Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) H epatic tumors with combined hepatocellular cholangiocarcinoma have been frequently described for instance, hepatoblastoma with cholangioblastic features in young patients 1 and HCCs with dual expression of hepatocyte and bile duct markers in adult patients suffering from diseases related to HCV and/or HBV infection. 2 Such tumors usually contain mature hepatocytes and so-called transitional areas that consist of undifferentiated cells that have morphological and immunological features of both hepatocytes and cholangiocytes. 3 Co-expression of hepatocytic and biliary markers suggests involvement of hepatic progenitor cells in development of these human tumors and supports the concept of genetic events to explain their abnormal growth during tumor formation. 4 However, mechanisms of occurrence of these progenitors and abnormal control of their expansion and differentiation are still unclear.Hepatic progenitor cells, also referred to as oval cells in rodents, have been defined as immature epithelial cells able to differentiate toward both biliary and hepatocytic lineages. The smallest ramification of the biliary tree in adult liver, the canal of Hering, may constitute the niche for these hepatic progenitor cells. 5 They are few in number, and because bile ductular and hepatocytic cells have a tremendous capacity to proliferate and differentiate, heAbbreviations: BrdU, bromodeoxyuridine; HNF, hepatocyte nuclear factor; RT, reverse transcription.

Background and aims Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings. Methods Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from ten patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or x-ray contrast solution on RNA quality. Results RNA was of variable quantity (100–1500 ng) and poor quality (Agilent RNA Integrity Number (RIN) <5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130bp. Differential gene expression by microarray analysis identified 1,140 up-regulated genes and 1,001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 upregulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3 and PNMA2 protein expression in cancer samples. Conclusions RNA isolated from biliary brushings, is suitable for molecular analysis of biliary diseases using qPCR and microarray.

The polyamine transport system (PTS) whose activity is up-regulated in cancer cells is an attractive target for drug design. Two heterocyclic (azepine and benzazepine) systems were conjugated to various polyamine moieties through an amidine bound to afford 18 compounds which were evaluated for their affinity for the PTS and their ability to use the PTS for cell delivery. Structure-activity relationship studies and lead optimization afforded two attractive PTS targeting compounds. The azepine-spermidine conjugate 14 is a very selective substrate of the PTS that may serve as a vector for radioelements used for diagnoses or therapeutics in nuclear medicine. The nitrobenzazepine-spermine conjugate 28 is a very powerful PTS inhibitor with very low intrinsic cytotoxicity, able to prevent the growth of polyamine depleted cells in presence of exogenous polyamines.

