41.16 Three Dimensional Confocal Imaging Analysis of Adipose Tissue

Posted on January 25, 2017

N. N. Chung1, C. P. Blackshear1, C. Ransom1, D. Irizarry1, D. Nguyen1, M. Longaker1,2, D. C. Wan1  1Hagey Laboratory For Pediatric Regenerative Medicine,Stanford University Medical Center,Stanford, CA, USA 2Institute For Stem Cell Research And Regenerative Medicine,Stanford University Medical Center,Stanford, CA, USA

Introduction:  Autologous fat grafting is frequently used to correct soft tissue defects, but outcomes remain unpredictable. Studies have shown stromal cell enrichment of fat grafts to improve volume retention, but the mechanism of this effect has been primarily explained through inferential gene expression analysis and two-dimensional histologic staining. In this study, we employed novel three-dimensional confocal imaging to determine how graft architecture changes with stromal cell enrichment.

Methods:  Confocal microscopy with optical sectioning was employed to reconstruct three-dimensional data of whole mounted tissue using Imaris. Unprocessed human abdominoplasty fat, as well as unenriched human fat grafts and stromal cell enriched fat grafts after two, four, six, and eight weeks of implantation in immunocompromised mice were evaluated. Supplemental stromal cells were fluorescently tagged to determine three-dimensional distribution throughout the fat graft. Isosurface rendering was employed to determine individual adipocyte size and variability, vascular density, and proximity of stromal cells to new vasculature.

Results: Preliminary data has shown that confocal imaging of adipose tissue results in superior images that elucidate tissue architecture and more closely approximates the graft in situ. Images allowed for clear visualization of supplemented ASCs within the graft environment, showing an intimate relationship with revascularization patterns in the fat graft. Heterogeneity in adipocyte size was also appreciated with fat grafts supplemented with stromal cells appearing more similar to unprocessed abdominoplasty fat.

Conclusion: Confocal imaging allows for direct three-dimensional visualization of the fat graft environment that approximates in situ, which holds promise for future study of CAL grafts and retention.

 

41.11 The Effects of Smooth Muscle Cells and Subpopulations of Fibroblasts on Breast Capsular Contracture

Posted on January 25, 2017

D. M. Irizarry1, C. D. Marshall1, D. Nguyen1, M. T. Longaker1,2, D. C. Wan1  1Stanford University Medical Center,Hagey Laboratory For Pediatric Regenerative Medicine,Palo Alto, CA, USA 2Stanford University Medical Center,Institute For Stem Cell Research And Regenerative Medicine,Palo Alto, CA, USA

Introduction:
Despite continued improvements in breast implant design, capsular contracture remains a significant problem. This is further worsened by adjuvant radiation therapy for breast cancer. Several studies have correlated fibroblast density with capsular contracture, and more recent reports have also shown a functional difference in fibroblasts from non-contracted and contracted breast implant capsules.  Emerging data has now shown specific fibroblast subpopulations to exist, and in particular, CD26+ fibroblasts have been implicated in fibrosis/scarring following radiotherapy. The role of this fibroblast subpopulation in radiation-induced contracture was thus evaluated. 

Methods:
Human breast capsule specimens were taken from patients following mastectomy with tissue expander placement and post-operative radiation therapy. The contralateral non-irradiated side served as a control. Capsules were mechanically and then enzymatically digested to obtain fibroblasts. Fluorescence-activated cell sorting and immunohistochemistry staining were performed. Cell surface marker expression and gene expression were compared between irradiated and non-irradiated specimens.  Proportions of CD26+ fibroblasts were compared between the irradiated side and non-irradiated side. 

Results:
Histology of the specimens revealed that irradiated, contracted capsule contained more dense eosinophilic material suggestive of smooth muscle tissue. Furthermore, irradiated, more severely contracted capsules were found to have more vimentin+/CD26+ cells by histology and flow cytometry. Gene analysis demonstrated differences in fibrotic gene expression between fibroblasts harvested from each side. 

Conclusion:
These results suggest a role for CD26-expressing fibroblasts and smooth muscle cells in breast capsule contracture following radiation. These are both potential future targets for intervention aimed at reducing capsular contracture severity.
 

41.10 Cellular Evidence of Polyethylene Glycol Fusion in Cultured Neuroblastoma Cells

Posted on January 25, 2017

A. N. Hoffman2, R. Bamba1, M. Stephanides1, A. C. Pollins1, W. P. Thayer1  1Vanderbilt University Medical Center,Plastic Surgery,Nashville, TN, USA 2Vanderbilt University School Of Medicine,Nashville, TN, USA

Introduction:
The degree of functional recovery after a peripheral nerve injury is rarely satisfactory.  Peripheral nerves have the capacity to regenerage, but unfortunately this regeneration is very slow, and there is usually significant loss of function. Current methods in nerve repair depend on the type of nerve injury and include direct repair, nerve conduits, nerve autografts and allografts. These techniques focus on optimizing the process of axonal regeneration. Polyethylene glycol (PEG) fusion is a new technique in peripheral nerve repair that aims to fuse damaged axons in order to provide some immediate restoration of axonal function. The PEG fusion technique involves reconnecting the cut ends of severed axons with microsutures and applying a sequence of solutions including PEG. It is thought to delay Wallerian degeneration, and animal studies have shown immediate restoration of compound action potentials and improved behavioral outcomes.

Methods:
Rat B35 neuroblastoma cells were grown using standard cell culture techniques. The cells were labeled separately with CellTracker Green CMFDA and CellTrace Violet fluorescent dyes. These dyes were chosen because they freely diffuse across cell membranes, and are not transferable between cells except to daughter cells. Equal concentrations and volumes of the green- and violet-labeled cells were then combined and PEG solutions were added to the cell pellet at concentrations of 50%, 75% or 100% PEG by weight with 5% DMSO. A control sample consisted of the two populations of fluorescently labeled cells combined without addition of PEG. Other controls included unstained and single stain groups. The samples were analyzed using the BD LSRFortessa flow cytometer. The amount of fusion was assessed by the percent of double positive cells.

Results:

Results show that there is increasing cell fusion with increasing concentrations of PEG. Viability tests were also performed with the viability factor PI, and they showed decreasing viability with increasing concentrations of PEG (Table 1).

Conclusion:
The results of this experiment provide evidence of PEG fusion at the cellular level. Using the double-positive population as an indicator of cell fusion is not a perfect measure, as cells are equally likely to fuse with other cells of the same color. Therefore, it is most likely an underestimate of the amount of fusion. Assessment of cell viability indicated that there is increasing cell death with increasing concentration of PEG. This trade-off between increased fusion and increased cell death represents a significant balance that must be assessed for applicability in patients with nerve injuries. Future directions of this experiment include studying the long term viability of fused cells.

