Tumor decellularization reveals proteomic and mechanical characteristics of the extracellular matrix of primary liver cancer.

Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development.

Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

Human branching cholangiocyte organoids recapitulate functional bile duct formation.

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Models behind the mystery of establishing enhancer-promoter interactions.

Enhancers and promoters are transcriptional regulatory elements whose facilitated interactions increase gene expression. Enhancer DNA sequences can be located far away from the promoter sequences that they regulate. Currently, the mechanism facilitating the establishment of enhancer-promoter interactions remains unclear. However, mutations causing errors in these interactions have been linked to cancer and disease, further conveying the need to understand the full mechanism. This review discusses multiple models that have been proposed to describe how enhancers go the distance to interact with promoters. Evidence supporting loop formation models is reviewed in addition to more complex hypotheses involving aspects of 3D chromatin organization and phase separation.