Direct Reprogramming of Hepatocytes Into JAK/Stat-Dependent LGR5+ Liver Cells Able to Initiate Intrahepatic Cholangiocarcinoma.

Somatic cells that have been partially reprogrammed by the factors Oct4, Sox2, Klf4, and cMyc (OSKM) have been demonstrated to be potentially tumorigenic in vitro and in vivo due to the acquisition of cancer-associated genomic alterations and the absence of OSKM clearance over time. In the present study, we obtained partially reprogrammed, SSEA1-negative cells by transducing murine hepatocytes with Δ1Δ3-deleted adenoviruses that expressed the 4 OSKM factors. We observed that, under long-term 2D and 3D culture conditions, hepatocytes could be converted into LGR5-positive cells with self-renewal capacity that was dependent on 3 cross-signaling pathways: IL6/Jak/Stat3, LGR5/R-spondin, and Wnt/ß-catenin. Following engraftment in syngeneic mice, LGR5-positive cells that expressed the cancer markers CD51, CD166, and CD73 were capable of forming invasive and metastatic tumors reminiscent of intrahepatic cholangiocarcinoma (ICC): they were positive for CK19 and CK7, featured associations of cord-like structures, and contained cuboidal and atypical cells with dissimilar degrees of pleomorphism and mitosis. The LGR5+-derived tumors exhibited a highly vascularized stroma with substantial fibrosis. In addition, we identified pro-angiogenic factors and signaling pathways involved in neo-angiogenesis and vascular development, which represent potential new targets for anti-angiogenic strategies to overcome tumor resistance to current ICC treatments.

Clinical Application of Infrared Spectroscopy in Liver Transplantation for Rapid Assessment of Lipid Content in Liver Graft.

Liver transplantation (LT) is a major treatment for patients with end-stage liver diseases. Steatosis is a significant risk factor for primary graft nonfunction and associated with poor long-term graft outcomes. Traditionally, the evaluation of steatosis is based on frozen section examination to estimate the percentage of hepatocytes containing lipid vesicles. However, this visual evaluation correlates poorly with the true lipid content. This study aimed to address the potential of infrared (IR) microspectroscopy for rapidly estimating lipid content in the context of LT and assessing its impact on survival. Clinical data were collected for >20 months from 58 patients who underwent transplantation. For each liver graft, macrovacuolar steatosis and microvesicular steatosis were evaluated through histologic examination of frozen tissue section. Triglycerides (TG) were further quantified using gas phase chromatography coupled with a flame ionization detector (GC-FID) and estimated by IR microspectroscopy. A linear relationship and significant correlation were observed between the TG measured by GC-FID and those estimated using IR microspectroscopy (R2 = 0.86). In some cases, microvesicular steatosis was related to high lipid content despite low levels of macrovacuolar steatosis. Seven patients experienced posttransplantation liver failure, including 5 deceased patients. All patients underwent transplantation with grafts containing significantly high TG levels. A concentration of 250 nmol/mg was identified as the threshold above which the risk of failure after LT significantly increased, affecting 35% of patients. Our study established a strong correlation between LT outcomes and lipid content. IR microspectroscopy proved to be a rapid and reliable approach for assessing the lipid content in clinical settings.

Patient-derived organoids identify an apico-basolateral polarity switch associated with survival in colorectal cancer.

The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies - MUC CRCs either display a conventional 'apical-in' polarity or, more frequently, harbor an inverted 'apical-out' topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFß and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.

Impact of the overexpression of the tyrosine kinase receptor RET in the hematopoietic potential of induced pluripotent stem cells (iPSCs).

INTRODUCTION: Previous studies have suggested that the tyrosine kinase receptor RET plays a significant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in human iPSC-derived hematopoiesis has not been tested so far. METHODS: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. The hematopoietic potential was tested using 2D cultures and evaluated regarding the phenotype and the clonogenic potential of generated cells. RESULTS: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of primitive hematopoietic cells (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific activated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. CONCLUSION: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related to MAPK activation.

CXCL-8 as a signature of severe Helicobacter pylori infection and a stimulator of stomach region-dependent immune response.

