23.07 Immunologic Changes in Regionally Treated Melanoma

Posted on January 18, 2018

J. A. Perone1, T. Tamesa1,2, M. Tsutsui2, R. E. Alvarado1, S. Moore1, P. Dolber1, I. Pinchuk1, K. Olino1, D. Tyler1  1University Of Texas Medical Branch,Galveston, TX, USA 2Durham Veterans Affairs Medical Center,Durham, NC, USA

Introduction:
Advanced melanoma remains a highly lethal disease with poor long-term survival in the majority of patients. Although systemic immunotherapy has improved outcomes, there is much room for improvement.  One strategy to help augment systemic immunotherapy has been regional therapeutics.  Regional therapies offer variable immunogenic effects on the microenvironment, usually with minimal toxicity, that could synergize with systemic treatments to improve antitumor responses. To better define the spectrum of immune effects produced by regional therapy, we picked four different chemotherapeutic agents to explore the range of immune mediated events that occur in the context of variable mechanisms of cell death.

Methods:
B16 F10.9-OVA melanoma cells were injected intradermally in the left leg of C57BL/6 female mice to create a solitary leg tumor.  At tumor volumes of ~50mm3 animals were randomized and isolated limb infusion (ILI) was performed using the maximally tolerated dose of saline, melphalan, doxorubicin, temozolomide, or oxaliplatin.  Following survival studies, immunologic evaluation was performed by harvesting Inguinal (ILN) and popliteal lymph nodes (PLN) on days 3 and 10 post ILI and immunologic evaluation was performed utilizing RT-PCR and flow cytometry, staining for CD4, CD8, FoxP3, Tbet, PD-1, and PD-L1. 

Results:
While regional chemotherapy was exceptionally effective at killing tumor, we were surprised to see the induction of immunosuppressive profiles in the microenvironment at days 3 and 10.  Increased expression of PD-L1 on CD4+ and CD8+ Tcells was observed not only in inguinal nodes but also in popliteal nodes and tumors on day 10 following treatment with either melphalan or doxorubicin (Figure 1).  Immunosuppression associated with temozolomide and oxaliplatin was primarily manifested as an increase in tregs(CD4+FoxP3+) in both the ILN and PLN between days 3 and 10, as well as an increase in exhausted Tcells (CD8+PD1+) and suppressor Tcells (CD8+FoxP3+) (Figure 2).  PCR analysis of tumors treated with doxorubicin also showed similar increase in tregs compared to naïve animals.

Conclusion:
Regional chemotherapy used in ILI creates an immunosuppressive environment around treated tumors that varies depending on the chemotherapeutic agent utilized.  Characterization of the immunosuppressive profile induced by regional treatment allows development of novel strategies, such as combining systemic anti-PD1 treatment with melphalan-based ILI, to augment both regional and systemic anti tumor response. 
 

23.04 CD40 signaling is required for expansion of terminally-differentiated CX3CR1+ CD8+ T cells

Posted on January 18, 2018

F. Ito1,2,3, T. Hoki2, C. A. Eppolito2, A. J. Francois2, K. Odunsi2,4,5, T. Yamauchi2  3State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA 4Roswell Park Cancer Institute,Gynecologic Oncology,Buffalo, NY, USA 5Roswell Park Cancer Institute,Immunology,Buffalo, NY, USA

Introduction: Successful immunotherapeutic treatment of chronic infectious diseases and cancer requires the generation of a strong cellular immune response. Combined CD40 antibody and toll-like receptor (TLR) (CD40/TLR) stimulation has been found to mediate potent cellular immunity in the context of tumor immunology and cancer immunotherapy. However, the mechanisms of enhanced antitumor efficacy by CD40/TLR stimulation remain elusive.

Methods: To investigate phenotype and function of CD40/TLR vaccine-stimulated antigen-specific T cells, we used MC38 colon adenocarcinoma and B16 melanoma models. To evaluate endogenous T cell response, MC38 tumor-bearing C57BL/6 mice were treated with mutated-neoantigen peptide, agonistic CD40 antibody, and poly I:C (TLR3). To assess phenotype and function of adoptively-transferred T cells, we used pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen expressed on B16 melanoma. In vitro–activated Pmel-1 CD8+ T cells were adoptively transferred into C57BL/6 mice bearing subcutaneous B16 melanomas. Systemic administration of IL-2 and vaccination with the gp100, anti-CD40 antibody, and Imiquimod (TLR7) were used to enhance antitumor immunity of transferred T cells. Tissues including blood, spleen, and tumors were harvested for further analysis. In both tumor models, control mice were treated with no vaccination, CD40 antibody alone or TLR agonist alone.

Results: In both tumor models, mice treated with combined CD40/TLR vaccination had significantly decreased tumor growth and improved survival compared to no vaccination, CD40 antibody alone or TLR agonist alone. Interestingly, vaccination with the cognate antigen and CD40/TLR not only expanded antigen-specific CD8 T cells in both tumor models, but also facilitated them to express the chemokine receptor, CX3CR1. CX3CR1+ CD8+ T cells were found to express higher levels of killer-cell lectin like receptor G1 (KLRG1), TNF-related apoptosis-inducing ligand (TRAIL), perforin, and granzyme, suggesting terminally-differentiated subset. The generation of CX3CR1+ CD8+ T cells was greatly facilitated by CD40 antibody while TLR agonist increases the expansion of total number of antigen-specific CD8 T cells. Importantly, total number and frequency of CX3CR1+ CD8+ T cells were significantly decreased in tumor-bearing CD40 knockout (KO) mice, indicating that host expression of CD40 is required for generation and expansion of CX3CR1+ CD8+ T cells.

Conclusion: Effective vaccination with the cognate antigen and CD40/TLR accompanies generation of tumor-specific terminally-differentiated CX3CR1+ CD8+ T cells dependent on CD40 signaling.

