Purpose: Overexpression of epithelial cell adhesion molecule (EpCAM) correlates with poor prognosis, therapeutic failure and early tumor recurrence in hepatocellular carcinoma (HCC) patients. The tumor microenvironment dictates the fate of tumor-initiating cancer stem cells (CSCs); however, very limited studies were attempted to evaluate CSC tumorigenesis in the liver microenvironment. Here, we have systemically investigated the role of EpCAM+ cancer cells in tumor initiation in orthotopic HCC models. Results: Control mice and the mice with bland steatosis failed to develop tumors. In the mice with steatohepatitis, EpCAM+ CSCs have shown significantly increased ability in terms of tumor initiation and growth, compared to that with EpCAM- non-CSCs inoculation (p < 0.005). For Hep3B inoculation, EpCAM-High group has shown significantly higher tumor growth compared with EpCAM-Low (p < 0.005). For HepG2 inoculation, both EpCAM-High and EpCAM-Low groups confirmed similar tumor incidence and growth. Methods: Diet-induced compromised microenvironments were established to mimic clinical fatty liver and non-alcoholic steatohepatitis (NASH) patients and the tumorigenic capabilities of Hepa1-6 cells were evaluated. CSCs were enriched by spheroid culture and labeled with copGFP for EpCAM+ CSCs and with mCherry for non-CSCs. FACS-sorted cells were inoculated into left liver lobes, and tumor growth was monitored by high-frequency ultrasound. The subpopulations of Hep3B and HepG2 cells in terms of EpCAM-Low and EpCAM-High were evaluated in the orthotopic model of athymic mice. Conclusions: NASH microenvironment promotes the EpCAM+ CSCs initiated tumorigenesis in immunocompetent mouse model. Differential EpCAM expression demonstrates distinct tumor biology in athymic mouse models.

Background: Nonalcoholic steatohepatitis (NASH) is the most severe form of non-alcoholic fatty liver disease (NAFLD) and a potential precursor of hepatocellular carcinoma (HCC). FGF21, with its metabolic benefits, was reported as the liver safeguard to protect from obesity, diabetes, and NAFLD. Recent study showed an exosome based FGF21 inhibition via organs communication to regulate hepatic metabolism. However, the role of the exosome-FGF21 axis in NASH-HCC has never been studied. Aim: To study the exosome based FGF21 metabolic regulation during NASH-HCC carcinogenetic transformation. Methods: NASH-HCC model was induced in FGF21 knockout (KO) mice and wild type (WT) control using methionine-choline-deficient diets (MCD) and diethylnitrosamine (DEN). The exosomes releasing, miRNA and protein contents were analyzed in the primary cultured hepatocytes from the liver tissues of NASH-HCC mice (KO and WT) as well as benign controls. FGF21 was knockdown (KD) by shRNA in a mouse hepatoma cell line (Hepa1-6) and a benign mouse hepatocyte cell line (FL83B) to investigate the potential carcinogenetic signaling related to exosome-FGF21 axis. Result: NASH-HCC model was successfully established with confirmation of the metabolic disorders and liver cancer development. Early HCC detection and increased HCC incidence were found in the KO-MCD mice (P<0.05 versus KO-CD and WT controls). The collected exosomes from primary cultured hepatocytes and cell lines were identified using the antibodies of Tsg101, CD63 and cytochrome C by western blotting and immunofluorescent staining. In the KO-MCD mice, exosomes retained in the cytosol of benign hepatocyte but released increasingly from cancer cell. Similar results were showed in the Hepa1-6KD cells and the FL83BKD cells. Aberrant EMT, and p53 and Wnt signaling was found in the KO-MCD mice as well as Hepa1-6KD cells challenged with free fatty acid. Conclusion: lack of FGF21 plays critical role for the exosome retention in hepatocytes of NASH mice but not in HCC mice. Abnormal traffic of exosomes contribute to the carcinogenetic signaling. This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226. Citation Format: Youxi Yu, Robert C. Martin, Qianqian Zheng, Xiaoju Shi, Xingkai Liu, Wei Guo, Suping Li, Ping Zhang, Yan Li. Lack of FGF21 promotes NASH-HCC transition via exosome-mediated carcinogenetic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4373.

