their lack of immunosuppressive effects. Additional studies in translational models are warranted to further validate this significant opportunity and support future clinical applications.

24.05 Myofibroblast-Derived Angiogenin Regulates Tumor Growth in a Mouse Model of Colorectal Cancer

Posted on January 31, 2019

R. Plummer1, T. Liu1, N. Ciomek2, J. Yoo1  1Tufts Medical Center,Surgery,Boston, MA, USA 2Tufts Medical Center,Pathology,Boston, MA, USA

Introduction:   The myofibroblast (MFB) is an inportant stromal cell of the GI tract that has been implicated in the pathophysiology of colorectal cancer (CRC).  Angiogenin (ANG), a secreted 14-kDa member of the ribonuclease superfamily, enhances cell growth and survival.  We recently reported that ANG regulates cell signaling events within the MFB, but the impact of MFB-derived ANG on CRC has not been previously studied.  Our goal was to evaluate the role of MFB-derived ANG on CRC growth in a novel mouse model involving orthotopic implantation of primary CRC and MFB cells via murine colonoscopy.  

Methods:   After isoflurane anesthesia, a colonoscopy was performed with endoscopic microinjection of a cell suspension that contained genetically defined (ApcΔ /Δ ;Kras G12D/+;Trp53Δ /Δ ) primary mouse CRC cells (1×104-4×104) into 8-10 week-old wild-type (WT) and ANG-knockout (ANG-KO) C57BL/6 mice.  In a second set of experiments, CRC cells (4×104) were injected with primary syngeneic WT or ANG-KO MFB (2×105) into WT and ANG-KO C57BL/6 mice.  A follow-up colonoscopy was performed weekly for 4 weeks with photo and video documentation.  Tumor size was graded based on tumor diameter relative to colon circumference (grade 1=just detectable; grade2=1/8; grade3=1/4; grade 4=1/2).  Endoscopic data was correlated with anatomic/histologic findings.  

Results:  A total of 37 mice were injected with a survival rate of 94%.  Injected CRC cells successfully implanted in colon submucosa of WT immune-competent mice and grew in a dose-dependent fashion (Week 1: 5×103 – grade 0, 1×104 – grade 1, 4×104 – grade 1.4, n=17 total) that persisted over 4 weeks.  However, injected CRC cells at all concentrations failed to grow in ANG-KO mice over 4 weeks (grade 0, n=8).  Compared to injection of CRC cells alone, co-injection of CRC with WT MFB in WT mice led to enhanced tumor growth (Week 1: grade 3.3, n=4).  Tumor growth was unchanged following co-injection of CRC with WT MFB in ANG-KO mice (Week 1: grade 3.1, n=4).  However, there was significantly diminished tumor growth following co-injection of CRC with ANG-KO MFB in ANG-KO mice (Week 1: grade 1.5, n=4).

Conclusion:  Orthotopic implantation of CRC leads to dose-dependent tumor growth in WT mice but no growth in ANG-KO mice, suggesting that ANG is required for tumor growth.  WT MFB enhances tumor growth when orthotopically co-injected with primary CRC in WT mice.  This effect can be sustained when host ANG is absent (ANG-KO mice) but MFB-derived ANG (WT MFB) is still present.  However, the loss of both host ANG (ANG-KO mice) and MFB-derived ANG (ANG-KO MFB) resulted in markedly reduced tumor formation and growth. These results suggest that MFB enhances CRC growth through a mechanism that involves MFB-derived ANG.

24.04 The First HSP90β Inhibitor Differentially Regulate Wild & Mutant p53 Expressing Breast Cancer Cells

Posted on January 31, 2019

C. Subramanian1, N. Zhang1, B. B. Blagg2, M. S. Cohen1  1University of Michigan,General Surgery,Ann Arbor, MI, USA 2University of Notre Dame,Department Of Chemistry And Biochemistry,Notre Dame, INDIANA, USA

Introduction: Heat shock protein 90 (Hsp90) is a molecular chaperone that is critical for several cellular processes including the folding, maturation and degradation of client proteins involved in the development of cancers (including the breast cancer) and their metastatic spread. Current Hsp90 inhibitors in clinical trials non-selectively target all four isoforms of Hsp90 and have shown detrimental toxicities. Through an innovative structure guided design, our collaborative group has recently developed the first Hsp90β isoform specific inhibitors. During the initial screens with our lead β  specific Hsp90 inhibitor, KUNB105, we noted that cancer cells with wild type (wt) p53 were more sensitive to the drug than p53 mutant (mt) cells. Therefore, we have hypothesized that it is the differential effect in p53 pathway proteins to Hsp90β  selective inhibition that leads to this selective effect on p53 wt breast cancer (BC) cells compared to mt p53 cell lines.  