Introduction: Chimeric antigen receptor (CAR) T cell therapies directed against B cell maturation antigen (BCMA) have shown significant activity in patients with RRMM, however single antigen targeting with CAR-T cells can result in antigen negative relapse. Dual antigen targeting increases targetable tumor antigens and may reduce the risk of antigen negative disease escape. 'A proliferation-inducing ligand' (APRIL) is a natural high affinity ligand for BCMA and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI). Like BCMA, TACI is also a tumor necrosis factor receptor and is involved in maturation of B-cells, including their maturation to plasma cells. Importantly, TACI is expressed on MM cells. In this study, we are evaluating the safety and efficacy of AUTO2, a CAR-T cell therapy designed to target BCMA and TACI. Methods: We designed a novel CAR construct using a truncated form of APRIL as the tumor-targeting domain which recognizes both BCMA and TACI on MM cells. AUTO2 is retrovirally transduced to express APRIL CAR and the RQR8 safety switch. The APRIL CAR construct is in a third-generation format with composite endodomains of CD28, OX40 and CD3 zeta. The cell product was manufactured on a semi-automated and closed process. Patients (≥ 18 years) with RRMM; Eastern Cooperative Oncology Group Performance Status <2, who have had at least 3 prior lines of therapy or have double refractory disease to proteasome inhibitors (PI) and immunomodulatory agents (IMiD), adequate hepatic and cardiac function and an absolute lymphocyte count ≥0.5 x 10e9/L are eligible. Patients with CNS disease, prior allogeneic stem cell transplant, are excluded. All patients receive lymphodepletion with 30 mg/m2/day fludarabine and 300 mg/m2/day cyclophosphamide for 3 days prior to AUTO2 infusion. Five dose levels are being explored (15 x 10e6; 75 x 10e6 ;225 x 10e6; 600 x10e6 x and 900x 10e6 transduced CAR-T cells). The primary endpoint of this phase 1 study is incidence of Grade 3 to 5 toxicity occurring within the DLT period (28 days post AUTO2 infusion), frequency of DLTs and the persistence of AUTO2. Key secondary endpoints include overall response rate, duration of response, and overall survival, as well as biomarker endpoints such as AUTO2 levels in blood. Results: As of the data cut-off date (July 03, 2019), 12 patients have been enrolled. Eleven patients have been dosed on study, 1 at 15 x10e6, 3 at 75x10e6, 3 at 225x10e6, 3 at 600x10e6 and 1 at 900x10e6 CAR-T cells. Two patients have been retreated. All patients were successfully manufactured and received target dose. Median age was 61 years (range 45-69 years), median 5 prior lines of treatment (range 3-6) ,73% had prior autologous transplant, 100% were refractory to a PI or IMiD, 80% were refractory to both and 45% were refractory to daratumumab. Eleven patients had a minimum of 4 week follow up and were evaluable for safety analysis. No AUTO2 related deaths were observed and no DLTs were observed. The most frequent ≥ Grade 3 adverse events (>30%) were anemia (82%), neutrophil count decreased (73%). Five patients (45%) experienced CRS, all were grade 1, no ≥ G2 CRS was noted. Tocilizumab was given to 3 patients (27%). No cases of neurotoxicity occurred. Seven patients were dosed, in the ≥ 225x10e6 dose cohorts, the ORR was 43% (28% PRs and 14% VGPRs). Interestingly the patient dosed at 15x10e6 CAR-T cells maintained stable disease (SD) for a year and was retreated at higher dose of 225x10e6 CAR-T cells and continues with SD without further treatment. This patient had the highest baseline levels of TACI and was previously primary refractory to treatment. Another patient initially treated at 75 x10e6 CAR -T cells was retreated with 225x 10e6 CAR-T cells and achieved a partial response. Updated data as well as cellular kinetics, product characteristics and additional biomarker analysis including BCMA and TACI will be presented. Conclusions: AUTO2 is a novel CAR-T therapy, with a manageable safety profile at doses up to 900x10e6 CAR-T cells. Disclosures Popat: Janssen: Honoraria, Other: travel support to meetings; GSK: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Other: travel, accommodations, expenses. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cavet:Amgen: Other: congress support , Research Funding, Speakers Bureau; Oncopeptide: Other: congress support , Research Funding, Speakers Bureau; EUSA: Other: congress support , Research Funding, Speakers Bureau; GSK: Other: congress support, Research Funding, Speakers Bureau; Celgene: Other: congress support , Research Funding, Speakers Bureau; Janssen: Other: Congress support , Research Funding, Speakers Bureau; Takeda: Other: congress support , Research Funding, Speakers Bureau. Yong:Autolus: Consultancy; Sanofi: Speakers Bureau; Amgen: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Takeda: Research Funding, Speakers Bureau. Lee:Autolus Therapeutics: Equity Ownership, Research Funding. Faulkner:Autolus Therapeutics: Employment, Equity Ownership. Kotsopoulou:Autolus Therapeutics: Employment, Equity Ownership. Al-Hajj:Autolus Therapeutics: Employment, Equity Ownership. Thomas:Autolus: Employment, Equity Ownership. Cordoba:Autolus: Employment, Equity Ownership. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Cerec:Autolus Therapeutics: Employment, Equity Ownership. Peddareddigari:Autolus Therapeutics: Employment, Equity Ownership. Khokhar:Autolus Therapeutics: Employment, Equity Ownership. Menne:Kyowa Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant.

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation.

ABSTRACT:Fatty acid microsomal -oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-

Arginine and ornithine are known to be important for various biological processes in the testis, but the delivery of extracellular cationic amino acids to the seminiferous tubule cells remains poorly understood. We investigated the activity and expression of cationic amino acid transporters in isolated rat Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids. We assessed the l-arginine uptake kinetics, Na(+) dependence of transport, profiles of cis inhibition of uptake by cationic and neutral amino acids, and sensitivity to trans stimulation of cationic amino acid transporters, and studied the expression of the genes encoding them by RT-PCR. Our data suggest that l-arginine is taken up by Sertoli cells and peritubular cells, principally via system y(+)L (SLC3A2/SLC7A6) and system y(+) (SLC7A1 and SLC7A2), with system B(0+) making a minor contribution. By contrast, system B(0+), associated with system y(+)L (SLC3A2/SLC7A7 and SLC7A6), made a major contribution to the transport of cationic amino acids in pachytene spermatocytes and early spermatids. Sertoli cells had higher rates of l-arginine transport than the other seminiferous tubule cells. This high efficiency of arginine transport in Sertoli cells and the properties of the y(+)L system predominating in these cells strongly suggest that Sertoli cells play a key role in supplying germ cells with l-arginine and other cationic amino acids. Furthermore, whereas cytokines induce nitric oxide (NO) production in peritubular and Sertoli cells, little or no upregulation of arginine transport by cytokines was observed in these cells. Thus, NO synthesis does not depend on the stimulation of arginine transport in these somatic tubular cells.

No abstract