Irreversible electroporation (IRE) has been demonstrated as an effective local method for locally advanced (stage 3) pancreatic adenocarcinoma. Immune regulatory T cells (Tregs) induce immunosuppression of tumors by inhibiting patients’ anti-tumor adaptive immune response. This study aimed to evaluate the immunomodulation effect of IRE to identify an ideal time point for potential adjuvant immunotherapy.

Abstract Introduction: Hepatocellular carcinoma (HCC) attains resistance to anti-cancer drugs which accounts for most of the therapeutic failures, and is one of the major factors for poor prognosis and poor overall survival of HCC patients. Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation, drug resistance and recurrence in tumor microenvironment. We hypothesize that canonical Wnt/β-catenin mediated CSC enrichment is responsible for acquired drug resistance in HCC. We aimed to study HCC CSCs for aberrant Wnt/β-catenin signaling, possible dedifferentiation, and drug resistance property. Methods: Total n=26 human HCC specimens with adjacent controls were analyzed for EpCAM and β-catenin expression by histology and western blot. In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. CSC enrichment was confirmed by analyzing for established surface markers (EpCAM, CD44, CD133, CD90) and functional markers (Aldeflour assay and Hoechst-33342 efflux). For possible dedifferentiation, we analyzed gene expression profile during enrichment process using qRT-PCR for dedifferentiation and stem cell specific genes. Drug resistance property of CSCs was studied for Doxorubicin and Sorafenib by XTT assay. To investigate CSC activation, Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets (C-Myc,Cyclin-D1,TCF1) and findings were confirmed by β-catenin knockdown, inhibitors of Wnt pathways (LiCl, XAV939, FH535) and TOP flash reporter assay. To study in vivo tumorigenesis of HCC CSCs, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells for possible lineage tracking. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Human specimens showed concomitant EpCAM and beta-catenin expression. Spheroid forming HCC CSCs showed significant higher β-catenin and EpCAM expression, and acquired drug resistance compared with control HCC cells. Silencing β-catenin by knockdown abolish drug resistance property of CSCs for Doxorubicin and reverse spheroids to adherent phenotype. Our in vivo findings confirmed EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p<0.005). Conclusions: Our findings suggest that possible dedifferentiation by canonical β-catenin confer drug resistance and CSC properties to HCC cells. Sorted EpCAM+ CSCs showed very aggressive in vivo tumor initiation and growth in immunocompetent orthotopic mouse model. This EpCAM+ CSCs population could potentially responsible for HCC recurrence and therapeutic failure. Citation Format: Harshul Pandit, Yan Li, Gouzhen Cui, Qianqian Zheng, Suping Li, Robert Martin. Wnt/β-catenin mediated enrichment of EpCAM positive cancer stem cells promote drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1120.

Background: The purpose of this study is to compare the ablation performance between a synchronous microwave ablation (MWA) system and a commercially available asynchronous system in ex vivo bovine liver and evaluate the efficacy of ablation at varying entrance angles. Materials and methods: Two 915-MHz MWA systems were used in bench top ex vivo bovine livers with various conditions (synchronous versus asynchronous). Using synchronous technology ablations to liver, kidney, or lung at angles of 0, 15, 30, and 90 degrees were evaluated. Results: Synchronous and asynchronous MWA systems created mean ablation zone volumes of 26.4 and 15.8 cm(3), 62.9 and 45.4 cm(3), 90.8 and 56.4, and 75.7 and 54.8 cm(3) with single, double (2 microwave probes in use simultaneous) (2 cm spacing), and triple (three probes in use simultaneously) (2 cm and 3 cm spacing) antennae configurations, respectively; adjusted P-values <= 0.006. Ablation defects were similar across all groups when evaluated for entrance angle. Specifically, when comparing 0-degree angle to all other angles, achieved zones of ablation (ZA) were similar (mean +/- standard deviation for 0-degree versus all other angles: 8.72 +/- 4.84 versus 9.38 +/- 4.11 cm(2), P = 0.75). The use of the long-tip probe resulted in a statistically significant increase in the achieved ZA when compared to the short tip probe (10.9 +/- 4.3 versus 6.5 +/- 2.4, respectively; P = 0.01). Conclusions: Newly developed synchronous microwave technology creates significantly larger ablation zones when compared to an existing asynchronous commercially available system. The angle of approach does not affect the resulting ZA. This is clinically relevant as true 0-degree angle is often difficult to obtain. (C) 2018 Elsevier Inc. All rights reserved.