Methods: Validated BC cell lines MCF7 (p53 wt) and MDA-MB-231 (p53 mt) were treated with KUNB105 and viability of cells were measured using cell TiterGlo. Changes in expression levels of Hsp90 clients were analyzed by western. Apoptosis was measured using RealTime-Glo Annexin V Apoptosis and Necrosis Assay. Changes in cancer pathway genes was analyzed after treatment of cells with KUNB105 using Qiagen Cancer Pathway Profiler assay. Prism software was used for statistical analysis and all experiments were repeated in triplicate.

Results: Viability of MDA-MB-231 and MCF7 cells after treatment with KUNB105 showed an IC50 value of 20.5 & 8.2 μ M respectively. Analysis of p53-pathway by Western showed dose dependent increase in p53 (3-5 fold) and mdm2 (2-3 fold) starting from 10  μ M KUNB105 treatment for MCF7 cells whereas the levels of p53 did not show observable change even at the highest concentration of 30 μ M KUNB105 (p<0.001) for MDA-MB-231. By contrast, the cell cycle dependent protein p21 showed dose dependent upregulation starting at 1μ M KUNB105 (2- fold, p<0.01) in both cell lines indicating that KUNB105 treatment leads to cell cycle arrest. Evaluation of the effect of KUNB105 treatment on apoptosis showed increase in luminescence starting from 2 h (30000-33000 compared to around 12000 for the control, p<0.001) and peaked at 10 h (50,000-54,000, p<0.001) for both the cells indicating significant induction of apoptosis. Evaluation of cancer pathway after KUNB105 treatment of BC cells showed differential expression of genes in cell cycle (AURKA, CCND2, E2F4, MK167, WEE1, STM1), DNA damage and repair (ERCC5 and GADD45G) as well as apoptosis (BCL2L11).

Conclusions: Our results indicate that KUNB105 has selectivity to p53 wt BC and induces apoptosis through p53 pathway-specific mechanistic differences between wt and mt BC cell lines. Further studies related to this unique targeting effect are needed for translational applications of this promising and novel Hsp90β  inhibitor.

24.03 Orthotopic implantation develop better triple negative breast cancer Patient-Derived Xenograft

Posted on January 31, 2019

M. Okano1, M. Oshi1, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA

Introduction: Patient-Derived Xenograft (PDX) has come into the limelight of breast cancer research to be used for pre-clinical studies. Some of its weaknesses are its poor engraftment rates and slow growths, which often limits its use as an avatar of the donor patient. Therefore, improvement of the models especially in engraftment and tumor growth are in urgent need. We hypothesized that orthotopically implanted tumors (Ortho) engraft better, grow faster and larger compared from subcutaneously implanted PDX (SQ), which is the standard model. 

Methods: NSG mice were used to generate PDX. 2 tumors were derived from brain metastasis (B-met), and the others were from primary breast cancers. 3 tumors were ER(+)HER2(-) and 7 tumors were triple negative (TN). Both of B-met tumors are ER(+)HER2(-). Tumor “engraftment” was defined as tumorigenesis of palpable tumor after implantation regardless of time it took.