41.09 Tracking CD Marker Drift of Fresh Adipose Derived Stem Cells Induced by 2-Dimensional Cell Culture

Posted on January 25, 2017

E. Brett1, D. Wan1  1Stanford University,Palo Alto, CA, USA

Introduction:  The influence of static, 2D culture on cell types has been investigated on cell lines and primary cultures. Long term subcultures of cell lines have shown susceptibility to ‘drift’, both phenotypically and genotypically. CD marker drift is pertinent to primary cell types which require fluorescent assisted cell sorting for isolation. Adipose derived stem cells (ASCs) are an example of such cells, relying on FACS selection of CD45-/CD31-/CD34+ cells from processed lipoaspirate. 

Methods:  Choosing CD90, CD105, and BMPR1b, we plated FACS-sorted ASCs positive and negative for these markers, and analysed the extent of CD marker drift over 0hr, 12hr, 24hr, 36hr, 48hr, D3, D5, and D7 after standard cell culture. It was consistently observed that ASCs which were sorted for a presence or absence of specific marker at 0 hour have a significantly different CD expression profile after 36 hours of cell culture (*p<0.05). We also analysed the effect of CD positive and negative populations on one another in a co-culture setting. A decrease of CD marker expression was largely observed across the cell populations, caused by originally CD-positive cells drifting towards a negative phenotype. The osteogenic potential of purified positive and negative populations was assessed using a standardized in vitro model of osteogenic differentiation. Significant differences were observed between the osteogenic capacities of fresh ASCs versus ASCs whose CD marker profiles had drifted over 36 hours of culture (*p<0.05). This drift was characterized further using qRT-PCR, which detected concomitant increases in expression of osteogenic and mechanotransductive genes in plated cells. 

Results: These observations allude to the volatile nature of CD expression by FACS-sorted ASCs once in culture, and suggest an influence of cell culture on capabilities of ASCs to commit to lineage. 

Conclusion: In vitro culture has governance on phenotypic and genotypic profile of ASCs. This has a significant downstream impact on the osteogenic capacity of the cells in vitro; an important consideration for all in vitro assays. 

 

25.08 Circulating Tumor Cell Phenotypic and Genotypic Profiling for Hepatocellular Carcinoma

Posted on January 25, 2017

C. M. Court1,5, S. Hou1,3, S. Sho1,5, P. Winograd1,5, Q. Li1, S. Sadeghi4, R. S. Finn4, F. W. Samuel3,6, B. V. Naini6, F. M. Kaldas1,2, R. W. Busuttil1,2, J. S. Tomlinson1,5, H. R. Tseng3, V. G. Agopian1,2  1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Liver Transplantation,Los Angeles, CA, USA 3University Of California – Los Angeles,Molecular And Medical Pharmacology,Los Angeles, CA, USA 4University Of California – Los Angeles,Hematology/Oncology,Los Angeles, CA, USA 5VA Greater Los Angeles,Surgery,Los Angeles, CA, USA 6University Of California – Los Angeles,Pathology,Los Angeles, CA, USA

Introduction: Treatment options available to patients with hepatocellular carcinoma (HCC) include potentially curative surgical therapy (resection and liver transplantation) for early stage patients and molecular targeted therapy for advanced/metastatic patients. Currently, treatment selection is based on clinical and radiological staging of disease, partly due to the lack of available biomarkers to inform treatment decisions. Circulating tumors cells (CTCs) are one such potential biomarker with proven efficacy in many cancers, but have not been well characterized in HCC. We sought to develop a novel, blood-based assay capable of detecting HCC CTC phenotypes and genotypes with prognostic importance that may impact treatment selection.

Methods: Utilizing the microfluidic NanoVelcro platform which allows for multichannel immunocytochemistry as well as single cell isolation and sequencing, we prospectively evaluated 4 milliliters of blood from 69 patients (50-HCC, 11-benign liver disease, 8-healthy controls) and developed assays to enumerate and characterize CTC phenotype (epithelial-type cytokeratin (CK+) CTCs, mesenchymal-type Vimentin (Vim+) CTCs, programmed death-ligand 1 (PDL1+) CTCs) and identify CTC genotype by sequencing for actionable somatic mutations associated with targeted drug therapy. CTC number and phenotype was correlated with clinicopathologic data. 

Results: CK+ CTCs were detected in 96% of HCC patients (Mean=6.3, range=1-27) and discriminated HCC and non-HCC patients (≥2 CTCs, sensitivity=90.0%, specificity=89.5%, AUROC=0.946, p<0.001). Mesenchymal-type Vim+ CTCs were detected nearly exclusively in advanced (stage III/IV) patients (Mean = 6.5, range = 1-20), with excellent discrimination between early, transplant-eligible and advanced stage, transplant-ineligible patients (AUROC = 0.970, p < 0.001; Fig). In advanced HCC patients, PDL1+ CTCs were detected in 5/8 (63%) of patients, and were noted to decrease following treatment with nivolumab, a PD-1 inhibitor. Using our single CTC isolation and sequencing method, we were able to perform gene panel sequencing in CTCs from 10 patients. In two of those patients, potentially actionable mutations (PIK3CA and TP53) were found in CTC-DNA. 

Conclusion:CTCs are a promising serum biomarker in HCC with potential treatment implications for both early and late stage patients. Vim+ CTCs accurately discriminated early and late stage HCC patients and may improve transplant candidate selection. For advanced stage patients, both CTC phenotype (PDL1+) and genotype analysis may allow for selection of specific targeted therapies. Longitudinal follow-up with evaluation of cancer-specific outcomes is necessary to establish whether CTCs may improve current treatment algorithms.

25.07 Mutant-Allele Tumor Heterogeneity Scores Correlate With Neoadjuvant Therapy Response in Rectal Cancer

Posted on January 25, 2017

A. Greenbaum2, S. Ness4, T. Bocklage3, J. Lee1, A. Rajput2  1University Of New Mexico HSC,Epidemiology, Biostatistics And Preventative Medicine/Internal Medicine,Albuquerque, NM, USA 2University Of New Mexico HSC,Surgery,Albuquerque, NM, USA 3University Of New Mexico HSC,Pathology,Albuquerque, NM, USA 4University Of New Mexico HSC,Internal Medicine,Albuquerque, NM, USA

Introduction:   Neoadjuvant chemoradiation is the standard of care for locally advanced adenocarcinoma of the rectum.  It is currently unknown which patients will respond to therapy.  We aimed to determine if Mutant-Allele Tumor Heterogeneity (MATH) scores, a novel bioinformatics tool, can predict response to neoadjuvant treatment in locally advanced rectal tumors.

Methods:    We performed high read-depth (“deep”) sequencing of >400 cancer-relevant genes on a group of 13 patients with locally advanced rectal adenocarcinoma.  Normal and tumor DNA were extracted from formalin-fixed, paraffin-embedded tissues.   DNA samples were analyzed using the Ion Ampliseq Comprehensive Cancer Panel™ assay. Sequencing was performed on the Ion Proton Next-Generation Sequencing™ instrument.   Mutant allele frequencies were determined and a calculated MATH score was used to quantify tumor heterogeneity.  Response to chemo therapy was determine by primary resection pathology report.

Results:  A total of 13 patients with locally advanced rectal cancer (T3/4 or N1/2) were analyzed.  The boxplot in Figure 1 shows the range of calculated MATH scores by neoadjuvant therapy response category. Four patients were noted to have complete response, 7 had minimal/moderate and 2 demonstrated poor response. Tumor heterogeneity (as shown in MATH scores) was found to be significantly different amongst the 3 response groups (p=0.026), with higher MATH scores correlating with poorer response to treatment.