Helicobacter pylori infection is involved in development of diverse gastro-pathologies. Our aim is to investigate potential signature of cytokines-chemokine levels (IL-17A, IL-1ß, and CXCL-8) in H. pylori-infected patients and their impact on immune response in both corpus and antrum. Multivariate level analysis with machine learning model were carried out using cytokines/chemokine levels of infected Moroccan patients. In addition, Geo dataset was used to run enrichment analysis following CXCL-8 upregulation. Our analysis showed that combination of cytokines-chemokine levels allowed prediction of positive H. pylori density score with <5% of miss-classification error, with fundus CXCL-8 being the most important variable for this discrimination. Furthermore, CXCL-8 dependent expression profile was mainly associated to IL6/JAK/STAT3 signaling in the antrum, interferons alpha and gamma responses in the corpus and commonly induced transcriptional /proliferative activities. To conclude, CXCL-8 level might be a signature of Moroccan H. pylori-infected patients and an inducer of regional-dependent immune response at the gastric level. Larger trials must be carried out to validate the relevance of these results for diverse populations.

Type I and III interferons are good markers to monitor COVID-19 pathophysiology.

The COVID-19 pandemic has caused millions of deaths and has resulted in disastrous societal and economic impacts worldwide. During SARS-CoV-2 infection, abnormal levels of pro-inflammatory cytokines have been observed and were associated to the severity of the disease. Type I (-α/ß) and Type III (IFN-λ) interferons are family members of cytokines that play an important role in fighting viral replication during the early phases of infection. The location and timing of the IFNs production have been shown to be decisive for the COVID-19 outcome. Despite the effectiveness of COVID-19 vaccines and with the emergence of new SARS-CoV-2 variants, a better understanding of the involvement of IFNs as players in antiviral immunity in the COVID-19 pathophysiology is necessary to implement additional potent prophylactic and/or therapeutic approaches. In this study, we investigated the role of type I and III IFN in COVID-19 pathophysiology. We first analyzed the IFN-α, IFN-ß and IFN- λ mRNA expression in nasopharyngeal swabs and blood samples from Moroccan patients infected with SARS-CoV-2 and secondly correlated these IFNs expressions with COVID-19 clinical and biological parameters. Our results showed that in the upper airways of patients with mild, non-severe, or severe COVID-19 manifestations, the IFN- α, - ß and - λ are expressed in the same manner as in controls. However, in blood samples their expression was downregulated in all groups. Univariate linear models with interferons as predictors to evaluate clinical-biological parameters highlighted that the main clinical-biological relations were found when testing: FiO2, Lymphocyte values and virus load. Furthermore, the multivariate models confirmed that quantifications of interferons during COVID-19 are good biological markers for tracking COVID-19 pathophysiology.

Intrinsic cancer cell phosphoinositide 3-kinase δ regulates fibrosis and vascular development in cholangiocarcinoma.

BACKGROUND & AIMS: The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation. APPROACH & RESULTS: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFß/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression. CONCLUSIONS: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFß/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients.

Deciphering Potential Molecular Signatures to Differentiate Acute Myeloid Leukemia (AML) with BCR::ABL1 from Chronic Myeloid Leukemia (CML) in Blast Crisis.

Acute myeloid leukemia (AML) with BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.

The Angiogenesis Inhibitor Isthmin-1 (ISM1) Is Overexpressed in Experimental Models of Glomerulopathy and Impairs the Viability of Podocytes.