Abstract Introduction: Combining gemcitabine (gem) with irreversible electroporation (IRE) has been shown to result in increased tumor cell apoptosis in a murine model of pancreatic adenocarcinoma (PDAC). However, it remains unclear whether this effect can be augmented locally and systemically by utilizing more active chemotherapies. The objective of this study was to evaluate the efficacy of IRE+FOLFIRINOX (FOL: 5-FU, leucovorin, irinotecan, and oxaliplatin). Methods: Athymic nude mice underwent intrapancreatic injection with S2-013 PDAC cells. After 7-10 days, tumors were confirmed with ultrasound and mice were treated with chemotherapy (gem or FOL), IRE, or chemotherapy+IRE. Four hours after last chemotherapy, pancreas was evaluated histologically. Levels of irinotecan and its active metabolite (SN-38) were evaluated in organs of mice treated with FOL and IRE+FOL. Experiments were repeated and survival analyses performed to evaluate long-term efficacy. Pancreas was harvested for histology and evaluation of apoptotic machinery, cell cycle proteins, and tumor cell proliferation. Results: IRE+FOL (ECT) resulted in increased tumor cells apoptosis compared to gem, gem+IRE, or FOL (apoptotic index (AI) 34.6% vs. 3.8%, 7.3%, 5.8%, respectively, p<0.001). High performance liquid chromatography (HPLC) demonstrated a 1.5-2-fold increase in irinotecan and SN-38 in the pancreas and a 4-fold increase of both compounds in the liver in mice treated with ECT vs. FOL. ECT significantly improved overall survival when compared to mice treated with IRE or FOL (median 7 days ECT vs. 4 days IRE vs. 3 days FOL, p=0.03). Histology demonstrated increased tumor cell apoptosis at time of death (AI = 34.2% ECT vs. 13.2% IRE vs. 4.4% FOL, p=0.026). Western blot demonstrated an increase in active caspase 3 (0.14 ECT vs. 0.00 IRE vs. 0.03 FOL fold change/GAPDH, p=0.02). ECT resulted in lower cell proliferation compared to IRE or FOL (0.2% vs. 0.3% vs. 0.9, p<0.001). Conclusions: ECT with IRE and FOL causes significant tumor cell apoptosis, decreases tumor cell proliferation, enhances systemic FOL exposure, and improves overall survival. Human Phase 1 trials have been initiated to evaluate safety and efficacy of this therapeutic modality. Citation Format: Neal Bhutiani, Harshul Pandit, Qianqian Zheng, Suping Li, Yan Li, Robert C. Martin. Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4393.

Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/β-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model.