Results:The overall engraftment rate was significantly better in Ortho than SQ (77.8% (n=137/176) vs. 50.7% (n=79/156), p<0.01). Ortho tumors grew remarkably larger than SQ tumors. The mean tumor weight was significantly heavier in Ortho than SQ (0.75g vs 0.14g, p<0.01). Ortho tumors demonstrated more abundant mitotic figures compared with SQ tumors (19.2 vs 7.9, p<0.01). Ortho tumors had more Ki-67 positive cells than SQ tumors (31.5 vs 21.8, p=0.015). The tumor weight was significantly larger when implantation was made to the 2nd or to the 4th mammary fat pad (0.73g vs 0.96g, p=0.02). Tumor engraftment of 1st generation was low (24.8% (n=32/129)), but the rate of 2nd (82.2% (n=46/56)) and 3rd (80.6% (n=58/72)) generation was significantly increased (p<0.001). The time it took for the 1st generation to grow was the longest between 3 generations (1st; 152days, 2nd; 66days, 3rd; 63days, p<0.01). The mean tumor weight was significantly higher in Ortho sites among all generations (1st -3rd) of TN cancer (0.2g, 1.1g vs 0.8g, respectively, p<0.01). ER positive cancer xenograft revealed significantly lower engraftment rate (26.7% (n=12/45) vs 65.1% (n=216/332), p<0.01), slower tumor growth, and lighter tumor weight (0.18g vs 0.47g, p<0.001) than TN xenograft. The xenograft from brain-metastasized breast cancer also showed higher engraftment rate in MP than SQ (94.4% (n=68/72) vs. 69.4% (n=50/72), p<0.01) although was not the organ that brain metastasis originally grew. The brain metastasis tumors also demonstrated higher tumor weight in MP than SQ (0.28g vs 0.54g, p<0.001). The brain metastasis tumors grew faster than primary tumor (52days vs 95days, p<0.01). 

Conclusion:Orthotopical implantation showed better take rate, greater tumor size and weight than heterotopic implantation, regardless of the cancer subtypes and their sources.

 

24.02 Plasminogen, collagenolytic bacteria and anastomotic leak: prevention via tranexamic acid enema

Posted on January 31, 2019

R. A. Jacobson1,2, A. Williamson2, S. Gaines2, K. Wienholts3, B. D. Shogan2, O. Zaborina2, J. C. Alverdy2  1Rush University Medical Center,Surgery,Chicago, IL, USA 2University Of Chicago,Surgery,Chicago, IL, USA 3Radboud University,Nijmegen, Netherlands

Introduction: The protease plasminogen (PLG) and its activator urokinase play a critical role in regulating collagen remodeling during gastrointestinal healing. We have previously shown that colonization of anastomotic tissue by collagenolytic Enterococcus faecalis can cause anastomotic leak (AL). We have recently discovered that E. faecalis can cause overactivation of PLG. The aims of this study were to: 1) determine the mechanism of PLG activation in bacterial-mediated AL pathogenesis, and 2) analyze the ability of tranexamic acid (TXA), a suppressor of PLG activation, to prevent AL caused by bacterial pathogens.

Methods: Activation of PLG and pro-urokinase (puPA) by collagenoltyic E. faecalis V583 and mutant strains lacking the collagenase genes gelE and sprE was measured using fluorogenic assays.  In an established model of colorectal AL caused by collagenolytic bacteria, mice received either TXA or vehicle control on postoperative days 1, 2 and 3 via enema. Anastomoses were evaluated on postoperative day 8 using anastomotic healing score (AHS – 0: perfectly healed; 1 – flimsy adhesions; 2 – dense adhesions; 3 – abscess; 4 gross disruption).  Fluorescence microscopy was used to demonstrate the temporospatial dynamics of PLG deposition and the impact of TXA on this process.

Results: Wild type E. faecalis activated puPA to a greater extent than mutants deficient in GelE, SprE, or both; activation was partially rescued when mutant strains were complemented with their deficient genes (Fig 1A). In the presence of puPA, the parent strain activated more PLG than mutants (Fig 1B). The presence of TXA inhibited the ability of E. faecalis to bind (flow cytometric data not shown) and activate PLG (497.8±20.1 RFU/s 0 TXA vs 272.1±7.3 10mM TXA, p<.05). When applied to our mouse model of E. faecalis-induced AL, TXA reduced the incidence of leak compared to vehicle (AHS 2.7±0.7 vehicle vs 1.4±0.5 TXA). Rescue of AL with TXA was redemonstrated in our model of P. aeruginosa-induced leak (AHS 1.6±0.5 vs 3.8±0.4, p<.05). Fluorescence microscopy demonstrated qualitatively less PLG at the anastomotic site when mice received TXA compared to vehicle control (Fig 1C).

Conclusion: To our knowledge this is the first mechanistic description of PLG activation by E. faecalis and its involvement in AL. Our data demonstrate microbial PLG activation is a novel, plausible and generalizable mechanism for pathogen-mediated AL. TXA limits the ability of pathogens to activate PLG and disrupt the anastomosis. TXA is inexpensive and FDA approved for use in elective surgery; its local delivery to the anastomotic site may be a promising approach to prevent AL in high risk anastomoses.

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