23.05 CRISPR/Cas9 Genome Editing of Tumor-specific CD8+ T cell-derived Induced Pluripotent Stem Cells

Posted on January 18, 2018

T. Yamauchi1, H. Saito2,3, T. Hoki1, F. Ito1,2,4,5  2University Of Michigan,Surgery,Ann Arbor, MI, USA 3Kanazawa Medical University,Biochemistry,Kanazawa, ISHIKAWA, Japan 4State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 5Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 1Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA

Introduction: Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics.  This limitation can be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. We and others have shown that iPSC-derived regenerated T cells have potent antitumor efficacy in vitro and in vivo. Recently, the type II bacterial CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system was developed as an efficient and versatile technology for genome editing in eukaryotic cells and whole organisms. The potential of iPSCs can be further enhanced by genome engineering and then used to study individual gene function, track cells or endogenous proteins with a knock-in reporter, and correct genetic defects for gene therapy. 

Methods: We reprogrammed T-cell receptor (TCR) transgenic CD8+ T cells into pluripotency, and established a syngeneic mouse model for evaluating in vitro and in vivo antitumor reactivity of regenerated T cells from iPSCs bearing a rearranged TCR of known antigen specificity. Plasmids encoding rtTA and tet-O-Cas9-2A-mCherry were obtained and co-transduced using the lentiviral system. The Cas9 expression was analyzed after the doxycycline treatment, and clonally-derived Cas9-iPSCs were obtained.

Results: Pluripotency of TCR transgenic T cell-derived iPSCs (TiPSCs) were confirmed with immunostaining of embryonic stem cell (ESC) markers, RT-PCR (reverse transcription-polymerase chain reaction) analysis of pluripotency-associated transcription factors, and whole genome expression profiling by microarray analysis that demonstrated a high degree of similarity in their gene expression patterns and close correlation values with ESCs, but distinct from parental CD8+ T cells. Cytogenetic analysis revealed derived TiPSCs maintained normal karyotype. The TiPSCs differentiated to embryoid bodies in vitro, and upregulation of marker genes for all three germ layers was detected by immunostaining. Their differentiation capacity was further confirmed by teratoma formation in immune-deficient mice in vivo. Moreover, we confirmed that the TiPSCs retained the same rearranged configuration of TCR chain genes as the original TCR transgenic T cells. TiPSCs were, then, subjected to the lentivirus-mediated transduction of tetracycline-inducible Cas9 vectors. The transduction efficacy was confirmed by the mCherry fluorescence and the RT-PCR against the Cas9 sequence.

Conclusion: Our results indicate successful reprogramming of antigen-specific T cells and lentivirus-mediated transduction of tetracycline-inducible Cas9 vectors into TiPSCs, which will allow us to generate an unlimited number of phenotypically defined, functional and expandable genome-edited autologous antigen-specific T cells.

23.02 Defining CD8+ T-cell Subsets that are Rescued by PD-1/PD-L1 Blockade in the Tumor Microenvironment

Posted on January 18, 2018

T. Yamauchi1, T. Hoki1, C. A. Eppolito1, A. Francois1, K. Odunsi1,2,3, F. Ito1,4,5  1Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA 2Roswell Park Cancer Institute,Gynecologic Oncology,Buffalo, NY, USA 3Roswell Park Cancer Institute,Immunology,Buffalo, NY, USA 4State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 5Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA

Introduction: Cancer immunotherapies that target the T-cell immune checkpoints, such as programmed cell death-1 (PD-1) and its ligand (PD-L1) have shown unprecedented success for the treatment of a variety of malignancies including melanoma. Although a significant number of cancer patients benefit from immune checkpoint inhibitors (CPIs), many fail to have clinical responses.   A better understanding of the mechanisms that regulate CD8+ T-cell responses in the tumor microenvironment is required to improve immunotherapies that restore function in exhausted CD8+ T cells.  Although heterogeneity of effector CD8+ T cells in the tumor microenvironment (TME) has been recognized, their functions and roles are ill-defined.

Methods: We have evaluated phenotypical and functional heterogeneity of tumor-infiltrating lymphocytes (TILs) after adoptive transfer of ex vivo primed pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen expressed on B16 melanoma. In vitro–activated Pmel-1 CD8+ T cells were adoptively transferred into C57BL/6 mice bearing subcutaneous B16 melanomas that had been established for 11-14 days. Systemic administration of IL-2 and vaccination with anti-CD40 antibody and toll-like receptor (TLR) agonist were used to enhance antitumor immunity of transferred T cells. Tumor and spleen were harvested for functional analysis of adoptively transferred T cells.

Results: We found that the chemokine receptor, CX3CR1 identified three distinct effector CD8+ T-cell subsets, CX3CR1 negative (-), intermediate (int), and high (hi) in blood, spleen and the TME.  A CX3CR1hi subset contained terminally-differentiated CD8+ T cells that expressed higher levels of killer-cell lectin like receptor G1 (KLRG1), TNF-related apoptosis-inducing ligand (TRAIL), perforin, and granzyme. Significantly more CX3CR1int CD8+ T cells expressed CD25 compared to other subsets, suggesting this is the subset that is rapidly proliferate and preferentially generate terminally-differentiated T cells. Unexpectedly, despite their terminally differentiated status, a CX3CR1hi CD8+ T-cell subset expressed significantly lower levels of co-inhibitory receptors, PD-1, LAG3, and TIGIT compared to CX3CR1– and CX3CR1int CD8+ T-cell subsets in the TME. In line with this, proliferation and cytokine production of CX3CR1– and CX3CR1int CD8+ T-cell subsets were significantly decreased in the TME compared to CX3CR1hi CD8+ T-cell subset. Importantly, PD-1/PD-L1 blockade significantly improved effector functions of CX3CR1– and CX3CR1int CD8+ T-cell subsets in the TME.