Conclusion:  The novel approach of applying the shape of a whole bioinformatics data set to analyze tumor heterogeneity may provide a useful biomarker for locally advanced rectal cancer.  MATH scores may allow a means of predicting response to neoadjuvant chemoradiation therapy.

 

25.05 Pomalidomide enhanced antitumor effects of gemcitabine and nab-Paclitaxel in pancreatic cancer cells

Posted on January 25, 2017

N. SAITO1, Y. Shirai1, T. Horiuchi1, H. Sugano1, R. Iwase1, K. Haruki1, Y. Fujiwara1, K. Furukawa1, H. Shiba1, T. Uwagawa1, T. Ohashi2, K. Yanaga1  1The Jike University School Of Medicine,Department Of Surgery,Minato-ku, TOKYO, Japan 2The Jikei University School of Medicine,Division Of Gene Therapy, Research Center For Medical Sciences,Minato-ku, TOKYO, Japan

Introduction:  NF-κB plays an important role in chemoresistance. Although gemcitabine and nab-paclitaxel therapy (GN) has been effective for pancreatic cancer, the therapeutic efficacy is attenuated by anticancer agents-induced activation of NF-κB. Meanwhile, pomalidomide is a novel immunomodulatory drug derived from thalidomide. Since thalidomide is a NF-κB inhibitor in digestive cancer, we hypothesized that pomalidomide also inhibits NF-κB activation, and enhances antitumor effects of GN for pancreatic cancer cell lines. 

Methods:  In vitro, we used human pancreatic cancer cell lines (MIA PaCa-2, PANC-1). We compared the antitumor effect of pomalidomide plus GN (GNP) with GN. Concentration of NF-κB, cell proliferation, cell cycle, and induction of apoptosis were evaluated by each assay. In vivo, we created xenograft orthotopic pancreatic cancer model (BALB/c with PANC-1). The animals were treated with oral polmalidomide five times a week and i.p. injection of GN once a week for 5 weeks. We evaluated sequential tumor volume by MRI and conclusive tumor weight and volume. We assessed the in vivo protein and apoptosis levels examined in vitro.

Results: 

Pomalidomide suppressed GN-induced NF-κB activation (MIA PaCa-2; GN : GNP = 9.57 ± 0.47 : 7.09 ± 0.20 ng/mg; p<0.01, PANC-1; GN : GNP = 69.58 ± 5.18 : 32.34 ± 13.61 ng/mg; p<0.05). Cell viability in GNP was significantly lower than that in GN (MIA PaCa-2; GN : GNP = 51.6 ± 9.0 : 24.1 ± 7.7 % ; p?0.01 , PANC-1; GN : GNP = 70.4 ± 7.5 : 41.7 ± 2.7 % ; p?0.01).

Moreover, in GNP, the levels of apoptotic protein (cleaved caspase 8, cleaved caspase 3, cleaved PARP) were higher than those in GN. Similar to nuclear NF-κB concentrations, phosphorylated IκBα was lower in GNP than that in GN. In addition, pomalidomide suppressed the expression levels of VEGF of pancreatic cancer in a dose-dependent manner. 

In vivo, the tumor weight (GN : GNP = 538 ± 36 : 385 ± 88 mg; p<0.05) and tumor volume (GN : GNP = 587 ± 51 : 313 ± 89 mm3; p=0.01) were significantly lower in GNP than those in the GN after five weeks of treatment. Moreover, immunohistochemical staining revealed down-regulation of VEGF and Ki-67 in GNP. 

Conclusion: Pomalidomide inhibited NF-κB activation and enhanced the antitumor effects of GN on pancreatic cancer cells.
 

25.04 Loss of miR-155 Upregulates WEE1 in Metastatic Melanoma

Posted on January 25, 2017

J. A. Campbell1, I. Huffnagle1, G. P. Robertson2, C. R. Pameijer1  1Penn State Hershey Medical Center,Department Of Surgery,Hershey, PA, USA 2Penn State Hershey Medical Center,Department Of Pharmacology,Hershey, PA, USA

Introduction:  WEE1 is a protein kinase in the BRAF pathway and is over-expressed in melanoma. It is involved in regulating cell cycle progression and is involved in tumor progression in several malignant tumors. In melanoma, experimentally induced down-regulation of WEE1 has been shown to inhibit cell growth in vitro and in vivo. Like many other proteins, WEE1 expression is regulated by microRNA. MicroRNAs are small, non-coding segments of RNA that are involved in post transcriptional regulation of gene expression and have been implicated in oncogenesis. Melanoma tumors have been shown to have very low levels of microRNA-155 (miR-155), although the downstream effect of this low expression is unknown. We evaluated a group of melanoma patient tumor samples for miR-155 expression, clinical outcome and possible mechanism of action of miR-155.

Methods:  RNA was extracted from melanoma patient tumor samples. miRNA microarray analysis was performed, with confirmation by qRT-PCR. Melanoma cell lines transfected with miR-155 were used for a mouse experimental metastasis model. Luciferase reporter assay was used to show interaction between miR-155 and the 3’UTR of WEE1. Western blotting was used to show the effect of miR-155 transfection on the protein levels of WEE1 in melanoma cells.

Results: In patient tumor samples miR-155 was lost in patients who develop metastatic disease. In the mouse model, transfection of cells from two metastatic melanoma cell lines with miR-155 mimic decreased the metastatic potential of melanoma cells, with a significant difference in the number of lung metastases between miR-155 and control mice (figure 1a). Transfection of melanoma cells with miR-155 reduced WEE1 protein levels (figure 1b) while inhibition of endogenous miR-155 up-regulated WEE1 expression.  Luciferase reporter assay demonstrated that miR-155 interacts with the WEE1 3’UTR and impaired gene expression in melanoma cells.

Conclusion: miR-155 is lost in patients who develop metastatic melanoma. Loss of miR-155 allows for upregulation of WEE1 in melanoma. Our in vivo studies showed significant decrease in metastasis with cells over expressing miR-155 suggesting that in melanoma miR-155 affects metastasis through WEE1 kinase.

25.03 Use of Circulating Microvesicles, Exosomes, as a Biomarker to Track Disease Burden in Melanoma

Posted on January 25, 2017

J. A. Cintolo-Gonzalez1, W. Michaud1, S. Cohen1, D. Plana1, D. J. Panka2, R. J. Sullivan3, G. M. Boland1  1Massachusetts General Hospital,Surgery,Boston, MA, USA 2Beth Israel Deaconess Medical Center,Hematology/Oncology,Boston, MA, USA 3Massachusetts General Hospital,Hematology/Oncology,Boston, MA, USA

Introduction:
Exosomes are extracellular microvesicles, which contain a variety of nucleic acids (DNA, messenger RNA (mRNA), microRNA (miRNA)) and proteins. Exosomes can be analyzed from archived serum/plasma samples making them ideal biomarkers to study tumor-based changes. We examined the feasibility and accuracy of exosomal mRNA analysis to assess disease burden in patients undergoing surgical resection for metastatic melanoma.