Focal segmental glomerulosclerosis (FSGS) is a major cause of end-stage renal disease and remains without specific treatment. To identify new events during FSGS progression, we used an experimental model of FSGS associated with nephroangiosclerosis in rats injected with L-NAME (Nω-nitro-L-arginine methyl ester). After transcriptomic analysis we focused our study on the role of Isthmin-1 (ISM1, an anti-angiogenic protein involved in endothelial cell apoptosis. We studied the renal expression of ISM1 in L-NAME rats and other models of proteinuria, particularly at the glomerular level. In the L-NAME model, withdrawal of the stimulus partially restored basal ISM1 levels, along with an improvement in renal function. In other four animal models of proteinuria, ISM1 was overexpressed and localized in podocytes while the renal function was degraded. Together these facts suggest that the glomerular expression of ISM1 correlates directly with the progression-recovery of the disease. Further in vitro experiments demonstrated that ISM1 co-localized with its receptors GRP78 and integrin αvß5 on podocytes. Treatment of human podocytes with low doses of recombinant ISM1 decreased cell viability and induced caspase activation. Stronger ISM1 stimuli in podocytes dropped mitochondrial membrane potential and induced nuclear translocation of apoptosis-inducing factor (AIF). Our results suggest that ISM1 participates in the progression of glomerular diseases and promotes podocyte apoptosis in two different complementary ways: one caspase-dependent and one caspase-independent associated with mitochondrial destabilization.

Indocyanine green fluorescence patterns of hepatocellular carcinoma correlate with pathological and molecular features.

BACKGROUND: Intraoperative Indocyanine Green Dye (ICG) routinely used in hepatobiliary surgery identifies different fluorescent patterns of hepatocellular carcinoma (HCC), a highly heterogeneous cancer. We aimed to correlate these patterns with gene mutations and extensive pathological features beyond the well-known tumor differentiation. METHODS: Between February 2017 and December 2019, 21 HCC in 16 consecutive patients who underwent intraoperative ICG fluorescence imaging were included. Pathological review was performed by one pathologist blinded to fluorescence features. Random forest machine learning algorithm correlated pathological features of the tumor, peritumoral and non-tumoral liver, and gene mutations from a 28 gene-panel with rim and intra-lesion fluorescence. RESULTS: Three HCC had negative intra-lesion and rim-like emission, 7 HCC had homogeneous pattern and 11 heterogeneous patterns in whom 3 with rim-like emission. Rim emission was associated with peritumoral vascular changes, lower differentiation and lower serum AFP level. Homogeneous intra-lesion fluorescence was associated with lower necrosis rate, thinner capsule, absence of peritumoral liver changes, and higher serum AFP level. Heterogeneous HCC without rim harbored lesser TP53 and ARID1A mutations. CONCLUSION: Tumoral and peri-tumoral fluorescence classification of HCC yielded a possible intraoperative pathological and molecular characterization. These preliminary observations could lead to intraoperative refinement in surgical strategy.

The proliferation of belatacept-resistant T cells requires early IFNα pathway activation.

Belatacept was developed to replace calcineurin inhibitors in kidney transplantation. Its use is associated with better kidney transplant function, a lower incidence of anti-donor antibodies and higher graft survival. However, it is also associated with a higher risk of cellular rejection. We studied the activation and proliferation mechanisms of belatacept-resistant T lymphocytes (TLs), to identify new pathways for control. We performed a transcriptomic analysis on CD4+ CD57+ PD1- memory TLs, which are responsible for a higher incidence of graft rejection, after allogeneic stimulation with activated dendritic cells (aDCs) in the presence or absence of belatacept. After six hours of contact with aDCs, the (CD4+ CD57+ PD1- ) (CD4+ CD57+ PD1+ ) and (CD4+ CD57- ) lymphocytes had different transcriptional profiles with or without belatacept. In the CD4+ CD57+ PD1- population, the IFNα-dependent activation pathway was positively overrepresented, and IRF7 transcript levels were high. IRF7 was associated with IFNα/ß and IL-6 regulation. The inhibition of both these cytokines in a context of belatacept treatment inhibited the proliferation of CD4+ CD57+ PD1- T cells. Our results show that IRF7 is rapidly upregulated in belatacept-resistant CD4+ CD57+ PD1- TLs. The inhibition of type I IFN or IL-6 in association with belatacept treatment reduces the proliferation of belatacept-resistant TLs, paving the way for new treatments for use in organ transplantation.

Donor HLA Class 1 Evolutionary Divergence Is a Major Predictor of Liver Allograft Rejection : A Retrospective Cohort Study.

