01.08 Potential of Liver Organoids as Implantation Therapy for Liver Insufficiency

Posted on January 18, 2016

V. X. Zhou1, M. Lolas1, T. T. Chang1 1University Of California – San Francisco,Surgery,San Francisco, CA, USA

Introduction: Liver transplantation is currently the only treatment for end-stage liver disease (ESLD) and is limited by the critical shortage of donor organs. Implantation of liver organoids generated ex vivo by three-dimensional (3D) culture techniques may serve as an adjunct or alternative to liver transplantation. Efficient high-engraftment methods of introducing organoids into the liver parenchyma have not been established. In this study, we aim to develop a reliable surgical technique to implant liver organoids into the liver and assess their ability to engraft and function.

Methods: Liver organoids composed of either hepatocytes alone or hepatocytes co-cultured with stellate cells were generated by 3D culture in rotating wall vessel bioreactors. Primary hepatocytes and stellate cells were isolated from ROSA26 C57BL/6 mice, in which β-galactosidase was expressed under a ubiquitous promoter, so that engrafted cells could be easily identified by X-gal staining when implanted into wild-type mice. Some recipient mice underwent simultaneous two-thirds partial hepatectomy to determine whether hepatocytes within implanted organoids proliferated in response to acute liver insufficiency. As control, freshly isolated primary hepatocytes were transplanted as single-cell suspensions. At various time-points after implantation, engraftment of liver organoids was qualitatively and quantitatively determined.

Results: Confocal microscopy analysis demonstrated that hepatocytes within liver organoids had cortical actin organization and produced extracellular matrix. Organoids maintained their 3D structure up to 7 days after implantation within recipient livers and engraftment efficiency was superior to that of single cells. Generating liver organoids with stellate cell co-culture and performing two-thirds partial hepatectomy on recipient mice did not appear to improve engraftment efficiency.

Conclusion: Implantation of liver organoids generated by 3D tissue culture is promising for development as a novel surgical therapy for ESLD. Further optimization of 3D culture and implantation techniques are required to increase engraftment efficiency.

01.04 SDF-1α Decreases Inflammation in Diabetic Dermal Fibroblasts by Upregulating miR-146a Expression

Posted on January 18, 2016

M. M. Hodges1, C. Zgheib1, J. Hu1, J. Xu1, K. W. Liechty1 1University Of Colorado Denver,Laboratory For Fetal And Regenerative Biology, Department Of Surgery,Aurora, CO, USA

Introduction: In 2014, the United States Center for Disease Control and Prevention (CDC) estimated that 29.1 million Americans (9.3% of Americans) were living with diabetes, with the economic burden of diabetes related wound care estimated as $38.6 billion in 2007. With the global prevalence of diabetes expected to double between 2000 and 2030, there will be an increasing need for innovative technologies to address the commensurate rise in chronic wounds. We have previously demonstrated that diabetic wounds exhibit markedly decreased levels of the anti-inflammatory microRNA miR-146a. Furthermore, we have shown improved healing of diabetic wounds after treatment with a lentiviral construct expressing stromal derived growth factor (SDF-1α). We hypothesize that the improved healing observed in diabetic wounds after SDF-1α treatment is partly due to increased miR-146a expression and the subsequent decrease in inflammation.

Methods: To test our hypothesis we isolated dermal fibroblasts from the skin of 10 week old diabetic (Db/Db) and non-diabetic (Db/+) mice. These fibroblasts were then cultured and transfected with a lentivirus expressing either SDF-1α or green fluorescent protein (GFP) as a control. RNA was extracted from these fibroblasts, and real time PCR analysis was utilized to quantify the gene expression of NFkB, TRAF6, IRAK1, IL-6, MIP2 (IL-8), and miR-146a.

Results: When compared to fibroblasts from the skin of non-diabetic mice, fibroblasts isolated from the skin of diabetic mice demonstrated significantly decreased levels of miR-146a and increased levels of NFkB, TRAF6, IRAK1, IL-6, and MIP2 (IL-8). When transfected with a lentivirus expressing SDF-1α, fibroblasts from the skin of diabetic mice demonstrated significantly increased expression of miR-146a, and significantly decreased expression of NFkB, TRAF6, IRAK1, IL-6, and MIP2 (IL-8).