Conclusion: The chemokine receptor, CX3CR1 defines distinct effector CD8+ T-cell subsets in periphery and in the TME. Tumor-infiltrating CX3CR1– and CX3CR1int CD8+ T-cell subsets express high levels of co-inhibitory receptors, PD-1, LAG3, and TIGIT, and their effector functions are improved by PD-1/PD-L1 blockade. 

23.03 Depletion of Gut Microbiome Lowers Cancer Burden in Murine Models by Modulating the Immune System

Posted on January 18, 2018

V. Sethi1, B. Giri1, B. Garg1, M. Tarique1, S. Lavania1, L. Hellmund1, Z. Malchiodi1, S. Kurtom1, A. Farrantella1, S. Banerjee1, S. Ramakrishnan1, S. Roy1, A. Saluja1, V. Dudeja1  1University Of Miami,Department Of Surgery,Miami, FL, USA

Introduction: Microbiome of the gut forms an important ‘hidden organ’ of our body, changing with diet, disease state, use of antibiotics, and even age. While literature abounds with associational studies of changed gut microbiome with disease states like inflammatory bowel disease, diabetes, colitis etc., precious little is known on how, if at all, the gut microbiome modulates cancer and metastases. The aim of this current study was to analyze this obscure cancer-gut microbiome relationship. 

Methods: The gut microbiome of C57BL/6 mice was depleted by daily oral administration of broad-spectrum antibiotics having minimal systemic absorption. Various cancers were modelled on antibiotic-given mice as well as control mice and tumor burden was compared between the two groups. These models included subcutaneous implantation of pancreatic cancer cells derived from KPC (Kras LSL.G12D/+; p53 R172H/+ ;Pdx::Cre) mice and melanoma cells derived from Braf-Pten (Tyr::CreER; Braf V600E/+;Ptenlox5/lox5  ) mice . Additionally hepatic metastases were induced by intrasplenic injections of KPC pancreatic cancer cells, B16-F10 melanoma cancer cells and MC38 colon cancer cells. Tumors were immunophenotyped through flow cytometry and immunostained for various antigens. Various ex-vivo cytotoxicity experiments were performed with cancer cells and splenocytes. To confirm the role of  adaptive immunity, similar experiments were performed in immunodeficient Rag1 knockout mice having a Rag1tm1Mom mutation.

Results:  Depletion of the gut microbiome significantly decreased primary cancer and metastases burden in all the studied murine models. Tumors in antibiotics-gavaged mice showed increased cleaved caspase 3 staining. Ex-vivo, splenocytes from antibiotics-gavaged mice caused a much higher cytotoxicity of cancer cells than control splenocytes. The role of immune system was confirmed when antibiotic administration in immunodeficient Rag1 KO mice failed to show a reduced cancer burden compared to control mice. 

Conclusion: Gut microbiome depletion causes reduced cancer burden by modulating the immune system. 

 

22.09 Modeling the Persistent Inflammation, Immunosuppression, and Catabolism Syndrome in Sepsis

Posted on January 18, 2018

V. Nomellini1, A. M. Pugh1, N. J. Auteri1, H. S. Goetzman1, C. C. Caldwell1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA

Introduction: Advancements in the management of sepsis have improved overall mortality, particularly in the acute phases of the disease. However, those that survive often have prolonged and complicated recovery periods. In the later stages of sepsis, patients frequently exhibit a Persistent Inflammation, Immunosuppression, and Catabolism Syndrome (PICS), which is associated with prolonged hospitalizations, multiple complications, poor wound healing, profound disability, and long-term neurocognitive deficits.

Methods: Our lab has developed a murine model of PICS utilizing a cecal ligation and puncture (CLP) method. Male CF-1 mice underwent 33% cecal ligation with a single 25-gauge needle puncture. Mice that survived 8 days after CLP demonstrated the signature characteristics of PICS. This model generates a mortality rate of about 25%, emulating that of humans.

Results:We observed that, mice that survive the CLP develop persistent inflammation with significant myelopoiesis in the blood, bone marrow, and spleen eight days after the initial insult. These mice also demonstrate ongoing immune suppression, marked by decreased total and naïve splenic CD4 and CD8 T cells with an increase in immature myeloid cells. The mice further display significant weight loss, decreased thigh muscle mass, and perform significantly worse on the Rotarod test of coordination, indicating a state of ongoing catabolism. When PICS mice are challenged with Pseudomonas aeruginosa intranasally, mortality is significantly increased and bacterial clearance in the lungs is compromised.

Conclusion: Altogether, we have created a sepsis model that concurrently exhibits all the characteristics of PICS and confirms their susceptibility to a secondary bacterial challenge. Future studies involving this mouse model will examine the mechanisms underlying PICS in order to identify these patients early in their clinical course and develop potential therapeutic targets to improve outcomes for this patient population.

 

22.10 Persistent Hypercatecholaminemia Drives Hematopoietic Progenitor Cell Mobilization following Trauma

Posted on January 18, 2018

G. D. Doobay1, E. S. Miller1, T. J. Loftus1, K. B. Kannan1, P. A. Efron1, A. M. Mohr1  1University Of Flordia College Of Medicine,Department Of Surgery,Gainesville, FL, USA

Introduction: Using a clinically relevant rodent model for persistent-injury associated anemia with lung contusion, hemorrhagic shock and chronic stress (LCHS/CS), persistent elevation of norepinephrine was shown to be associated with prolonged mobilization of hematopoietic progenitor cells (HPC) to the peripheral blood and sites of injury. The use of propranolol has been shown to decrease prolonged HPC mobilization following trauma. Norepinephrine induces secretion of high mobility growth box 1 (HMGB1) as well as granulocyte colony stimulating factor (G-CSF). Along with neutrophil elastase, all are key factors in the mobilization of HPC via cleavage of homing protein, stromal cell-derived factor 1 (SDF-1), and adhesion protein, vascular cell adhesion molecule 1 (VCAM1). We hypothesize that prolonged HPC mobilization is due to the destruction of anchors in the bone marrow mediated by persistent hypercatecholaminemia following LCHS/CS.