BACKGROUND: The HLA evolutionary divergence (HED), a continuous metric quantifying the peptidic differences between 2 homologous HLA alleles, reflects the breadth of the immunopeptidome presented to T lymphocytes. OBJECTIVE: To assess the potential effect of donor or recipient HED on liver transplant rejection. DESIGN: Retrospective cohort study. SETTING: Liver transplant units. PATIENTS: 1154 adults and 113 children who had a liver transplant between 2004 and 2018. MEASUREMENTS: Liver biopsies were done 1, 2, 5, and 10 years after the transplant and in case of liver dysfunction. Donor-specific anti-HLA antibodies (DSAs) were measured in children at the time of biopsy. The HED was calculated using the physicochemical Grantham distance for class I (HLA-A or HLA-B) and class II (HLA-DRB1 or HLA-DQB1) alleles. The influence of HED on the incidence of liver lesions was analyzed through the inverse probability weighting approach based on covariate balancing, generalized propensity scores. RESULTS: In adults, class I HED of the donor was associated with acute rejection (hazard ratio [HR], 1.09 [95% CI, 1.03 to 1.16]), chronic rejection (HR, 1.20 [CI, 1.10 to 1.31]), and ductopenia of 50% or more (HR, 1.33 [CI, 1.09 to 1.62]) but not with other histologic lesions. In children, class I HED of the donor was also associated with acute rejection (HR, 1.16 [CI, 1.03 to 1.30]) independent of the presence of DSAs. There was no effect of either donor class II HED or recipient class I or class II HED on the incidence of liver lesions in adults and children. LIMITATION: The DSAs were measured only in children. CONCLUSION: Class I HED of the donor predicts acute or chronic rejection of liver transplant. This novel and accessible prognostic marker could orientate donor selection and guide immunosuppression. PRIMARY FUNDING SOURCE: Institut National de la Santé et de la Recherche Médicale.

Tumors located in the central column of the liver are associated with increased surgical difficulty and postoperative complications following open liver resection for colorectal metastases.

BACKGROUND: To assess the impact of difficult location (based on preoperative computed tomography) of liver metastases from colorectal cancer (LMCRC) on surgical difficulty, and occurrence of severe postoperative complications (POCs). METHODS: A retrospective single-centre study of 911 consecutive patients with LMCRC who underwent hepatectomy by the open approach between 1998 and 2011, before implementation of laparoscopic surgery to obviate approach selection bias. LMCRC with at least one of the following four features on preoperative imaging: tumor invading the hepatocaval confluence or retro-hepatic inferior vena cava, centrally located (Segments 4,5,8) and >10 cm in diameter, abutting the supra-hilar area, or involving the paracaval portion or caudate process of Segment 1; were considered as topographically difficult (top-diff). Independent predictors of surgical difficulty assessed by number of blood units transfused, duration of ischemia, and number of sessions of pedicle clamping during surgery and of severe POCs were identified by multivariate analysis before, and after propensity score matching. RESULTS: Top-diff tumor location independently predicted surgical difficulty. Severe POCs were associated with the tumor location [top-diff vs. topographically non difficult (non top-diff)], preoperative portal vein embolization, and variables related to surgical difficulty. CONCLUSION: LMCRC in difficult location independently predicts surgical difficulty and severe POCs.

HLA-dependent heterogeneity and macrophage immunoproteasome activation during lung COVID-19 disease.

BACKGROUND: The worldwide pandemic caused by the SARS-CoV-2 virus is characterized by significant and unpredictable heterogeneity in symptoms that remains poorly understood. METHODS: Transcriptome and single cell transcriptome of COVID19 lung were integrated with deeplearning analysis of MHC class I immunopeptidome against SARS-COV2 proteome. RESULTS: An analysis of the transcriptomes of lung samples from COVID-19 patients revealed that activation of MHC class I antigen presentation in these tissues was correlated with the amount of SARS-CoV-2 RNA present. Similarly, a positive relationship was detected in these samples between the level of SARS-CoV-2 and the expression of a genomic cluster located in the 6p21.32 region (40 kb long, inside the MHC-II cluster) that encodes constituents of the immunoproteasome. An analysis of single-cell transcriptomes of bronchoalveolar cells highlighted the activation of the immunoproteasome in CD68 + M1 macrophages of COVID-19 patients in addition to a PSMB8-based trajectory in these cells that featured an activation of defense response during mild cases of the disease, and an impairment of alveolar clearance mechanisms during severe COVID-19. By examining the binding affinity of the SARS-CoV-2 immunopeptidome with the most common HLA-A, -B, and -C alleles worldwide, we found higher numbers of stronger presenters in type A alleles and in Asian populations, which could shed light on why this disease is now less widespread in this part of the world. CONCLUSIONS: HLA-dependent heterogeneity in macrophage immunoproteasome activation during lung COVID-19 disease could have implications for efforts to predict the response to HLA-dependent SARS-CoV-2 vaccines in the global population.