Methods:

Melanoma cell lines (A375, RPMI 7951, SK-MEL-30, and MeWo) were purchased directly from ATCC. Serial tumor and blood samples were collected from melanoma patients under IRB approved protocols.  Exosomes were isolated using combined filtration and ultracentrifugation. Exosomal RNA was isolated using the exoRNA serum/plasma kit (Qiagen Inc). 

Quantitative PCR (qPCR) was used to assess concordance of gene expression between paired exosomes and cell lines.  Paired exosomal and tumor mRNA from patient samples underwent whole transcriptome sequencing using Affymetrix Whole Transcriptome Pico Array.  Patient plasma-derived exosomes were analyzed via qPCR at pre-resection and post-resection time points.

Results:
Paired exosomes and their parental cell lines demonstrated concordance in gene expression as assessed by qPCR.  Specifically, in cell lines harboring a BRAF V600E mutation, BRAF V600E was detected by qPCR in cell lines and paired exosomes.   Likewise, whole transcriptome analysis of patient-derived exosomes and paired tumors demonstrated 80% concordance of gene expression. In patients undergoing resection of BRAF V600E mutant metastatic lesions, there was a significant decrease in exosomal BRAF V600E mRNA in patients rendered NED or with minimal residual disease but not in patients undergoing palliative resection with multiple residual sites of disease (Figure 1).  Of the patients undergoing definitive resection, patient 410, who showed ongoing, albeit decreased, exosomal BRAF V600E expression, recurred 3 months following resection.

Conclusion:
Exosomal mRNA reflects parental tumor mRNA expression and accurately predicts the presence of residual disease after resection in melanoma.  These findings support the use of exosomes as a biomarker for assessing tumor burden after surgery. Work is ongoing to assess the utility of exosomal RNA to assess minimal residual disease and risk of recurrence in high risk melanoma patients.

25.02 PI3K/mTOR Inhibition Suppresses Pancreatic Cancer by Reprogramming Cancer-Associated Fibroblasts

Posted on January 25, 2017

J. L. Williams2, I. A. Elliott1, A. H. Nguyen1, C. Matsumura1, R. Ghukasyan1, P. A. Toste1, J. R. Capri3, S. G. Patel1, L. Li1, N. Wu1, C. G. Radu3, T. R. Donahue1,3  1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2Harbor-UCLA Medical Center,Department Of Surgery,Torrance, CA, USA 3David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, with an overall survival of less than one year.  Contributing to this poor prognosis is PDAC’s characteristically dense stroma, which is comprised predominantly of cancer-associated fibroblasts (CAFs). CAFs promote tumor growth, metastasis, and treatment resistance because when activated by environmental factors such as cancer cell (CC) exposure, hypoxia, and cytotoxic chemotherapy, they secrete protumorigenic cytokines, growth factors, and extracellular matrix components.  However, elimination of CAFs also increases tumor growth and invasion.  Therefore, rather than ablation, reprogramming of CAFs to an inactive or quiescent state is a potential PDAC treatment strategy.  In this study, we aimed to determine the effect of PI3K/mTOR inhibition, a pathway known to be involved in fibroblast activation, on PDAC tumorigenicity.

Methods:  Immortalized CAFs were treated with NVP-BEZ235 (BEZ), a dual PI3K/mTOR inhibitor, and exposed to gemicitabine (GEM), hypoxia (1% O2), or PDAC CC conditioned media (CM) for 48 hours.  After treatment, markers of CAF activation were determined via reverse transcription polymerase chain reaction (RT-PCR), Western blot, and mass spectrometry proteomics analysis of CAF CM.  Additionally, PDAC CCs (Mia PaCa-2 and PANC-1) exposed to CM from treated CAFs were tested with in vitro viability, migration, and invasion assays. PDAC CCs were also co-injected with treated CAFs in vivo, and tumor size was assessed.  A 3D co-culture model was used to assess the effect of BEZ treatment on CCs grown together with CAFs.

Results: After treatment with BEZ, gene expression of known markers of CAF activation, including alpha-smooth muscle actin (αSMA) and type I collagen, were decreased, even after exposure to activating stimuli.  Similarly, αSMA protein levels were reduced in BEZ-treated CAFs. CM from BEZ-treated CAFs contained lower levels of extracellular matrix components, proinflammatory cytokines, and growth factors. CCs exposed to CM from BEZ-treated CAFs were less viable, migratory, and invasive in vitro. Pre-treatment of PDAC CAFs with BEZ restricted PDAC growth in our in vivo co-implantation model (Figure 1).  In CC-CAF 3D co-culture, combination treatment of BEZ with GEM was more effective at suppressing CC growth than GEM treatment alone.

Conclusion: Inhibition of PI3K/mTOR decreases CAF activation, resulting in reduced PDAC CC tumorigenesis in vitro and in vivo. Additionally, combination treatment with BEZ, a dual PI3K/mTOR inhibitor, and GEM synergistically inhibits PDAC CC growth in the presence of CAFs. These data suggest that the addition of BEZ may improve the effectiveness of cytotoxic chemotherapy in PDAC.
 

25.01 Notch1—WISP-1 axis determines the regulatory role of MSC-derived fibroblasts in melanoma metastasis

Posted on January 25, 2017

P. P. Parikh1, H. Shao1, L. Cai1, M. Moller1, B. Issac2, R. Vazquez-Padron1, M. Owyong1, Z. Liu1,2  1University Of Miami,Surgery,Miami, FL, USA 2University Of Miami,Sylvester Comprehensive Cancer Center,Miami, FL, USA

Introduction:  Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis.

Methods:  MSC-DF were generated from bone marrow (BM)-derived MSC obtained from Notch1Flox/Flox and ROSAN1IC mice and transduced with Cre-GFP/Lentivirus or GFP/Lentivirus (control).  Various MSC-DF were co-cultured or co-grafted with human melanoma cells (labeled with Luc2) in vitro or in vivo. Melanoma growth and metastasis in vivo were examined by measuring tumor size and IVIS scanning. cDNA microarray was employed to identify downstream targets of Notch1 signaling.

Results: We demonstrated that the Notch1 pathway’s activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1-/- selectively promoted, while MSC-DFN1IC+/+ preferentially suppressed melanoma metastasis, but not growth, in mouse models. When co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1-/- supported, but MSC-DFN1IC+/+ inhibited melanoma cells to form spheroids. We also identified Wnt-induced secreted protein-1 (WISP-1) from MSC-DF as a key downstream secretory mediator of Notch1 signaling on melanoma metastasis. 

Conclusion: These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We demonstrate that the Notch1—WISP-1 axis functions as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis.