Methods: Male Sprague-Dawley rats (n=6/group) were subjected to LCHS/CS ± daily propranolol (10mg/kg) (LCHS/CS+BB) and compared to naïve controls. Animals underwent two hours of daily restraint stress until the day of sacrifice (day 7).  Bone marrow mRNA was analyzed with RT-PCR for HMGB1, G-CSF, neutrophil elastase, SDF-1 and VCAM1 expression. Neutrophil elastase enzymatic activity in bone marrow was also assayed. All data were presented as mean±SD indexed to naïve control mRNA expression. Data significance was defined as bp<0.05 relative to untreated groups.

Results: Bone marrow expression of HMGB1, G-CSF, and neutrophil elastase was significantly elevated in LCHS/CS relative to naïve control (Table). The use of propranolol following LCHS/CS significantly reduced expression of HMGB1, G-CSF and neutrophil elastase when compare to LCHS/CS alone (Table). Bone marrow expression of SDF-1 and VCAM1 were significantly decreased on day seven following LCHS/CS and LCHS/CS+BB. Neutrophil elastase enzyme activity (units/L) in bone marrow was significantly elevated following LCHS/CS when compared to naïve control (2.5±0.6 vs. 0.9±0.7 units/L). The use of propranolol following LCHS/CS significantly reduced neutrophil elastase expression (1.2±0.8b units/L).

Conclusion: Increased bone marrow expression of HMGB1 and G-CSF following LCHS/CS is associated with elevated neutrophil elastase. Decreased expression of bone marrow anchoring molecules, SDF-1 and VCAM1, following LCHS/CS, likely mediates prolonged HPC mobilization. Propranolol’s ability to reduce HMGB1, G-CSF, and neutrophil elastase expression to that of control levels suggests that this is a pro-inflammatory proteolysis-mediated mobilization of HPC driven by persistent hypercatecholaminemia.

23.01 SMAD4 Loss in Patient-Derived Colorectal Cancer Tumoroids Confirms Chemoresistance.

Posted on January 18, 2018

B. Szeglin1, C. Wu9,11, I. Wasserman2, S. Uppada3, X. Chen6, K. Ganesh8, A. Elghouayel7,11, J. Shia5, A. Barlas10, P. B. Paty11, M. R. Weiser11, J. G. Guillem11, G. M. Nash11, K. Manova-Todorova10, P. Dhawan3, R. Beauchamp4, N. E. Kemeny8, J. Garcia-Aguilar11, C. L. Sawyers9, J. Smith9,11  1Albert Einstein College Of Medicine,Bronx, NY, USA 2Icahn School Of Medicine At Mount Sinai,New York, NY, USA 3University Of Nebraska Medical Center,Department Of Biochemistry And Molecular Biology,Omaha, NE, USA 4Vanderbilt University Medical Center,Section Of Surgical Sciences,Nashville, TN, USA 5Memorial Sloan-Kettering Cancer Center,Department Of Pathology,New York, NY, USA 6University Of Miami Miller School Of Medicine,Department Of Bioinformatics And Biostatistics,Miami, FL, USA 7College Of William And Mary,Williamsburg, VA, USA 8Memorial Sloan-Kettering Cancer Center,Department Of Medical Oncology,New York, NY, USA 9Memorial Sloan-Kettering Cancer Center,Human Oncology And Pathogenesis Program,New York, NY, USA 10Memorial Sloan-Kettering Cancer Center,Department Of Molecular Cytology,New York, NY, USA 11Memorial Sloan-Kettering Cancer Center,Colorectal Service, Department Of Surgery,New York, NY, USA

Introduction:
Loss of SMAD4, the central node of the TGF-β  superfamily, occurs in 10-20% of colorectal cancer (CRC) cases. SMAD4 loss in the context of activated Wnt signaling may play a role in disease progression and resistance to standard 5-fluorouracil (5-FU) based chemotherapy, but the underlying mechanisms are poorly understood. Development of relevant CRC models to better study SMAD4 biology and associated chemoresistance is needed.

Methods:
Fresh CRC specimens were obtained at time of resection. Tumors were dissociated to individual cells and seeded within a Matrigel matrix in our 3D tumoroid cell culture model. SMAD4 mutant versus SMAD4-wildtype (wt) tumoroids were injected subcutaneously into immunocompromised mice. The mice were treated with systemic 5-FU and tumors weighed at necropsy. In addition, CRISPR/Cas9 was used to knockdown (kd) SMAD4 in patient-derived tumoroids ex vivo.  SMAD4 expression was restored in SMAD4 mutant SW480 CRC cells. The SMAD4-kd tumoroids, SMAD4-wt tumoroids, and CRC cells with restored SMAD4 expression were treated with 5-FU or FOLFIRI (5-FU, leucovorin, and irinotecan) to determine dose-response differences. In a parallel, exploratory analysis, microarray expression data from 250 CRC patients was used to generate a SMAD4 signature (FDR < 10-7). The Illumina BaseSpace Correlation Engine was used to correlate this signature with compounds that could be used in synergy with 5-FU based chemotherapy in the context of SMAD4 loss.