Roles of tetraspanins during trophoblast development: bioinformatics and new perspectives.

Tetraspanins are a superfamily of membrane proteins found in all eukaryotic organisms. They act as scaffold molecules that regulate the traffic and function of other membrane/signaling proteins, resulting in important downstream cellular consequences. The aim of this work was to use transcriptomes and bioinformatics analysis to identify the tetraspanins (and their partners) involved in trophoblast differentiation. We built a protein-protein interaction network around tetraspanins which revealed that tetraspanins CD9, CD81, and CD82 show a specific expression during trophoblast differentiation. These proteins appeared to be interconnected and to recruit several membrane partners which include integrins, immune-related molecules, and a variety of receptors. During weeks 8 to 24, a CD9 expression trajectory was identified in extravillous trophoblasts, and a website was developed: ( https://extravillous.shinyapps.io/CD9humanEVT/ ). In conclusion, CD81 may, together with CD9 and CD82, be interconnected in controlling trophoblast invasion in the endometrium. CD9 expression trajectory in extravillous trophoblast between weeks 8 and 24 shows the involvement of CD9 in cell adhesion and migration.

Identification of Four Immune Subtypes Characterized by Distinct Composition and Functions of Tumor Microenvironment in Intrahepatic Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a severe malignant tumor in which the standard therapies are mostly ineffective. The biological significance of the desmoplastic tumor microenvironment (TME) of ICC has been stressed but was insufficiently taken into account in the search for classifications of ICC adapted to clinical trial design. We investigated the heterogeneous tumor stroma composition and built a TME-based classification of ICC tumors that detects potentially targetable ICC subtypes. APPROACH AND RESULTS: We established the bulk gene expression profiles of 78 ICCs. Epithelial and stromal compartments of 23 ICCs were laser microdissected. We quantified 14 gene expression signatures of the TME and those of 3 functional indicators (liver activity, inflammation, immune resistance). The cell population abundances were quantified using the microenvironment cell population-counter package and compared with immunohistochemistry. We performed an unsupervised TME-based classification of 198 ICCs (training set) and 368 ICCs (validation set). We determined immune response and signaling features of the different immune subtypes by functional annotations. We showed that a set of 198 ICCs could be classified into 4 TME-based subtypes related to distinct immune escape mechanisms and patient outcomes. The validity of these immune subtypes was confirmed over an independent set of 368 ICCs and by immunohistochemical analysis of 64 ICC tissue samples. About 45% of ICCs displayed an immune desert phenotype. The other subtypes differed in nature (lymphoid, myeloid, mesenchymal) and abundance of tumor-infiltrating cells. The inflamed subtype (11%) presented a massive T lymphocyte infiltration, an activation of inflammatory and immune checkpoint pathways, and was associated with the longest patient survival. CONCLUSION: We showed the existence of an inflamed ICC subtype, which is potentially treatable with checkpoint blockade immunotherapy.

Diffuse Versus Localized Caroli Disease: A Comparative MRCP Study.