 

24.10 ECM and CEACAM Proteins Characterize Pancreatic Duct Exosomes in Patients with Pancreatic Cancer

Posted on January 25, 2017

J. Zheng1, J. Hernandez1, A. Doussot1, L. Bojmar2, B. Costa-Silva2, E. VanBeek2, C. Zambirinis1, M. Tesic Mark3, H. Molina3, M. Gonen1, T. Kingham1, P. Allen1, M. D’Angelica1, R. DeMatteo1, D. Lyden1,2, W. Jarnagin1  1Memorial Sloan-Kettering Cancer Center,New York, NY, USA 2Weill Cornell Medical College,New York, NY, USA 3Rockefeller University,New York, NY, USA

Introduction: Exosomes have emerged as important vehicles for intercellular communication between cancer cells and their microenvironment and may facilitate tumor progression. Exosomes in pancreatic duct fluid have not been previously studied. We hypothesize that exosomes can be detected in the pancreatic duct fluid and that exosomal proteins may be useful as diagnostic and/or prognostic biomarkers in patients with pancreatic ductal adenocarcinoma (PDAC).

Methods: Pancreatic duct fluid was collected from 22 patients at the time of partial pancreatectomy for PDAC (n=13), intraductal papillary mucinous neoplasm (IPMN) (n=4) and other benign pancreatic diseases (n=5). Serial ultra centrifugation was used to isolate exosomes. Exosomes were quantified using the DS500 nanoparticle characterization system (NanoSight), and confirmed by transmission electron microscopy (TEM). Exosomal proteins were identified and analyzed using liquid chromatography-mass spectrometry (LC-MS). Expression levels of exosomal proteins were compared between patients with PDAC and benign diseases using Wilcoxon rank-sum test. Survival analyses among patients with PDAC were performed using Kaplan-Meier with log rank test.

Results: Exosomes were successfully isolated from the pancreatic duct fluid of all patients. The mean concentration of exosomes isolated was 5.2 +/- 1 x 108 particles/mL, with a mean vesicle size of 186 +/- 4nm. The presence of exosomes was confirmed for each patient using TEM. Characteristic exosome surface markers, including tetraspanins CD9 and CD63, were identified. No differences in either size or concentration of EVs were detected when patients were stratified by diagnosis, but significant differences in protein composition were found. Laminin β3, Laminin C2, Tenascin C, and matrix metalloproteinases 1 and 7 were significantly overexpressed among patients with PDAC as compared to patients with IPMN and benign diseases (Table). Similarly, CEACAM1, CEACAM5, and CEACAM6 were significantly overexpressed among patients with PDAC (p=<0.001, 0.025, and 0.030, respectively). Among patients with PDAC, patients having expression of all three CEACAMs (n=6) had significantly worse overall and recurrence-free survival compared to patients lacking any of the CEACAMs (n=7) (p=0.01 and 0.005, respectively).

Conclusion: Exosomes can be isolated from pancreatic duct fluid. Exosomal ECM-associated proteins and CEACAMs appear to possess diagnostic and prognostic utility for patients with PDAC. Further evaluation of the mechanistic significance of these proteins in exosomes is warranted.

 

24.09 Tumor Homing Peptide Display on AAV2 Surface Enhances Viral Targeting of Pancreatic Cancer

Posted on January 25, 2017

S. Liu1, J. Lee3, J. Yu1, R. Sanchez1, M. Van Dam3, E. Rozengurt2, F. Brunicardi1  1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Division Of Digestive Disease,Los Angeles, CA, USA 3David Geffen School Of Medicine, University Of California At Los Angeles,Crump Institute For Molecular Imaging,Los Angeles, CA, USA

Background: Recombinant adeno-associated virus type 2 (AAV2) vectors transduce a wide variety of tissues in vivo and provide long-term gene expression with minimal immune responses and no pathological responses to the host, thus making AAV2 an attractive vector for gene delivery in vivo. However, its broad host range limits its usefulness in which transgene expression needs to be limited to a specific organ or cell type, such as cancer. Furthermore, low efficiency of wild type AAV2 (AAV2wt) transfection of pancreatic cancer (PDAC) cells further limits its effectiveness. In this study, we explored the possibility of directing rAAV2 transduction by incorporating a panel of tumor homing peptides to target human PDAC cells and tumors in mice.

Methods: 18 tumor homing peptides (TumorHoPe) were selected from literature and TumorHoPe database for AAV2 capsid display. Synthetic oligos were inserted into the rAAV2 capsid at R588. scAAV-eGFP and scAAV2-Gaussia Luciferase (GLuc) reporters were used. Real time-PCR was used to quantify the AAV2 titers. AAV infection of human PDAC cell lines (PANC-1, Mia PaCa2, Capan-2 and AsPC-1) and benign HPDE cells were performed, followed by peptide competition assays. GLuc secretory activity was determined by bioluminescence assay. In vivo imaging was performed in PANC-1 subQ xenograft using systemic delivery of AAV2TumorHoPe-GLuc imaging vector via tail vein.

Results: 18 TumorHoPe displayed AAV2  and one mutated AAV2 were created (AAV2TumorHoPe). The greatest viral infection was obtained in PANC-1 by AAV2RGD (CDCRGDCFC), Mia PaCa2 by AAV2RGR (CRGRRST), Capan-2 by AAV2RGD and AsPC-1 by AAV2LGL (RGDLGLS), with no infection of benign HPDE cells (Fig. Upper). There was no significant GFP expression in AAV2WT transfections of PDAC cells. To further identify whether the TumorHoPe display virus targeted PDAC cells and enhancing the infection efficiency, competition assays were performed on each cells infected by virus in the presence of excess exogenous TumorHoPe peptides; infection efficiency dropped to 42.5%, 34.3%, 25.6% and 36.9% in PANC-1, Mia PaCa2, Capan-2 and AsPC-1 cells, respectively. Systemic delivery of AAV2TumorHoPe-GLuc imaging vector in PANC-1 tumor model resulted in highly specific imaging of tumors without toxicity (Fig. Bottom A), whereas control AAV2 vector showed only non-specific transfection with none seen in the PANC1 tumor (Fig. Bottom B).

Conclusion: This study demonstrates that TumorHoPe display on AAV2 surface enhances viral targeting of PDAC cells and tumor, but not benign HPDE cells. The unique type of TumorHoPe on the AAV2 surface contributes to specific efficiency in PDAC cells. These preclinical data suggest that TumorHoPe AAV2 gene delivery could be used for targeted imaging of PDAC.

24.08 Cancer Stemness In Bone Marrow Micrometastases Of Human Breast Cancer

Posted on January 25, 2017

M. C. Kuo1, A. N. Kothari1, Z. Mi1  1Loyola University Chicago Stritch School Of Medicine,Surgery,Maywood, IL, USA

Introduction: Cancer cells metastasize to the bone marrow (BM) to create the pre-metastatic niche. Cancer stemness (or expression of stem cell characteristics) is regulated by the tumor microenvironment (TME) and associated with self renewal, clonal maintenance, and poor clinical outcomes. Osteopontin (OPN) induces mesenchymal stem cells (MSC) in the TME adopt a cancer-associated fibroblast (CAF) phenotype to potentiate cancer growth and metastasis. The mechanisms by which cancer cells and TME regulate stemness in the BM pre-metastatic niche are unknown.  