Results:
Engrafted SMAD4-deficient tumors did not respond to 5-FU treatment, while SMAD4-retained tumors demonstrated decreased tumor weight compared to vehicle (p < 0.02). SMAD4-kd tumoroids treated with either 5-FU or FOLFIRI ex vivo were significantly more resistant to treatment than SMAD4-wt tumoroids (p < 0.01). Conversely, restoration of SMAD4 expression in CRC cells mutant for SMAD4 was associated with significant response to 5-FU based therapy (p < 0.01).  Finally, the SMAD4 signature implicated 3-3-diindolylmethane, a putative Wnt pathway inhibitor, as a lead candidate for use in the context of SMAD4 deficiency and 5-FU based chemotherapy.

Conclusion:
We demonstrate that loss of SMAD4 is associated with chemoresistance to 5-FU and FOLFIRI treatment in in vivo and ex vivo CRC tumoroid models, thereby establishing relevant biological systems to study patient-specific resistance mechanisms. Furthermore, in silico analysis of a SMAD4 gene expression signature reveals 3-3-diindolylmethane as a possible therapeutic compound to target activated Wnt signaling in the context of SMAD4 loss in CRC patients undergoing 5-FU based chemotherapy. 
 

22.07 The Effects of Glucose and B27 Deprivation on Astrocyte Expression of Erythropoietin Receptor

Posted on January 18, 2018

M. Eldeiry1, K. Yamanaka1, L. S. Foley1, X. Meng1, M. Aftab1, M. J. Weyant1, J. C. Cleveland1, D. A. Fullerton1, T. B. Reece1  1University Of Colorado Denver,Aurora, CO, USA

Introduction:

Spinal cord ischemia is a dreaded complication of complex aortic repairs that can result in paraplegia in up to 10-20% of cases. Erythropoietin plays an important, but still unclear role in protection of the spinal cord during ischemic injury. This role is elucidated by heterodimerization of the erythropoietin receptor (EpoR) with the beta common receptor (βCR) as previously demonstrated in a murine model of spinal cord ischemia-reperfusion injury. However, the cell-specific role and mechanism of βCR and EpoR upregulation and signaling have yet to be elucidated. The current model of neuronal injury employs oxygen and glucose starvation along with B27 neuronal supplement deprivation from the cell culture media. We wanted to evaluate the effects of glucose starvation and B27 deprivation on the support cell in the neuronal network, the astrocytes.

Methods:

Spinal cords were isolated from 3 day old neonatal mice. Following enzymatic tissue digestion and cell separation utilizing a density gradient, astrocytes were seeded onto a tissue culture until reaching confluence. The cells were subsequently enzymatically separated and re-seeded onto individual wells in a 24-well plate. After reaching maturity in 4-5 days the cells were treated with media that was either glucose depleted, deprived of B-27 neuronal supplement, or lacking both for 1, 2, and 4 hours. Cells were lysed and Western Blot analysis was used to evaluate the βCR and EpoR expression in the lysate.

Results:

Glucose starvation with or without B-27 deprivation led to a time-dependent increase in βCR expression with a peak at 2 hours (1.8 ± 0.7 and 1.9 ± 0.6 vs. 1.0 ± 0.1, p < 0.05, Figure a). Alternatively, glucose starvation alone did not seem to affect EpoR expression (0.8 ± 0.2, 1.0 ± 0.2, 1.0 ± 0.3 vs. 1.0 ± 0.2 , p > 0.99). However, B27 deprivation caused EpoR expression to be undetectable whether glucose was present during the treatment (Figure b).

Conclusion:

Creating in vitro models to gain a better understanding of the cellular biology of spinal cord ischemia reperfusion requires certain assumptions to be made.  In this study, we evaluated the role of glucose starvation along with B27 supplement deprivation. Two patterns were observed from this study. Glucose starvation causes a time-dependent increase in βCR expression without affecting EpoR expression. On the other hand, B27 deprivation doesn’t affect βCR expression but does seem to cause EpoR expression to be undetectable. These data give insight into the model of neuronal injury and its effect on the astrocytes that can be used to further guide studying of this complex neuronal network.

22.08 Chloroquine Ameliorates Pancreatitis through Inhibition of Neutrophil Extracellular Traps

Posted on January 18, 2018

B. A. Boone1, P. Murthy1, X. Liang1, A. D. Singhi1, R. Kang1, D. Tang1, H. J. Zeh1  1University Of Pittsburgh,Surgical Oncology,Pittsburgh, PA, USA

Introduction:  Neutrophil extracellular traps (NETs) occur when activated neutrophils release their intracellular contents, including histones, DNA, elastase and other proteins, into the extracellular space, tissues or circulation.  Recently, NETs have been implicated in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction.  We have previously shown that the autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore we sought to determine if CQ could improve severity and outcome in murine pancreatitis through NET inhibition.  

Methods:  Acute pancreatitis was induced in C57/Bl6 mice through two hourly injections of L arginine (4g/kg) into the peritoneal cavity.  Sham control mice were injected with saline.  Mice were sacrificed 48 hours after injection to collect serum.  Neutrophils were harvested from bone marrow using density gradient centrifugation and stimulated with platelet activating factor to induce NET formation.  Hoechst staining of DNA was then utilized to visualize NETs using fluorescence microscopy.  Citrullinated histone H3 (Cit H3), which allows for unwinding and expulsion of neutrophil DNA during NET formation and is a critical marker of NETs, was measured in murine serum using ELISA.  Serum DNA was measured using Quant-It picogreen.  For survival experiments, mice were injected with L arginine weekly x 3 weeks.  Animals were treated with CQ in the drinking water (100 mg/kg/day PO), beginning 1 hour after induction of pancreatitis. 