Abstract Background: Hepatocellular carcinoma (HCC) has been increasingly recognized in nonalcoholic steatohepatitis (NASH) before cirrhosis stage and the estimated annual HCC incidence in NASH patients is about 0.3%. Accumulating evidence shows that T helper (Th) 17-Interleukin (IL)-17 axis mediates the transition from NASH to HCC, while fibroblast growth factor 21 (FGF21) exerts pharmacologic efficacy in downregulation of Th17- IL-17 axis. In fact, FGF21 has recently emerged as a promising therapeutic candidate for the treatment of obesity and diabetes. However, the NASH-related carcinogenesis and role of FGF21 played during NASH-HCC transition are largely unknown. Aim: To investigate the effect of FGF21 on Th17-IL-17 axis during the NASH-HCC carcinogenetic transformation and to reveal the potential NASH-HCC mechanism. Methods: NASH-HCC was induced in FGF21 knockout (KO) mice treated with DEN at age 2 weeks at 100 mg/kg body weight (i.p.), and followed feeding with high-fat diet (HFD,60% kcal% fat) or methionine/choline-deficient diet (MCD). In an orthotopic HCC model with inoculation of 106 hepal-6 cells into 10 week-old male C57L/J mice, fenofibrate (50 mg/kg) was administered to induce FGF21. In vitro studies were performed using shRNA-FGF21 knockdown hepatocyte and HCC cell lines. The cellular and molecular events, in turn, white adipose tissue (WAT) lipolysis, insulin resistance (IR), release of free fatty acids (FFAs), Th17 cell infiltration and IL-17A production, and NASH-HCC initiation, were determined both in vivo and in vitro. Results: Early HCC detection and increased HCC incidence were found in the FGF21 knockout (KO) mice (P<0.05 versus wild-type controls). Increased WAT lipolysis, hepatic FFAs uptake, Th17 cells/IL-17A, and aberrant NF-κB and p53 signaling were found in NASH and HCC tissues (adipose, spleen and liver) from in FGF21-KO mice by flow cytometry, immunohistochemistry (IHC) and Western blot. IL-17A significantly induced FFAs uptake in the shRNA-FGF21 knockdown hepal-6 cells and FL83B cells. IL-17A promoted the shRNA-FGF21 knockdown hepal-6 cells spheroid formation through Wnt/β-catenin pathway. Fenofibrate treatment significantly increased PPAR-α and native FGF21 protein levels in the liver tissues, and decreased the tumor volumes. Conclusions: 1) Lack of FGF21 played a key role in hepatic lipid accumulation and subsequent upregulated Th17-IL-17 signaling in liver; 2) lack of FGF21 accelerated the Th17-IL-17 axis promoted NASH-HCC transition; 3) upregulation of native FGF21 not only attenuates NASH, but also prevents HCC carcinogenetic transformation via downregulation of Th17-IL-17 axis. This work was supported by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226. Citation Format: Qianqian Zheng, Harshul Pandit, Xingkai Liu, Youxi Yu, Suping Li, Shelby Turner, Xin Meng, Robert Martin, Yan Li. Lack of FGF21 accelerates the Th17-IL-17 axis-mediated transition from nonalcoholic steatohepatitis to hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5747.

We developed a protocol to investigate and optimize the application of contrast-enhanced ultrasound (CEUS) to non-invasive diagnosis of progressing fatty liver disease in mouse models. Eighteen 4-wk-old male C57 L/J mice were randomly assigned to one of the three groups and placed on a control diet, high-fat diet or non-alcoholic steatohepatitis diet for the next 10 wk. After 14 wk, B-mode imaging and CEUS imaging using a VisualSonics Vevo2100 system were performed. CEUS imaging and data analysis using three different parameters—peak enhancement, wash-in rate and wash-in perfusion index—revealed a significant decrease in representative blood flow in the high-fat diet group versus controls and a further significant decrease in the non-alcoholic steatohepatitis group (p < 0.001; n = 6/group). In conclusion, compared with B-mode imaging, non-targeted CEUS imaging was more sensitive in diagnosing early-stage fatty infiltration-mediated vascularity changes in liver parenchyma and provided a more accurate steatohepatitis diagnosis in mouse models.