Methods: Human breast cancer cell lines, MB231-Luc (OPN+) and MCF7-Luc (OPN-), were used in an orthotopic murine xenograft model. 6-week-old female NOD scid mice were implanted with 2×106 tumor cells in the presence and absence of human MSC-GFP cells in the R4 position of the mammary fat pad. In selected instances,  MCF7-Luc transfected to express OPN (MCF7[lvOPN]) were co-implanted instead of MCF7-Luc. OPN aptamer (APT), which blocks and inactivates extracellular OPN, and/or inactive mutant APT (muAPT) were utilized. After 8 weeks, animals were sacrificed and femoral BM isolated. Stem cell markers, Sox2, Oct4 and Nanog, and CAF markers, SMA and vimentin, were measured by RT-PCR using human specific primers and normalized to Luc and GFP mRNA, respectively. Relative cell number was determined by titrating cell number to Ct value of GFP or Luc in vitro. Each treatment had 3-5 mice. Statistical analysis was performed using Student’s t-test; p-values < 0.05 were considered significant.

Results: 
In BM from MB231+MSC, expression of Sox2, Oct4 and Nanog was 4x greater than MB231 alone.(p<0.05) Administration of APT to block OPN decreased Sox2, Oct4 and Nanog to levels equivalent to MB231 alone and MB231+MuAPT. Total MB231 cell numbers were not different. In contrast, although CAF markers were not different among all treatments, CAF numbers in BM were 15-fold greater in MB231+MSC vs MB231 alone and MB231+MSC+APT.(p<0.05) In parallel gain-of-function studies, MCF7-Luc (which do not express OPN) co-implanted with MSC did not express increased stem cell markers in BM when compared to MCF-Luc alone. In contrast, using MCF7 expressing OPN, MCF7[lvOPN]+MSC had 5x greater Sox2, Oct4 and Nanog expression.(p<0.05) APT with MCF7[vOPN]+MSC ablated the increase in stem cell markers. Again, CAF markers were unchanged among treatments, but CAF numbers were 70x higher in MCF7[lvOPN] + MSC vs MCF7+MSC and MCF7[lvOPN]+MSC+APT.(p<0.05)

Conclusion:   

In this xenograft model of human breast cancer, our results indicate: 1) both breast cancer cells and accompanying CAF from the primary site metastasize to the BM to form the pre-metastatic niche, 2) tumor-derived OPN mediates CAF migration to the BM, and 3) cancer cells exhibit significantly increased stemness in the presence of CAF in the BM. We conclude that OPN-dependent migration of CAF is required for increased cancer cell stemness in the BM pre-metastatic niche.   

24.07 Nucleotide Depletion by Autophagy Inhibition Sensitizes Kras-driven PDAC to Replication Stress

Posted on January 25, 2017

I. A. Elliott1, J. L. Williams2, R. Ghukasyan1, C. C. Matsumura1, N. Wu1, L. Li1, W. Kim3, S. Poddar3, E. R. Abt3, A. M. Dann1, H. DeRubertis1, D. Braas4, T. M. Le3, C. G. Radu3, T. R. Donahue1,3  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2Harbor-UCLA Medical Center,Department of Surgery,Torrance, CA, USA 3University Of California – Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA 4University Of California – Los Angeles,Metabolomics Center,Los Angeles, CA, USA

Introduction:
Autophagy is a critical source of nucleotides, which are rate-limiting for Ras-driven cancer cell proliferation. Genetically disabling autophagy impairs energy and redox homeostasis by depletion of nucleotide pools, and sensitizes cancer cells to radiation. We hypothesized that the autophagy inhibitor chloroquine (CQ) would deplete deoxyribonucleotide triphosphate (dNTP) availability for incorporation into DNA, thus sensitizing cancer cells to replication stress and revealing a targetable liability in Ras-driven pancreatic ductal adenocarcinoma. 

Methods:
Human (MiaPaCa-2) and murine (KPC) PDAC cells were treated with CQ +/- the replication stress response (RSR) inhibitor VE822. S-phase cells were labeled by pulsing with 5’-ethynyl-2’-deoxyuridine (EdU) and cell cycle progression measured by flow cytometry. Newly synthesized dNTP incorporation into DNA was measured via [13C6]glucose labeling and our novel liquid chromatography mass spectrometry (LC-MS) platform. Global metabolomics analyses were performed by detection of relative amounts of metabolites using LC-MS. Western blots (WB) were done on cell lysates. Cell viability was measured by Cell-Titer-Glo assay. MiaPaCa-2 or KPC cells were injected s.q. in the flanks of NSG or C57BL/6 mice. Mice were treated 5x/week with CQ+/-VE822 (60mg/kg p.o.). Immunohistochemistry (IHC) was performed on explanted KPC tumors after 3 days of CQ+VE822.

Results:
We found that CQ caused S-phase arrest in MiaPaCa-2, and impaired incorporation of newly synthesized dNTPs into replicating DNA(Fig.1a), indicating nucleotide pool insufficiency. Global metabolomic profiling of MiaPaCa-2 revealed that this was due to depletion of purine and pyrimidine substrates for dN synthesis under CQ, and this was exacerbated by addition of the RSR inhibitor VE822(Fig.1b). We then tested the impact of this nucleotide depletion on cell fate; CQ+VE822 treatment led to synergistic induction of DNA damage (reflected by pH2A.X WB), and decreased viability of MiaPaCa-2 and KPC in vitro (Fig.1c). We also observed increased IHC staining for pH2A.X in KPC tumors and impaired growth of MiaPaCa-2 tumors after CQ+VE822 treatment in vivo. Finally, we confirmed the ability of CQ to induce oxidative stress as indicated by HO-1 levels on WB. Management of redox balance and DNA damage are critical to recovery from radiation; accordingly, we found that CQ+VE822 profoundly impaired survival after radiation of MiaPaCa-2 and KPC cells.

Conclusion:
Pharmacologic inhibition of autophagy by CQ impairs incorporation of dNTPs into DNA by depleting substrates for nucleotide biosynthesis. When combined with an RSR inhibitor, this leads to induction of DNA damage and synthetic lethality in PDAC cells in vitro and in vivo.

23.09 Stored Erythrocytes Alter Endothelial Cell Tissue Factor Expression in Mice

Posted on January 25, 2017

A. D. Jung1, Y. Kim1, W. Abplanalp1, A. T. Makley1, M. D. Goodman1, T. A. Pritts1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA

Introduction:  Transfusion with packed red blood cells (pRBCs) remains essential for resuscitation after hemorrhage. During storage, pRBCs undergo a series of physical and biochemical changes that are collectively known as the red blood cell storage lesion. Previous work from our laboratory shows that this is associated with harmful effects in the transfusion recipient, including alterations in the coagulation cascade. Tissue factor, a constitutively expressed cell-surface glycoprotein, plays a key role in initiating the coagulation cascade, but the effect of transfusion of stored pRBCs on endothelial tissue factor expression is unknown. We hypothesized that aged pRBCs alter endothelial cell tissue factor expression.