Results:  Injection of L arginine resulted in increased serum amylase and trypsin compared with sham controls, consistent with induction of pancreatitis. Neutrophils harvested from mice with pancreatitis were more prone to NET formation than sham injected controls.  Induction of pancreatitis resulted in a significant increase in serum DNA and citrullinated histone H3, suggesting upregulated in vivo NET formation.  CQ treatment decreased the propensity to form NETs from neutrophils harvested in mice with pancreatitis.  Both serum DNA and Cit H3 were significantly decreased with CQ treatment, suggesting a decrease in NETs in response to CQ.  CQ lessened the severity of acute pancreatitis, resulting in a reduction in serum amylase and trypsin with CQ treatment.  CQ treatment improved survival from pancreatitis (Figure 1, median survival 15 days vs. not reached, p<0.05). 

Conclusion:  CQ treatment decreases the severity of L arginine induced murine pancreatitis through inhibition of neutrophil extracellular traps, resulting in improved survival.  NET inhibition represents a novel treatment strategy in acute pancreatitis.  Further study to translate these findings into treatment of patients with severe acute pancreatitis is warranted.
 

22.06 Loss of c-Kit Function Impairs NO Mediated Vascular Relaxation in Mutant Mice

Posted on January 18, 2018

D. R. Hernandez1, L. Song1, Z. M. Zigmond1, Y. Wei1, L. Martinez1, R. M. Lassance-Soares1, R. I. Vazquez-Padron1  1University Of Miami,Surgery,Miami, FL, USA

Introduction:

The receptor tyrosine kinase c-Kit has been recently found to have a role in vasculoprotection. Genetic deletion of c-Kit in smooth muscle cells increases aortic plaque accumulation and atherosclerosis burden in conditional hyperlipidemic mice. The present study tests whether a c-Kit deficiency in mutant mice (KitW/Wv) leads to hypertension and impaired vascular relaxation.

Methods:

Female and male c-Kit mutant (KitW/Wv) and littermate (Kit+/+) control mice were commercially available (Jackson lab). Blood pressure was determined in conscious mice using the tail-cuff method. The first-order branch of mesenteric artery was mounted into a pressure myograph (DMT, Aarhus, Denmark) to assess endothelium-dependent and -independent vasoreactivity under isobaric conditions. A search for differentially expressed genes between c-Kit mutant and control SMC was performed using the Mouse MI-Ready microarray (Ocean Ridge Biosciences).

Results:

The deficiency of c-Kit causes hypertension in mutant mice with respect to their control littermates (mean arterial pressure: 137.5 ± 5.0 vs. 113.06 ± 0.0, p=0.007). Acetylcholine (ACh) induced a concentration-dependent relaxation of norepinephrine pre-contracted vessels in both mutant and littermate mice. The Cox inhibitor indomethacin compromised ACh-induced vasorelaxation only in c-Kit deficient vessels but not in those from control mice, which highlighted the importance of endothelium-derived prostaglandins for vasorelaxation in the absence of c-Kit. Conversely, arteries of mutant mice were poor responders to a NO agonist (SNP). The secondary messenger, cyclic guanosine monophosphate, vasodilated both types of vessels in a similar manner, which indicated that defective NO activity in mutants was due to deficiencies in soluble guanylyl cyclase (sGC) or NO synthesis. A significant downregulation of smooth muscle sGC beta 1 subunit was further found in c-Kit mutant cells using transcriptomics analysis.

Conclusion:

These results suggest the existence of a novel c-Kit/sGC signaling axis in SMC that may be relevant for the control of vascular reactivity and hypertension.
 

22.04 Novel Development of Gastric Cancer Organoids from Endoscopic Biopsy Tissues

Posted on January 18, 2018

M. Lin1, K. Hirai1, M. Choi1, D. Tzimas1, J. C. Bucobo1, J. Buscaglia1, G. V. Georgakis1, A. Sasson1, M. Gao1, J. Kim1  1Stony Brook University Medical Center,Stony Brook, NY, USA

Introduction:  Comprehensive testing of drugs for advanced gastric cancer is limited by the lack of models that recapitulate human disease. Patient-derived, 3-dimensional organoids have been developed and may accurately model in vivo disease. Diagnostic upper endoscopy is a potential avenue for gastric cancer tissue sampling and organoid creation. Here, we report the first successful creation of organoids from endoscopic biopsy in a patient with gastric adenocarcinoma.

Methods:  With IRB approval and written informed consent, upper endoscopy was performed on a patient with large gastric adenocarcinoma. Standard biopsy forceps were used to obtain tissue from three different tumor locations to create three sets of gastric organoids using a modified technique. Biopsy tissues were placed in prepared organoid medium, then washed and isolated to preserve glandular architecture. Gastric glands were collected and plated in 24-well plates. To confirm gastric origin, we performed immunofluorescent staining for LGR5 and TROY protein markers. To assess potential tumor heterogeneity within this patient’s tumor, we also created gastric cancer organoids from surgical resection tissues and obtained whole tumor lysates. We extracted DNA from all organoids and tissues and performed low-coverage, whole genome sequencing. 

Results: We successfully obtained upper endoscopy gastric cancer biopsy tissues for creation of organoids. Immediately following resection of the primary tumor, we also obtained tissues for organoid creation and genomic testing. For all organoids, we observed an initial coalescence of gastric glandular tissues into cystic structures. Some cysts developed into spheroids, whereas others remained cystic. Budding structures formed on the periphery of the spheroids after 5-7 days in culture. Gastric origin of the organoids was confirmed based on positive LGR5 and TROY stains. Comparison of whole genome sequencing between endoscopic derived and surgical organoids and whole tumor DNA assessed the tumor homogeneity. 

Conclusion: We report the first successful creation of organoids from endoscopic biopsy of gastric adenocarcinoma. The organoids appear to accurately portray human disease and have tremendous potential to be used for personalized drug testing. 