Abstract Introduction: Non-alcoholic Steatohepatitis (NASH) is one of the major risk factors for cirrhosis and its progression to Hepatocellular Carcinoma (HCC). Cancer stem cells (CSCs) are sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. Understanding role of CSCs in tumorigenesis was limited to immunocompromised nude/SCID mouse models, and should be investigated in immunocompetent mouse model(s) representing clinical conditions. In this study, (1) we have investigated and confirmed CSCs properties in HCC cells lines, and (2) investigated tumor initiation capability of EpCAM positive CSCs in novel immunocompetent mouse model of HCC. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM,CD90,CD44,CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. To study in vivo tumorigenesis, immunocompetent mouse model was established using C57L/J mouse and copGFP expressing Hepa1-6 cells. Capability of EpCAM CSCs for tumor initiation was tested in 3 diet induced groups, i.e. control (10% dietary fat), high-fat (60% dietary fat), and NASH (60% dietary fat MCD diet with 0.1% methionine). 2 million FACS sorted EpCAM+/+ or EpCAM-/- cells were orthotopically injected into left liver lob. Tumor growth was monitored using high-frequency ultrasound and animals were euthanized after 18 days. Histology and ICC analysis was performed to confirm the tumors and findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression and significant higher chemoresistance compared with control HCC cells. Our in vivo findings confirmed that EpCAM CSCs required NASH microenvironment. In NASH group mice, EpCAM+/+ CSCs have shown significant tumorigenesis compared with no tumors in EpCAM-/- (n=9, p<0.005). Histology analysis of NASH liver tissue confirmed lobular and portal inflammation, hepatocellular ballooning, and fibrosis. Control and high-fat diet group mice failed to develop tumors. Conclusions: We have developed a novel mouse model to study CSCs to overcome confounding limitations of immunocompromised mouse models for lacking functional immune system components. Our findings using immunocompetent mouse model suggest that, in HCC, (1) EpCAM+ CSCs cannot initiate tumor by itself within normal liver microenvironment in the presence of functional immune components, (2) NASH microenvironment promotes EpCAM+ CSCs mediated tumorigenesis while EpCAM-/- non-CSCs subset failed to develop tumor. Citation Format: Harshul Pandit, Yan Li, Guozhen Cui, Suping Li, Salina Li, Robert C. Martin. Non-alcoholic steatohepatitis promotes EpCAM positive cancer stem cells mediated tumorigenesis in immunocompetent mouse model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1909. doi:10.1158/1538-7445.AM2017-1909

Abstract Introduction: Recurrence of Hepatocellular carcinoma (HCC) after definitive tumor treatment occurs in over 70% of patients within 14 months of initial treatment. The recurrent HCC cells demonstrate a more aggressive tumor growth pattern when compared with the initial HCC cells. Recurrent HCC attains resistance to chemotherapy which accounts for most of the therapeutic failures and is one of the major factors for poor overall survival (OS) in this disease. Underlying molecular mechanism(s) responsible for acquired chemotherapy resistance and tumor recurrence has not been well defined. Cancer stem cells (CSCs) are a sub-population of cells that bear stem-like properties, and are believed to contribute in tumor initiation and recurrence in tumor microenvironment. We hypothesize that in HCC, Wnt/β-catenin mediated CSC activation is responsible for acquired chemoresistance. Here, we aimed to study that enriched culture of EpCAM positive HCC cells with cancer stem-cell like properties could become resistant to anti-cancer drugs. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. To confirm CSCs, we analyzed CSC biomarkers (EpCAM, CD90, CD44, CD133) using flow-cytometry and Immunocytochemistry (ICC), and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance property of CSCs was studied using Doxorubicin (anthracycline antibiotic) and Sorafenib (multikinase inhibitor) by MTT assay. Tumorigenic potential of enriched CSCs was studied in orthotopic HCC mouse models. Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets i.e. C-Myc, Cyclin-D1, TCF1. Lentivirus mediated overexpression and knockdown of Wnt/β-catenin signaling components confirmed effect of Wnt/β-catenin signaling on activation of CSCs and drug resistance. Total 24 paired human HCC specimens were obtained and analyzed to confirm in vitro and in vivo findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression (EpCAM+) and significant higher chemoresistance compared with control HCC cells. ABCG2 expression (and its efflux activity) was found to be increased in CSCs, which further supports acquired chemoresistance. EpCAM+ CSCs have shown significant higher tumor proliferation rate in the mouse models compared with control. Wnt/β-catenin signaling activity was found to be increased in EpCAM+ CSCs compared with control, and associated with chemoresistance. Human HCC specimens confirmed concomitant increase/decrease in EpCAM and β-catenin expression. Conclusions: Subpopulation of EpCAM+ CSCs, (1) contribute to acquired chemoresistance in HCC, and (2) showing increased Wnt/β-catenin signaling. We believe, this EpCAM+ CSCs population is primarily responsible for HCC recurrence and therapeutic failure. Citation Format: Harshul Pandit, Yan Li, Guozhen Cui, Xuanyi Li, Suping Li, Yingbin Yang, Jack Rostas, Robert C.G. Martin. EpCAM-positive cancer stem cells acquired chemoresistance in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2535.