Methods:  Blood was harvested from murine donors, then separated into components. Leukoreduced and platelet-depleted pRBC units were stored in additive solution-3 at 4°C for 14 days. Microparticles were isolated from aged pRBC units by ultracentrifugation. In a simulated in vitro transfusion model, murine lung endothelial cells were incubated with fresh or 14-day-old erythrocytes suspended in media at 37°C for 1 h and endothelial cell tissue factor expression was analyzed via immunofluorescence. In a separate experiment, endothelial cells were exposed to aged vs fresh erythrocytes, then tissue factor expression was analyzed by Western blot. The third experiment involved exposing endothelial cells to media or isolated microparticles from aged pRBC units, and soluble tissue factor was determined using ELISA at 0.5, 6, 12, and 24h. 

Results: Whole cell immunofluorescent analysis of endothelial cells exposed to aged pRBCs demonstrated significant decreases in tissue factor expression vs baseline (11.7±0.7 vs 16.9±0.6 RFI, p<0.05). Western Blot analysis also demonstrated a significant decrease in tissue factor expression as compared to baseline. Treatment of endothelial cells with microparticles isolated from stored PRBC units resulted in a significant attenuation in tissue factor concentration at 30 minutes (4.3±0.4 vs 6.2±0.8, p<0.05), 6 hours (3.63±0.3 vs 5.5±0.6, p<0.05), and 24 hours (2.8±0.6 vs 4.7±0.5, p<0.05) as determined by ELISA.

Conclusion: Treatment of endothelial cells with aged pRBCs attenuates expression of tissue factor. Additional experiments indicate that this process is driven by red blood cell microparticles. Together, these data provide further evidence that transfusion with stored pRBC units causes endothelial cell dysfunction.  

 

23.08 CitH3: a Long-Lasting Blood Biomarker for Diagnosis and Treatment of Endotoxic Shock in Mice

Posted on January 25, 2017

B. Pan1, B. Liu1, E. Chen1, Y. Wang2, Y. Liang1, Y. Li1, H. B. Alam1  1University Of Michigan Hospital,General Surgery/Surgery/Medical School,Ann Arbor, MI, USA 2Penn State University,Department Of Biochemistry And Molecular Biology,University Park, PA, USA

Introduction: Usefulness of many biomarkers is limited by their short half-life in the circulation. We previously showed that citrullinated histone H3 (CitH3) is a potential diagnostic biomarker and mediator in sepsis, but it is unclear if it persists in blood long enough to be clinically useful. The present study was designed to test the hypothesis that circulating CitH3 is a long-lasting biomarker, and that treatment with YW3-56, a peptidylarginine deiminase-4 (PAD4) inhibitor, can improve survival in a lethal lipopolysaccharide (LPS) induced shock model while attenuating blood levels of CitH3.

Methods: Three experiments were carried out. In experiment I, CitH3 specific enzyme-linked immunosorbent assay (ELISA) was established by our laboratory. A 96-well plate was coated with CitH3 monoclonal antibody overnight, incubated with mouse serum for 2 hours (h) followed by incubation with CitH3 polyclonal antibody and HRP-linked anti-rabbit antibody.  Synthesized CitH3 was used to generate a standard curve (0–20 ng/ml). In experiment II, C57BL/6J mice were randomized to the following 3 groups with intraperitoneal injection of different reagents (n=7/group): (1) Dimethyl sulfoxide (DMSO), (2) LPS (35 mg/kg) +DMSO, and (3) LPS+YW3-56 (5 mg/kg) dissolved in DMSO (post LPS). Survival was monitored for 10 days. In experiment III, mice were subjected to the same treatment as the experiment II, and blood samples were collected at 0, 0.5, 3, 12, and 24 h after treatment. Plasma CitH3 were measured by both Western blot and ELISA. ANOVA was used for multiple comparisons and Kaplan-Meier curves for survival.

Results: All mice in the DMSO group survived. The LPS injection was universally lethal, with the majority of deaths within 24 h. YW3-56 treatment significantly improved survival compared to the LPS alone group (p<0.0001). No circulating CitH3 was found in DMSO group. LPS injection was associated with elevated plasma CitH3 at 0.5, 3, 12, and 24 h (87pg/ml, 245 pg/ml, 1180 pg/ml, and 318 pg/ml, respectively) with peak level at the 12-h time point. YW3-56 treatment significantly attenuated LPS-induced CitH3 levels in blood (13pg/ml, 73pg/ml, 183pg/ml, and 3pg/ml, respectively), compared to the untreated LPS group.  The elevated CitH3 was detectible by Western blot and ELISA, but only ELISA could accurately quantify the concentration of blood CitH3 (Figure 1).  

Conclusion: Our study demonstrated for the first time that (1) the CitH3 specific ELISA developed by us is a more reliable and accurate quantitative method than Western blot; (2) CitH3 protein in the peripheral blood is an ideal biomarker for sepsis because it responds to an insult early (0.5 h), lasts long, and is responsive to therapeutic interventions.

 

23.04 Post-Shock Mesenteric Lymph Exosomes are Key Mediators of Acute Lung Injury

Posted on January 25, 2017

M. Kojima1, T. Chan1, J. Gimenes1, B. Eliceiri1, A. Baird1, T. Costantini1, R. Coimbra1  1University Of California – San Diego,Division Of Trauma, Surgical Critical Care, Burns And Acute Care Surgery/Department Of Surgery,San Diego, CA, USA

Introduction: Acute lung injury (ALI) is a common cause of morbidity in patients following severe injury. Studies have shown that mesenteric lymph (ML) carries gut-derived inflammatory mediators to the lung and serves as the inciting event in ALI. Alveolar macrophages (AM), a lung resident macrophage, play a critical role in the development of ALI. We have recently demonstrated that exosomes, nano-sized extracellular vesicles, are present in ML and have the ability to trigger NF-κB activation in human monocytic cells in vitro. We hypothesized that exosomes in post-injury ML induce pro-inflammatory cytokine production in AM and contributes to post-injury ALI. 

Methods: Male rats underwent cannulation of the femoral artery, jugular vein and ML duct prior to trauma/hemorrhagic shock (T/HS; mean arterial pressure 35 mmHg for 60 min), followed by resuscitation with shed blood and two times normal saline. The ML was collected before hemorrhagic shock (pre-shock) and after T/HS (post-T/HS) for isolation of exosomes by differential centrifugation. In vitro AM were stimulated with exosomes harvested from pre-shock or post-T/HS ML for 6 or 12 hours for measurement of cytokine production by quantitative reverse transcription PCR (qRT-PCR). ML Exosomes from each experimental group (2 x 109 exosomes/g) were also intravenously injected into male naïve C57BL/6 mice to assess in vivo biologic activity. Lung injury was evaluated by measuring histologic lung injury, vascular permeability and immune cell recruitment in bronchoalveolar lavage (BAL) and lung tissue. 