 

22.05 ST266 Attenuates Neointima Hyperplasia and Luminal Stenosis After Arterial Balloon Angioplasty.

Posted on January 18, 2018

S. Rahimpour1, X. Liu2, X. Wang2, R. Banas3, S. M. Pham1  3Noveome Biotherapeutic, Inc.,Pittsburgh, PA, USA 1Mayo Clinic – Florida,Cardiothoracic Surgery,Jacksonville, FL, USA 2University Of Maryland,Surgery,Baltimore, MD, USA

Introduction: Inflammation plays an essential role in post-angioplasty neointima formation and in-stent restenosis. ST266 (Noveome Biotherapeutics, Inc.) a novel secretome derived from cultured human Amnion-Derived Multipotent Progenitor (AMP) cells, has been shown to be anti-inflammatory and to promote wound healing. This study examined the therapeutic potential of AMP cells and ST266 in a post balloon-angioplasty arterial restenosis model in rats.

Methods: Animals were randomly divided in the following groups (N=7): no-treatment (noTx), systemic ST266, systemic AMPs and local AMP implants. Neointima hyperplasia was induced in the iliac artery of Sprague-Dawley male rats using a 2F Fogarty embolectomy catheter. After surgery, the animals in ST266 groups received 0.1, 0.5 or 1ml IV ST266 daily. In the systemic AMP groups, single-dose (SD) of 0.5 million (M) or 1.0M AMPs was injected via inferior vena cava after the angioplasty. In local AMP experiment 1M, 5M or 20M AMPs were implanted in 300 µL Matrigel (MTG) around the iliac artery after balloon angioplasty. 

28 days after the surgery, the iliac arteries were removed for histologic analysis. Re-endothelialization index was measured 10 days after balloon angioplasty. We also performed Smooth Muscle Cell (SMC) migration and proliferation assay and Western Blot for SIRT1 expression. 

Results: Compared to noTx group, ST266 (1 ml) group decreased Neointima/Neointima+Media ratio (N/NM) (0.34±0.01 vs 0.54±0.04; p=0.004), and luminal stenosis (LS) (18.18±1.86 vs 39.23±5.75%; p=0.008). AMPs (at 1M dose) decreased LS compared to noTx group (18.56 ± 2.50 vs 35.92 ± 5.75; p=0.006). Significant reduction in N/NM were found between implanted AMPs (at 20 M cell dose) and noTx groups (0.35±0.02 vs 0.54±0.04; p=0.003) and the MTG-only group (0.53±0.05, p=0.007). 20M implanted AMPs decreased the LS (16.78±2.47%) compared to the both noTx (39.23±5.75%, p=0.001) and MTG-only groups (37.51±8.55%, p=0.016). ST266 (at 1 ml dose) significantly increased the re-endothelialization index compared to noTx group (0.40±0.04 vs 0.14±0.037, p=0.002).  SIRT1 expression significantly increased in SMCs 24 hrs. after treatment with 40% ST266 compared to SMC growth media and PBS (4.22 ± 0.23 vs 1.90 ± 0.17 and 2.56 ± 0.62, p=0.005).

Conclusion:ST266 reduces neointima formation and luminal stenosis after balloon angioplasty. It is a potential novel therapeutic agent to prevent vascular restenosis in human.

 

22.02 The Gut-Liver Axis: The Source of Inflammation in Aging

Posted on January 18, 2018

F. Adiliaghdam1, F. Kuehn1, S. R. Hamarneh1, R. Vasan1, J. M. Ramirez1, E. Samarbafzadeh1, E. Liu1, Y. Liu1, R. A. Hodin1  1Massachusetts General Hospital,General Surgery,Boston, MA, USA

Introduction: Inflammaging, a chronic low-grade inflammatory state linked to persistent endotoxemia, is thought to play a crucial role in human aging and age-related diseases. The portal system resides at the interface between the liver and gut and is likely the main entry port for the intestinal inflammatory mediators into the systemic circulation. Based on our previous discovery that mice lacking the brush border enzyme intestinal alkaline phosphatase (IAP) have an accelerated aging phenotype, we used this as a leaky gut model to understand the contribution of the portal system in the inflammaging process.  

 

Methods: C57/BL6 WT or IAP KO mice of different ages (3-22 months) were used for the aging model. Liver inflammation was assessed as an indirect proinflammatory characteristic of portal vein. To study the direct effects of portal vein serum, primary mouse macrophages were incubated with portal or systemic serum from different aged mice and inflammatory gene expression measured. LPS levels were measured in both systemic and portal serum using the LAL assay.

 

Results: We found an age-dependent increase in the inflammatory cytokine levels in the livers of WT mice (p<0.05 for TNF- α and p<0.001 for IL-6). IAP-deficient mice showed significantly higher cytokine levels in their liver compared to their WT littermates (mRNA fold change for TNF-α:2.23, p<0.05 and 3.89 for IL-6, p<0.01) The amount of LPS in portal and systemic serum increased as a function of age, but were > 1000 times higher in portal compared to systemic serum, regardless of age or genotype (p<0.001).  The absence of IAP was associated with significantly more LPS in both portal and systemic blood circulations. In young mice, serum LPS was higher in KO vs WT mice, 2.32 fold systemically and 1.32 fold portally. A similar trend was seen in old mice, where systemically ratios increase by 3.33 and portally 1.86 fold. Upon incubation of target cells, we found that both systemic and portal serum from old animals induced a significantly higher inflammatory response than serum derived from young animals (2.89 and 2.73 fold increase, respectively, p<0.01 and <0.05). There was also a highly significant difference between the magnitude of TNF-α expression induced by portal compared to systemic serum (4.06 and 6.02 fold increase in young and old group, respectively. p<0.01). Finally, portal serum from IAP KO mice resulted in a more pronounced inflammatory response than serum from their WT counterparts (1.89 and 3.44 fold increase in young and old group, respectively. p<0.001).

 

Conclusion: Portal vein serum contains proinflammatory characteristics that increase with aging. Targeting the “leaky gut” with IAP treatment could prevent the entry of gut-derived inflammatory mediators into the portal system, thus representing a novel therapy to prevent a variety of gut-derived systemic diseases.

22.03 Caspase-11 Mediated HMGB1 Release from the Liver Drives Immune Cell Pyroptosis in Sepsis

Posted on January 18, 2018

W. Li1,2, M. Deng1, B. Lu3, Y. Tang3, M. Scott1, Q. Wang1, T. R. Billiar1  1University Of Pittsburgh,Surgery,Pittsburgh, PA, USA 2The 3rd Xiangya Hospital Of Central South University,Surgery,Changsha, HUNAN, China 3The 3rd Xiangya Hospital Of Central South University,Department Of Hematology And Research Institute Of Immunology,Changsha, HUNAN, China

Introduction: Caspase-11, a recently described intracellular receptor for endotoxin (LPS) initiates the noncanonical inflammasome and induces pyroptotic cell death in immune cells during endotoxemia. It is unknown how LPS is delivered to cytosolic caspase-11 in endotoxemia or sepsis. We hypothesis that HMGB1, a known LPS-binding protein that contributes to lethality in sepsis, would deliver LPS to the cytosol of macrophages.

Methods: Experiments were carried out in cultured murine macrophages exposed to LPS in vitro and in a CLP sepsis model in vivo. We generated global caspase-11-/- , TLR4-/- and RAGE-/- mice as well as mice with selective deletion of caspase-11, HMGB1 or TLR4 from myeloid cells or hepatocytes. All in vivo experiments included 6-20 mice per group.

Results: In vitro experiments established that HMGB1 efficiently delivered LPS to the cytosol of cultured macrophages through RAGE-dependent uptake of HMGB1-LPS complexes. This in turn, led to LPS delivery to caspase-11 in the cytosol and pyroptosis.

In vivo, hepatocyte specific deletion of TLR4, caspase-11 or HMGB1 suppressed circulating HMGB1 levels, improved survival and bacterial clearance, and prevented immune cell death in the spleen during sepsis. In contrast, myeloid cell specific deletion of TLR4 or HMGB1 had no effect on circulating HMGB1 levels or immune cell death in sepsis, while caspase-11 deletion prevented immune cell death.

 

Conclusion: These findings indicate that hepatocytes are the dominant source of HMGB1 in sepsis. Surprisingly, the release of HMGB1 from the liver requires both TLR4 and caspase-11 in hepatocytes. The systemic release of HMGB1 drives caspase-11 dependent pyroptosis in immune cells. These studies identify a novel circuit that involves the LPS-sensing pathway in the liver for the regulation of systemic release of HMGB1 in sepsis. This liver-derived HMGB1 is required for caspase-11 dependent immune cell pyroptosis and lethality. 

 

22.01 Sleeve Gastrectomy Reverses Obesity-Induced Intestinal Immune Dysfunction

Posted on January 18, 2018

D. A. Harris1, R. Subramaniam1, K. Heshmati1, A. Tavakkoli1, E. Sheu1  1Brigham And Women’s Hospital,General Surgery,Boston, MA, USA

Introduction: Obesity is associated with chronic inflammation driven in part by disruption in intestinal immune integrity leading to insulin resistance. Sleeve gastrectomy (SG) is the most performed bariatric surgery in the US, but its mechanisms remain incompletely defined. We hypothesized that SG improves glucose metabolism through normalization of obesity-induced immune dysregulation.

Methods:  C57Bl/6J mice were placed into four groups – normal chow diet (lean; n=6); high fat diet (Obese; n=6); Obese Sham (n=7); Obese SG (n=6).  Glucose tolerance, insulin tolerance, and immune phenotype were measured through 6 weeks. Liver, jejunum, and spleen were harvested for time of flight mass cytometry (CyTOF), which is a novel platform allowing quantitative immune profiling. A 24-antibody panel directed against key markers of innate and adaptive lymphocytes was developed. Visualization of stochastic neighbor embedding (ViSNE) was used for unbiased profiling. 

Results: Obese mice weighed more (40.7±2.4 vs 27.9±2.7g; p<0.001) and had worse fasting glycemia (139±14 vs 99±3 mg/dL; p<0.001) compared to Lean mice. CyTOF was used to compare splenic, hepatic, and jejunal immune populations in these groups. Obese mice have organ-specific perturbations of multiple immune populations compared to Lean mice such as changes in splenic B-cells, hepatic CD4+ TH1 cells, and jejunal CD8+CD103+ tissue resident memory (TRM) T-cells (p<0.05).

Obese SG mice exhibited significant weight loss, improved oral glucose tolerance (AUC, Obese Sham: 34693 [30338 to 39048], Obese SG: 25630 [21984 to 29257]), and increased insulin sensitivity (p<0.05 at 0, 15, 30 mins) compared to Obese Sham mice. Again, CyTOF was used to evaluate the impact of SG on splenic, hepatic, and jejunal immunity. Of the 40 distinct lymphocyte populations assessed, ViSNE revealed that only changes in jejunal CD8+CD103+ TRM correlated with improved weight and insulin sensitivity following SG (Figure 1A). TRM are responsible for rapid pathogen defense at mucosal surfaces. Obese mice have reduced jejunal TRM compared to Lean mice (85.2±7 vs 36.8±10, p=0.003, Figure 1B). SG repairs this obesity-induced mucosal immune defect by restoring jejunal TRM to levels seen in healthy, Lean mice (Figure 1B), a finding validated by flow cytometry. Changes in TRM were confined to jejunum and not seen in the liver or spleen.

Conclusion: Obesity is associated with a disruption in intestinal immunity leading to insulin resistance. SG reverses the deficit in jejunal TRM cells seen in obesity and thereby restores protective intestinal immune function, which may contribute to its metabolic benefits.
 

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