Background Human fibroblast growth factor 19 (FGF19), its receptor (FGFR4) and EpCAM play an important role in cell proliferation, differentiation, motility, and overexpression have been linked to hepatocellular carcinoma (HCC). The aim of this study was to evaluate the FGF19 signals responsible for the progression of HCC arising from fatty liver. Results FGF19 level was significantly increased in the HCC patients' serum compared to non-HCC controls. The IHC results demonstrated significant increases of protein expressions of FGF19, FGFR4 and EpCAM in specimens with fatty liver, NASH, cirrhosis, and HCC compared to healthy liver tissue. There was a significant positive correlation between the protein expressions (FGF19, FGFR4, and EpCAM) and histopathologic changes from FL to HCC. Furthermore, FGF19 was positively correlated with FGFR4 and with EpCAM. Materials and Methods FGF19 protein levels in serum and tissues were determined by ELISA assay. The FGFR4, and EpCAM expression and tissue distribution were further evaluated by immunohistochemical staining in tissue array samples. FGF19, FGFR4 and EpCAM expressions between the different histologic stages of fatty liver steatohepatitis-cirrhosis-HCC carcinogenesis sequence were compared to healthy hepatic tissue. Conclusions Overexpression of FGF19/FGFR4 significantly correlated with EpCAM as a marker of hepatic cancer stem cells within the fatty liver-steatosis-cirrhosis-HCC sequence. Impact This is the first study to elucidate FGF19/FGFR4 signaling in favor of HCC cells developing as indicated by increased EpCAM within the carcinogenesis sequence from fatty liver to hepatocellular carcinoma. Our study has the potential to yield novel and cost effective screening strategies for HCC patients.

Introduction: Irreversible electroporation (IRE) utilizes short, high-voltage pulses to irreversibly permeabilize the cell membrane, resulting in apoptotic cell death. In addition to the irreversible zone, IRE creates a reversible zone that could be utilized for enhanced drug delivery. The hypothesis of this study is that a zone of reversible electroporation exists and allows for increased chemotherapy delivery.Methods: Ten immunocompromised mice with orthotopic human pancreatic adenocarcinoma tumors (Panc1) were treated with either IRE between two doses of gemcitabine (15 mg/kg) (ECT) (N = 5) or gemcitabine alone (N = 5). Gemcitabine levels in the serum, liver, and pancreas were analyzed with liquid chromatography/mass spectrometry (LC/MS).Results: Concentration of gemcitabine within reversibly electroporated pancreatic tissue was higher in mice receiving ECT compared to those receiving gemcitabine alone (13,567 ng/ml vs. 4,126 ng/ml; P = 0.0009). Pancreatic gemcitabine levels were 5.52 and 5.96 times higher than liver and serum levels, respectively, in the ECT group compared to 2.85 and 2.53 times higher (P = 0.117, P = 0.058), respectively, in mice receiving gemcitabine alone.Conclusion: IRE can potentially reduce local recurrence by allowing increased drug delivery to the tissue in the reversible electroporation zone. This holds significant potential in augmenting efficacy of gemcitabine in treatment of locally advanced and borderline resectable pancreatic adenocarcinoma. (C) 2016 Wiley Periodicals, Inc.