OBJECTIVE. The purpose of this multicenter retrospective study was to assess the MRCP features of Caroli disease (CD). MATERIALS AND METHODS. Sixty-six patients were identified from 2000 to 2019. The inclusion criteria were diagnosis of diffuse or localized CD mentioned in an imaging report, presence of intrahepatic bile duct (IHBD) dilatation, and having undergone an MRCP examination. The exclusion criteria included presence of obstructive proximal biliary stricture and having undergone hepatobiliary surgery other than cholecystectomy. Histopathology records were available for 53 of the 66 (80%) patients. Diffuse and localized diseases were compared by chi-square and t tests and Kaplan-Meier model. RESULTS. Forty-five patients had diffuse bilobar CD ((five pediatric patients [three girls and two boys] with a mean [± SD] age of 8 ± 5 years [range, 1-15 years] and 40 adult patients [26 men and 14 women] with a mean age of 35 ± 11 years [range, 20-62 years]) and 21 patients had localized disease (12 men and 9 women; mean age, 54 ± 14 years). Congenital hepatic fibrosis was found only in patients with diffuse CD (35/45 [78%]), as was a "central dot" sign (15/35 [43%]). IHBD dilatation with both saccular and fusiform features was found in 43 (96%) and the peripheral "funnel-shaped" sign in 41 (91%) of the 45 patients with diffuse CD but in none of the patients with localized disease (p < .001). Intrahepatic biliary calculi were found in all patients with localized disease but in only 16 of the 45 (36%) patients with diffuse CD (p < .001). Left liver atrophy was found in 18 of the 21 (86%) patients with localized disease and in none of the patients with diffuse CD (p < .001). The overall survival rate among patients with diffuse CD was significantly lower than that among patients with localized disease (p = .03). CONCLUSION. Diffuse IHBD dilatation with both saccular and fusiform features associated with the peripheral funnel-shaped sign can be used for the diagnosis of CD on MRCP. Localized IHBD dilatation seems to be mainly related to primary intrahepatic lithiasis.

iPSC-Derived Hereditary Breast Cancer Model Reveals the BRCA1-Deleted Tumor Niche as a New Culprit in Disease Progression.

Tumor progression begins when cancer cells recruit tumor-associated stromal cells to produce a vascular niche, ultimately resulting in uncontrolled growth, invasion, and metastasis. It is poorly understood, though, how this process might be affected by deletions or mutations in the breast cancer type 1 susceptibility (BRCA1) gene in patients with a lifetime risk of developing breast and/or ovarian cancer. To model the BRCA1-deleted stroma, we first generated induced pluripotent stem cells (iPSCs) from patients carrying a germline deletion of exon 17 of the BRCA1 gene (BRCA1+/- who, based on their family histories, were at a high risk for cancer. Using peripheral blood mononuclear cells (PBMCs) of these two affected family members and two normal (BRCA1+/+) individuals, we established a number of iPSC clones via non-integrating Sendai virus-based delivery of the four OCT4, SOX2, KLF4, and c-MYC factors. Induced mesenchymal stem cells (iMSCs) were generated and used as normal and pathological stromal cells. In transcriptome analyses, BRCA1+/- iMSCs exhibited a unique pro-angiogenic signature: compared to non-mutated iMSCs, they expressed high levels of HIF-1α, angiogenic factors belonging to the VEGF, PDGF, and ANGPT subfamilies showing high angiogenic potential. This was confirmed in vitro through the increased capacity to generate tube-like structures compared to BRCA1+/+ iMSCs and in vivo by a matrigel plug angiogenesis assay where the BRCA1+/- iMSCs promoted the development of an extended and organized vessel network. We also reported a highly increased migration capacity of BRCA1+/- iMSCs through an in vitro wound healing assay that correlated with the upregulation of the periostin (POSTN). Finally, we assessed the ability of both iMSCs to facilitate the engraftment of murine breast cancer cells using a xenogenic 4T1 transplant model. The co-injection of BRCA1+/- iMSCs and 4T1 breast cancer cells into mouse mammary fat pads gave rise to highly aggressive tumor growth (2-fold increase in tumor volume compared to 4T1 alone, p = 0.01283) and a higher prevalence of spontaneous metastatic spread to the lungs. Here, we report for the first time a major effect of BRCA1 haploinsufficiency on tumor-associated stroma in the context of BRCA1-associated cancers. The unique iMSC model used here was generated using patient-specific iPSCs, which opens new therapeutic avenues for the prevention and personalized treatment of BRCA1-associated hereditary breast cancer.

BMAL1 Knockdown Leans Epithelial-Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells.

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial-mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial-mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.