Conclusions: The significantly increased expression of inflammatory mediators NFkB, TRAF6, IRAK1, IL-6, and MIP2 (IL-8) observed in diabetic fibroblasts is nearly reversed with lenti-SDF-1α transfection. Similarly, the decreased level of miR-146a observed in diabetic fibroblasts is completely reversed when diabetic fibroblasts are transfected with lenti-SDF-1α. The accelerated wound healing previously observed in diabetic skin after lenti-SDF-1α treatment may be due in part to the increased expression of miR-146a and decreased expression of inflammatory mediators NFkB, TRAF6, IRAK1, IL-6, and MIP2 (IL-8). These results support further investigation of the role of SDF-1α in the pathogenesis of diabetic wound healing impairment and as a possible therapeutic target in the treatment of diabetic wounds.

01.05 Mechanistic Analysis: Alcohol Induces Apoptosis and Autophagy in the Liver of Hypercholesterolemic Swine

Posted on January 18, 2016

I. J. Lawandy1, B. A. Potz1, N. Y. Elmadhun1, A. D. Lassaletta1, J. A. Feng1, F. W. Sellke1 1Brown University,Surgery/Cardiothoracic Surgery/ Warren Alpert Medical School,Providence, RHODE ISLAND, USA

Introduction: Autophagy serves as a cellular protective mechanism against alcohol induced tissue injury but can also be detrimental leading to apoptosis. Our lab has previously shown moderate alcohol consumption in a swine model of metabolic syndrome worsens glucose metabolism by altering activation of the insulin signaling pathway in the liver. We examined the effect of alcohol consumption on apoptosis and autophagy signaling in the liver in our clinically relevant animal model of chronic myocardial ischemia and hypercholesterolemia.

Methods: Twenty-six Yorkshire swine were fed a hypercaloric, high-fat diet for 4 weeks then split into 3 groups: hypercholesterolemic diet alone (HCC, n= 9), hypercholesterolemic diet with vodka (HCV, n= 9), and hypercholesterolemic diet with wine (HCW,n = 8) for 7 weeks. Animals underwent euthanasia and liver tissue samples were harvested for analysis.

Results: There were significant increases in the pro-apoptotic proteins caspase 3, caspase 8, caspase 9 and cleaved caspase 9 in the HCV group and a trend increase in the HCW group compared to control. There was a significant decrease in pro-apoptotic protein Bad in the HCW and HCV groups compared to control. There was a significant increase in anti-apoptoic signal BCL-2 in the HCW group compared to the HCV group and trend increase in the HCW group compared to control. There were significant increases in pro-survival proteins AKT and p-AKT in the HCW and the HCV group compared to control. There was significant increase in MTOR in HCW compared to HCV and a trend increase in compared to control. There was a significant increase in p-MTOR in the HCV group compared to the control. There was a significant decrease in autophagy protein LCB-3 in the HCW and a trend decrease in HCV compared to the control. (See table)

There was a trend increase in anti-apoptotic signal p-BCL-2 in the HCW and HCV groups (p=0.151) compared to control. There were trend increases in the HCV group in AMPKA (p= 0.071) and TNF-alpha (p=0.071) compared to control. There was a trend increase pro- autophagy protein Beclin 1 in the HCW group (p=0.431) compared to control. There were trend increases in pro-autophagy proteins Lamp-1 and Lamp-2 in the HCW group and a trend decrease in the HCV group (p=0.120) compared to control. There were no significant differences in cleaved Caspase 3 (p=0.296), Bax (p= 0.169), p-Bad (p =0.675), LKB-1 (p=0.523), P-AMPKA (p=0.758) or ATG5 (p=0.319) compared to the control.

Conclusions: Moderate Alcohol consumption altered cell survival protein expression related to apoptosis and autophage in pig liver in the setting of hypercholesterolemia. Vodka may induce more pro apoptotic pathways and wine may induce more pro survival pathways in pig liver tissue.

01.02 Novel Nano-Liposomes for Multi-Modal Image-Guided Lung Cancer Surgery

Posted on January 18, 2016

P. Patel1, T. Kato1, H. Ujiie1, D. Lee1, J. Ahn1, H. Hu1, J. Zheng2,3, K. Yasufuku1,2,3 1University of Toronto,Division Of Thoracic Surgery,Toronto, Ontario, Canada 2University Health Network,TECHNA Institute,Toronto, Ontario, Canada 3University of Toronto,Institute Of Biomaterials & Biomedical Engineering,Toronto, Ontario, Canada

Introduction:
To investigate the feasibility of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice.

