Revzin, Alexander

The Fusarium fungi is found in cereals and feedstuffs and may produce mycotoxins, which are secondary metabolites, such as the T-2 toxin (T-2). In this work, we explored the hepatotoxicity of T-2 using microfluidic 3D hepatic cultures. The objectives were: (i) exploring the benefits of microfluidic 3D cultures compared to conventional 3D cultures available commercially (Aggrewell plates), (ii) establishing 3D co-cultures of hepatic cells (HepG2) and stellate cells (LX2) and assessing T-2 exposure in this model, (iii) characterizing the induction of metabolizing enzymes, and (iv) evaluating inflammatory markers upon T-2 exposure in microfluidic hepatic cultures. Our results demonstrated that, in comparison to commercial (large-volume) 3D cultures, spheroids formed faster and were more functional in microfluidic devices. The viability and hepatic function decreased with increasing T-2 concentrations in both monoculture and co-cultures. The RT-PCR analysis revealed that exposure to T-2 upregulates the expression of multiple Phase I and Phase II hepatic enzymes. In addition, several pro- and anti-inflammatory proteins were increased in co-cultures after exposure to T-2.

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.

Abstract Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions – to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut’s neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate.