Expression Microdissection for the Analysis of miRNA in a Single-Cell Type.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

Therapeutic Response of miR-145 Micelles on Patient-Derived Vascular Smooth Muscle Cells.

During atherosclerosis, vascular smooth muscle cells (VSMCs) undergo a phenotypic transition from a healthy contractile state into pathological phenotypes including a proliferative and migratory, synthetic phenotype and osteochondrogenic-like phenotype that exacerbate plaques. Thus, inhibiting the transition of healthy, quiescent VSMCs to atherogenic cell types has the potential to mitigate atherosclerosis. To that end, previously, we reported that delivery of microRNA-145 (miR-145, a potent gatekeeper of the contractile VSMC phenotype) using nanoparticle micelles limited atherosclerotic plaque growth in murine models of atherosclerosis. Building on this preclinical data and toward clinical application, in this study, we tested the therapeutic viability of miR-145 micelles on patient-derived VSMCs and evaluated their effects based on disease severity. We collected vascular tissues from 11 patients with healthy, moderate, or severe stages of atherosclerosis that were discarded following vascular surgery or organ transplant, and isolated VSMCs from these tissues. We found that with increasing disease severity, patient-derived VSMCs had decreasing levels of contractile markers (miR-145, ACTA2, MYH11) and increasing levels of synthetic markers (KLF4, KLF5, and ELK1). Treatment with miR-145 micelles showed that an increase in disease severity correlated with a more robust response to therapy in VSMCs. Notably, miR-145 micelle therapy rescued contractile marker expression to baseline contractile levels in VSMCs derived from the most severely diseased tissues. As such, we demonstrate the use of miR-145 micelles across different stages of atherosclerosis disease and present further evidence of the translatability of miR-145 micelle treatment for atherosclerosis.

Oral delivery of metformin by chitosan nanoparticles for polycystic kidney disease.

Nanoparticle drug delivery has many advantages over small molecule therapeutics, including reducing off-target side effects and increasing drug potency. However, many nanoparticles are administered parenterally, which is challenging for chronic diseases such as polycystic kidney disease (PKD), the most common hereditary disease worldwide in which patients need continuous treatment over decades. To address this clinical need, we present the development of nanoparticles synthesized from chitosan, a widely available polymer chosen for its ability to improve oral bioavailability. Specifically, we optimized the synthesis parameters of chitosan nanoparticles and demonstrate mucoadhesion and permeation across an intestinal barrier model in vitro. Furthermore, when administered orally to mice, ex vivo imaging of rhodamine-loaded chitosan nanoparticles showed significantly higher accumulation in the intestines compared to the free model drug, as well as 1.3 times higher serum area under the curve (AUC), demonstrating controlled release and improved serum delivery over 24 h. To test its utility for chronic diseases such as PKD, we loaded the candidate PKD drug, metformin, into chitosan nanoparticles, and upon oral administration to a PKD murine model (Pkd1fl/fl;Pax8-rtTA;Tet-O cre), a lower cyst burden was observed compared to free metformin, and was well tolerated upon repeated dosages. Blood urea nitrogen (BUN) and creatinine levels were similar to untreated mice, demonstrating kidney and biocompatibility health. Our study builds upon previous chitosan-based drug delivery approaches, and demonstrates a novel, oral nanoformulation for PKD.

A microfluidic cell-migration assay for the prediction of progression-free survival and recurrence time of patients with glioblastoma.

Clinical scores, molecular markers and cellular phenotypes have been used to predict the clinical outcomes of patients with glioblastoma. However, their clinical use has been hampered by confounders such as patient co-morbidities, by the tumoral heterogeneity of molecular and cellular markers, and by the complexity and cost of high-throughput single-cell analysis. Here, we show that a microfluidic assay for the quantification of cell migration and proliferation can categorize patients with glioblastoma according to progression-free survival. We quantified with a composite score the ability of primary glioblastoma cells to proliferate (via the protein biomarker Ki-67) and to squeeze through microfluidic channels, mimicking aspects of the tight perivascular conduits and white-matter tracts in brain parenchyma. The assay retrospectively categorized 28 patients according to progression-free survival (short-term or long-term) with an accuracy of 86%, predicted time to recurrence and correctly categorized five additional patients on the basis of survival prospectively. RNA sequencing of the highly motile cells revealed differentially expressed genes that correlated with poor prognosis. Our findings suggest that cell-migration and proliferation levels can predict patient-specific clinical outcomes.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.