Abstract Background: Fibroblast growth factor 19 (FGF19), a member of the fibroblast growth factor (FGF) family, is a hormone that regulate glucose, lipid, and energy homeostasis in human. It has been shown that FGF19 and its specific receptor FGFR4 play an important role in non-alcoholic steatohepatitis (NASH) which is closely related hepatocellular carcinoma (HCC). Recently, we found FGF19 overexpressed in the HCC tissue and serum in patients. The high level of FGF19 was positively related to cancer stem cells (CSCs) specific surface markers such as CD133 and EpCAM. The aim of this study is to identify potential mechanism in which FGF15 (the FGF19's orthologous protein in mouse)/FGFR4 associated abnormal lipid metabolism initiates HCC progression in a NASH-HCC mouse Model. Methods: In vivo study, male C57L/J mice were injected intraperitoneally with N-nitrosodiethylamine(DEN) when 2 weeks old. Both the DEN and control mice were divided into two groups based on receiving high fat diet (HFD) or control diet (CD). Tumor nodules in the liver were monitored by ultrasound. Body weight, glucose tolerance test (GTT) and insulin tolerance test (ITT) were recorded during the experimental period. The liver weights, tumor volume, histology, alanine transaminase (ALT), alpha fetoprotein (AFP), and triglyceride (TG) were determined at 8, 20 and 32weeks. In vitro study, human HCC cell lines (Hep3B and Huh7) as well as mouse HCC cell lines (Hepa1-6 and FL83B,) were exposed to a long-chain mixture of free fatty acid(FFA) at different concentrations (UT, 0.5mM and 1mM FFA) for 24 and 48 hours, respectively. EpCAM and CD133 were determined by flow cytometery in the isolated tumor cells from HCC tissues. FGF19/15, FGFR4, CSCs surface markers and Wnt signaling pathway (β-catenin, GSK3β) were analyzed by RT-PCR and Western blot. FGFR4 signal was blocked to elucidate the role of FGF19/15 during the CSCs activation in vitro. In addition, FGF19, FGFR4 and EpCAM were also analyzed in human HCC samples. Results: Metabolic disorders were observed when DEN mice fed with HFD. HCC incidence and tumor volume were significantly increased in DEN+HFD group compared to that in DEN+CD group. DEN mice fed with HFD showed increased levels of FGF15/FGFR4 and β-catenin as well as the CSCs surface makers. Increased expression of CSCs surface makers were also found in the HCC cell lines when exposure to FFA in vitro. Blockage the FGF19 signal can abolish Wnt/β-catenin pathway and cell stemness features in the FFA environment. Increased levels of FGF19, FGFR4 and EpCAM were found in human HCC samples. Conclusion: Up-regulated FGF19/15 and FGFR4 promoted the development of HCC by active CSCs signaling in metabolic disorder microenvironment. FFA enhanced the CSCs activation was partially dependent on Wnt/β-catenin signaling. FGF19/FGFR4 could be a potential therapeutic target in HCC patients. Citation Format: Guozhen Cui, Xingkai Liu, Harshul Pandit, Yingbin Yang, Suping Li, Lu Cai, Robert C Martin, Hengjun Zhao, Wei Li, Yan Li. Metabolic hormones FGF19/15 promote hepatocellular carcinoma through activation of CSCs in fatty liver. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1039.

