Choi, Daheui

Abstract Accurate, noninvasive biomarkers for diagnosis and longitudinal monitoring of metabolic dysfunction–associated steatohepatitis (MASH) remain an urgent unmet clinical need. Here, we describe an electrochemical immunoassay that involves capturing hepatic extracellular vesicles (EVs) on antibody-modified electrodes followed by detecting EV-borne biomarkers using redox active gold nanoparticles (AuNPs). The intensity of electrochemical redox signal represents a quantitative measure of biomarker abundance. Three MASH-associated biomarkers—S100A11, TNF-α, and TGF-β1—were analyzed on hepatic EVs to assess steatotic, inflammatory, and fibrotic states of the diseased liver. The electrochemical signals for individual biomarkers were added into a composite EV MASH score. Plasma samples from 28 patients with MASH, 22 healthy controls, and 27 patients with non-MASH liver disease were scored in this manner, revealing 100% accuracy in the training cohort and >90% accuracy in an independent validation cohort. Our electrochemical immunosensor represents a minimally invasive liquid liver biopsy, offering a promising alternative to solid liver biopsies for screening and monitoring of MASH. One Sentence Summary Circulating hepatic extracellular vesicles carry biomarkers of liver injury enabling accurate, minimally invasive MASH diagnosis in blood.

ABSTRACT Encapsulation of human pluripotent stem cells (hPSC) has high relevance for biomedical applications ranging from tissue engineering to drug screening and cellular therapies. Microcapsules serve a multitude of purposes—from promoting hPSC organization into spheroids to enabling scalable differentiation in vitro and providing immunoisolation in vivo. While multiple encapsulation strategies have been reported, there is typically a tradeoff between the structural complexity of capsules and throughput of their generation. Our paper describes a novel 3D printed microfluidic encapsulation device for fabricating structurally complex microcapsules with a hydrogel shell and aqueous core at rates of up to 1825 Hz. The use of 3D printing allowed to dramatically decrease device footprint when compared to standard soft lithography‐based microfluidic encapsulation devices. Leveraging decreased footprint and the ability to arrange fluidic networks in a 3D space, we fabricated a 10‐nozzle microencapsulation device that generated microcapsules at a rate 10‐times that of the single‐nozzle device. The novel microfluidic device was used to encapsulate hPSCs, human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs). Hydrogel microcapsules with aqueous core promoted hPSCs aggregation into spheroids or embryoid bodies, which maintained high levels of pluripotency. Our technology enables safe and effective encapsulation of hPSCs on a scale (billions of cells) required to treat patients.

Metabolic dysfunction–associated steatohepatitis (MASH) is a progressive liver disease characterized by complex interactions between lipotoxicity, ER stress responses, and immune-mediated inflammation. We identified enrichment of the proinflammatory alarmin S100 calcium-binding protein A11 (S100A11) on extracellular vesicles stimulated by palmitate-induced lipotoxic ER stress with concomitant upregulation of hepatocellular S100A11 abundance in an IRE1A-XBP1s–dependent manner. We next investigated the epigenetic mechanisms that regulate this stress response. Publicly available human liver ChIP-Seq GEO datasets demonstrated a region of histone H3 lysine 27 (H3K27) acetylation upstream of the S100A11 promoter. H3K27 acetylation ChIP-qPCR demonstrated a positive correlation between lipotoxic ER stress and H3K27 acetylation of the region, which we termed the lipotoxicity-influenced enhancer (LIE) domain. CRISPR-mediated repression of the LIE domain reduced palmitate-induced H3K27 acetylation and corresponding S100A11 upregulation in Huh7 cells and immortalized mouse hepatocytes. Silencing of the murine LIE in 2 independent steatohepatitis models demonstrated reduced S100a11 upregulation and attenuated liver injury. We confirmed H3K27 acetylation and XBP1s occupancy at the LIE domain in human MASH liver samples and an increase in hepatocyte-derived S100A11-enriched extracellular vesicles in MASH patient plasma. Our studies demonstrate a LIE domain that mediates hepatic S100A11 upregulation. This pathway may be a potential therapeutic target in MASH.

Purpose: To investigate the molecular characteristics of and potential for precision medicine in KRAS wildtype pancreatic ductal adenocarcinoma (PDAC). Patients and Methods: We investigated 27 patients with KRASWT PDAC at our institution. Clinical data were obtained via chart review. Tumor specimens for each subject were interrogated for somatic single nucleotide variants, insertion and deletions, and copy number variants by DNA sequencing. Gene fusions were detected from RNA-seq. A patient-derived organoid (PDO) was developed from a patient with a MET translocation and expanded ex vivo to predict therapeutic sensitivity prior to enrollment in a phase 2 clinical trial. Results: Transcriptomic analysis showed our cohort may be stratified by the relative gene expression of the KRAS signaling cascade. The PDO derived from our patient harboring a TFG-MET rearrangement was found to have in vitro sensitivity to the multi-tyrosine kinase inhibitor crizotinib. The patient was enrolled in the phase 2 SPARTA clinical trial and received monotherapy with vebrelitinib, a c-MET inhibitor, and achieved a partial and durable response. Conclusions: KRASWT PDAC is molecularly distinct from KRASMUT and enriched with potentially actionable genetic variants. In our study, transcriptomic profiling revealed that the KRAS signaling cascade may play a key role in KRASWT PDAC. Our report of a KRASWT PDAC patient with TFG-MET rearrangement who responded to a cMET inhibitor further supports the pursuit of precision oncology in this sub-population. Identification of targetable mutations, perhaps through approaches like RNA-seq, can help enable precision-driven approaches to select optimal treatment based on tumor characteristics.

AbstractPatient‐derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non‐resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold‐standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.