Methods:
CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human non-small cell lung cancer. At 48 h post-injection (peak liposome accumulation time), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure the fluorescence intensity of tumor and adjacent lung tissue. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation was performed using Hematoxylin & Eosin, and immunofluorescence macrophage marker F4/80 and endothelial cell marker CD31, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections.

Results:
The fluorescence TBR measured when the lung is in the inflated state (2.0 ± 0.58) was significantly greater than in the deflated state (1.42 ± 0.38) (p<0.05). Mean fluorescence signal in tumor was highly variable across samples, (49.0 ± 18.8 AU). CT image analysis revealed greater contrast enhancement in lung tumors (a mean increase of 110 ± 57 HU) when CF800 is administered compared to the non-contrast enhanced tumors (p <0.05).

Conclusion:
This preliminary data suggests that the high fluorescence TBR demonstrated with the SPY system, and CT tumor contrast enhancement provided by CF800 may have clinical utility in aiding delineation and localization of lung cancer during CT and NIR image-guided surgery.

01.03 Tumor-Targeting Nanotheranostic Micelles for Neuroendocrine Cancer Therapy

Posted on January 18, 2016

R. Jaskula Sztul1,3, G. Chen2, A. Harrison1, S. Gong2, H. Chen1,3 1University Of Wisconsin,Surgery,Madison, WI, USA 2University Of Wisconsin,Wisconsin Institutes For Discovery,Madison, WI, USA 3University Of Alabama,Surgery,Birmingham, Alabama, USA

Introduction: Although neuroendocrine tumors (NETs) are slow growing, they are frequently metastatic at the time of their discovery and no longer amenable to curative surgery. Therefore, there is a great need to develop novel therapeutic strategies both to reduce tumor burden and control the release of hormones. To address this need, we developed and optimized a family of novel multifunctional upconversion nanoparticle (UCNP)-based theranostic unimolecular micelles capable of delivering a newly reported anticancer drug AB3 for NET-targeted combination chemotherapy, photodynamic therapy (PDT), and bioimaging. These UCNP-based micelles conjugated with photosensitizer (Rose Bengal, RB) specifically target NET cells using somatostatin analog (KE108). In the current study we assessed the antitumor effects of the nanotheranostic micelles both in vitro and in vivo.

Methods: Stable UCNP-based micelles were prepared in an aqueous solution using multi-arm star amphiphilic block copolymer. KE108 was conjugated for active tumor-targeting. AB3 was loaded into the photosensitive hydrophobic core of the resulting micelles. The effect of 980nm on singlet oxygen generation and in vitro drug release of the UCNP-based micelles were studied. Cell proliferation was assessed by MTT assay in human medullary thyroid cancer cell line (MZ-CRC-1) treated with a family of AB3-loaded micelles (AB3 mM) for 48h with or without 980nm irradiation. The effect of the KE108 targeting ligands on the cellular uptake of the nanotheranostic micelles was measured by flow cytometry and confocal laser scanning microscope (CLSM). The antitumor efficacy of AB3-loaded micelles was determined in GI neuroendocrine (BON) xenografts after two intravenous injections performed with 7 day interval with a dose of 20 mg/kgBW. The group treated with UCNP-based micelles containing RB was irradiated for 15 min with 980nm laser at 4h post injection.

Results: The family of UCNP-based NET-targeting unimolecular micelles was developed for targeted delivery of AB3 and RB to NETs. UCNP-based micelles for targeted and combined chemotherapy with PDT exhibited the strongest antiproliferative effect. Moreover, the targeted micelles exhibited a much higher cellular uptake than non-targeted micelles based on flow cytometry and CLSM analyses. Additionally, AB3 loaded micelles conjugated with RB and KE108 (i.e., T-RB-AB3), enabling combined chemo-therapy and PDT, induced the best antitumor efficacy (82% reduction in tumor volume) and did not cause any significant changes in body weight or survival. Pathological assessment of H&E-stained sections of different organs of mice treated with T-RB-AB3 micelles did not indicate any signs of inflammation or necrotic regions.

Conclusion:The AB3-loaded UCNP-based micelles conjugated with both RB and KE108, offering combination chemotherapy and PDT, are more effective at suppressing NET cell growth while having minimal toxicity to non-NET cells.

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