Results:  Exosomes released into post-T/HS ML increased the gene expression of pro-inflammatory cytokines (TNF-a and CINC-1; see figure), NF-kB (5-fold increase at 6h vs. pre-shock; p<0.001) and iNOS (4-fold increase at 12h vs. pre-shock; p<0.001) in AM in vitro. Compared to pre-shock ML exosomes, the in vivo injection of post-T/HS ML exosomes resulted in increased histological lung injury score, a 2-fold increase in Evan’s blue dye lung permeability (0.058 ± 0.003 to 0.117 ± 0.011 mg/g tissue; p<0.05) as well as an increase of wet-to-dry ratio (4.608 ± 0.071 to 5.202 ± 0.149; p<0.05). Exosomes in post-T/HS ML also induced increased recruitment of neutrophils (CD45+Ly-6G+) and macrophages (CD45+CD11c+) in BALF and lung parenchyma determined by flow cytometry (p<0.05 vs. pre-shock).   

Conclusion:  Exosomes released into post-T/HS ML caused an inflammatory response from AM in vitro and ALI in vivo. Our findings define the critical role of ML exosomes as a biologically active mediator of ALI after severe injury.

 

23.03 FER Gene Delivery Improves Survival by Enhancing Lung Immune Response Against Lethal Pneumonia

Posted on January 25, 2017

L. K. McCandless1, Y. S. Yin1, V. Dolgachev1, S. Panicker1, M. Suresh1, M. Hemmila1, K. Raghavendran1, D. Machado-Aranda1  1University Of Michigan,Division Of Acute Care Surgery/Department Of Surgery,Ann Arbor, MI, USA

Introduction: Alternative treatments against antibiotic-resistant bacteria are being strongly investigated. Potentially, transient manipulations of the genome could induce a more efficient immune response. A recent Genome-wide Association Study demonstrated correlation of FER gene expression with survival among pneumonia (PNA) patients. This was confirmed by electroporation-mediated delivery of FER gene in a lethal murine model of combined lung contusion (LC) and PNA. Neither study was conceived to determine the mechanisms for these favorable outcomes. We propose that FER improves survival by recruitment of activated immune cells primed to remove bacteria from the lung.

Methods: C57/BL6 mice were inoculated with 500 CFU of Klebsiella pneumoniae. At 1-hr they received plasmid DNA encoding human (pFER) gene via pharyngeal drop followed by 8 electroporation pulses (EP)  inducing its expression in the lung. We recorded survival data for pFER-EP and control groups (PNA-only; PNA/EP-empty vector and PNA/EP-Na+/K+-ATPase gene). In parallel experiments, animals were sacrificed at specific time points (24, 48, 72 hr), bronchial alveolar lavage (BAL) fluid and lung tissues were harvested; cellular subpopulations counted by flow cytometry; specific genes and signaling pathways were assessed by TaqMan/Western Blot and finally cytokines by ELISA.

Results: After pFER-EP; 5-day survival was markedly improved compared to PNA-only control (80% vs 20%; p < 0.05). Early significant numbers of inflammatory monocytes were only detected in BAL fluid from pFER-EP animals exhibiting known antibacterial markers Toll Receptor- 2 and 4 (TLR2 and TLR4) respectively; being the later more predominant. Both BAL cells and lung tissues had higher protein expression of phosphorylated STAT3 (p-STAT3), a transcription factor critical in bacterial removal. The increased levels of p-STAT3 correlated with the decreased expression of its chaperone Heat-Shock Protein 90 (HSP90). However at 72 hr, HSP90 and other proteosome genes (NRLP2, NRF2) dramatically increased to counter-regulate p-STAT3, inducing monocyte apoptosis and avoiding damage to surrounding tissues.  BAL TLR4+ monocytes were found to highly express Nitric oxide synthase-2 (Nos-2), Resistin-like molecule α1 (Fizz1), Tumor Necrosis Factor-α (TNFα) and Interferon-γ  (IFNγ) all important against bacterial infection. Additionally, pFER-EP was able to rescue neutrophilic response in TLR4-/- mice, via increased production of strong chemoattractant KC and counterbalanced by decoy receptor for advance glycosylation (sRAGE), and independent from TNFα  and IFNγ levels.

Conclusion: Lung gene delivery of the FER improved pneumonia survival by early STAT3 phosphorylation and suppression of HSP90, in turn enhancing antibacterial TLR4+ monocytes. Additionally, FER expression can modulate neutrophilic response via KC and sRAGE cytokines independently from TLR4, TNFα and IFNγ, thus constituting a novel therapeutic strategy.  

22.09 Effect of collagen substrates on oxidative stress-induced changes in SMC phenotype in BAV aortopathy.

Posted on January 25, 2017

P. G. Chan1, M. Billaud1, J. Phillippi1, T. Gleason1  1University Of Pittsburgh,Cardiothoracic Surgery,Pittsburgh, PA, USA

Introduction: Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation and is associated with ascending aortopathy in form of dissection or aneurysmal disease which involve extensive collagen remodeling.  Our prior studies have uncovered that the smooth muscle cells (SMC) from the ascending aorta of BAV specimens have decreased oxidative stress defense compared to SMCs from patients with tricuspid aortic valve (TAV).  We hypothesize that oxidative stress-induced alterations in SMC phenotype can be alleviated by collagen substrates in TAV SMCs but not in BAV SMCs.

Methods: Aortic tissue was obtained from patients undergoing aortic surgery or heart transplantation with IRB approval and informed patient consent.  SMCs from TAV (n=2) and BAV (n=3) patients were harvested using previously established protocols.  SMCs were seeded at a density of 2.8×103/cm2 on tissue culture polystyrene (TCP) in the presence or absence of type 1 collagen substrate.  SMCs were cultured in the presence or absence of 20µM tert-butyl hydroperoxide (tBHP) and monitored in three random fields per well using phase contrast time-lapse microscopy in a stage-top incubation chamber for up to 12 hours.  Post-imaging, the aspect ratios (long axis/short axis) of cells (>17 cells/well) were measured at period time points as a means to assess SMC phenotype.

Results: Aspect ratio for SMCs isolated decreased in the presence of tBHP from 4.4±0.81 to 2.4±0.17 (p<0.001) for TAV SMCs and from 5.0±0.46 to 2.3±0.52 (p<0.001) for BAV SMCs.  When cultured on Type 1 collagen coated wells, TAV SMCs maintained a high aspect ratio despite exposure to oxidative stress.  TAV SMCs increased in aspect ratio on collagen to 6.2±1.55 which was relatively maintained at 4.8±0.83 (p=0.557) when exposed to tBHP.  The aspect ratio of BAV SMCs cultured on collagen was found to be decreased under tBHP treatment (3.88±0.38 vs. 2.1±0.32, p<.001) (Figure 1).

Conclusion: Oxidative stress was found to alter SMC phenotype by decreasing aspect ratio. Culture on collagen prevented oxidative stress induced alteration for TAV SMCs, but not for BAV SMCs.  These results provide further support that BAV SMCs have impaired basal oxidative stress response compared to TAV SMCs despite modulating the extracellular milieu.  Ongoing work is focused on how the extracellular matrix interplays with oxidative stress mediated effects on SMC function in BAV aortopathy.  Ultimately, with this knowledge, we hope to develop therapeutics for patients who are at risk for aneurysmal progression, delineate the molecular mechanisms associated with BAV aortopathy, and improve rupture risk predictions and recommendations for surgical or medical intervention.

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