Abstract Background: Cancer progenitor/stem cells (CSCs), a small subpopulation with stem-like property, are known as a key factor for tumor initiation. We have reported that the turmeric derived volatile constitutes can inhibit mouse hepatoma cell growth in vivo and in vitro. Although the mechanism is largely unknown, the potential effects of the turmeric constitutes could come from either intervention of the tumor microenvironment, or directly abolishing the CSCs. This concept is supported by current studies in which the phyto-chemicals from natural products present a fundamentally different paradigm for cancer therapy though modulating the CSC and microenvironment signaling pathways thereby eliminating CSCs. The ingredient analysis of turmeric derived volatile constitutes by GCxGC/TOF-MS showed that there were more than twenty phyto-chemicals considered to be biologically active, including anti-inflammation, anti-virus, anti-oxidation and anticancer. Among these ingredients, the anti-cancer compounds known as turmerones are rich (46%). In this study, we are going to study the effects of turmerones on the CSCs from mouse hepatoma hepal-6 cells. Methods: A spheroid assay was performed to test the effect of turmericones on sphere formation of hepal-6 cells. The CSC surface antigens including EpCAM, CD133, CD90, and the functional marker aldehyde dehydrogenase were determined by flow cytometery. We further established a diabetic model in C57L/J mouse to create the metabolic disorder microenvironment in the liver to facilitate hepatocellular carcinoma (HCC) initiation. The hepal-6 spheres were pretreated with turmerones and inoculated into the liver lobs of diabetic mouse. The animals were continued to treat with turmerones by i.p. every 3 days for 6 weeks. The liver weights, tumor volume and histology were determined. The cells from tumor tissues were isolated and CSCs were identified by the surface/functional markers. To investigate the diabetes compromised liver microenvironment, IL-6, IGF-1, TGF-β, TNF-α, and FGF-21 were determined in the liver tissues. To investigate the CSC signaling, CSC self-renewal and Wnt signaling pathway (β-catenin, EpCAM, E-Cadherin, TGF-β1, FZD-7, and LRP5/6) are analyzed by RT-PCR and Western blot. Results: Turmerones inhibits hepal-6 cells sphere formation and abolish the CSCs in vitro. The tumor size was significantly decreasd by turmerones treatment compare to control. Although turmerones did not change the serum levels of glucose and insulin, the increased levels (interleukin-6, IGF-1, TGF-β, TNF-α, and FGF-21) and aberrant Wnt pathway components in diabetic liver tissues were ameliorated by turmerones at varying extents. Conclusion: Turmerones treatment can inhibit the HCC formation in diabetic liver. The intervention of tumor microenvironment and Wnt pathway components by turmerones may contribute to the inactivation of CSCs thereby inhibiting the HCC formation. Citation Format: Yan Li, Harshul Pandit, Xuanyi Li, Suping Li, Jingwen Zhang, Guozhen Cui, Robert C. Martin. Abolish cancer progenitor cells in a mouse model of diabetes associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4069. doi:10.1158/1538-7445.AM2015-4069

Aim: Provide an enhanced local drug delivery, nanoparticle(s) to minimize systemic effects and achieve enhanced permeability and drug retention into abnormal cells and stroma. Materials & methods: Here a simultaneous loading of lipophilic gold nanorods (GNRs) and curcumin into polymeric nanomicelles made of biocompatible PLGA-b-PEG copolymer through a double re-emulsification process has been developed. Results: Initial results in vitro on Barrett's esophagus and esophageal adenocarcinoma cell lines demonstrated a significant reduction in cell viability with curcumin and GNRs exposure (p < 0.05). In vivo Barrett's-associated animal model confirmed these results with successful in vivo demonstrated eradication of all high-grade dysplastic premalignant cancer cells. Conclusion: The synthesis of this novel nanosystem containing GNRs and curcumin is safe and effective in treating and eradicating premalignant esophageal adenocarcinoma.

A novel highly biocompatible nanosystem made up of a chitosan matrix and filled with magnesium nanoparticles was synthesized using a simple and one-pot strategy, and tested as a promising, well-tolerated tool for photothermal therapy. Moreover, in vivo a proof of concept on hepatocarcinoma-bearing mice is presented.