Patient-derived response estimates from zero-passage organoids of luminal breast cancer.

Background: Primary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations. Methods: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 µl replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these "zero-passage" organoids by withdrawing ß-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times. Results: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined. Conclusions: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.

Identification and Evaluation of Brominated Carbazoles as a Novel Antibiotic Adjuvant Scaffold in MRSA.

Antibiotic-resistant infections are a pressing global concern, causing millions of deaths each year. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections in healthcare settings and is increasingly responsible for community-acquired infections that are often more difficult to treat. Antibiotic adjuvants are small molecules that potentiate antibiotics through nontoxic mechanisms and show excellent promise as novel therapeutics. Screening of low-molecular-weight compounds was employed to identify novel antibiotic adjuvant scaffolds for further elaboration. Brominated carbazoles emerged from this screening as lead compounds for further evaluation. Lead carbazoles were able to potentiate several ß-lactam antibiotics in three medically relevant strains of MRSA. Gene expression studies determined that these carbazoles were dampening the transcription of key genes that modulate ß-lactam resistance in MRSA. The lead brominated carbazoles represent novel scaffolds for elaboration as antibiotic adjuvants.

Adaptable Xerogel-Layered Amperometric Biosensor Platforms on Wire Electrodes for Clinically Relevant Measurements.

Biosensing strategies that employ readily adaptable materials for different analytes, can be miniaturized into needle electrode form, and function in bodily fluids represent a significant step toward the development of clinically relevant in vitro and in vivo sensors. In this work, a general scheme for 1st generation amperometric biosensors involving layer-by-layer electrode modification with enzyme-doped xerogels, electrochemically-deposited polymer, and polyurethane semi-permeable membranes is shown to achieve these goals. With minor modifications to these materials, sensors representing potential point-of-care medical tools are demonstrated to be sensitive and selective for a number of conditions. The potential for bedside measurements or continuous monitoring of analytes may offer faster and more accurate clinical diagnoses for diseases such as diabetes (glucose), preeclampsia (uric acid), galactosemia (galactose), xanthinuria (xanthine), and sepsis (lactate). For the specific diagnostic application, the sensing schemes have been miniaturized to wire electrodes and/or demonstrated as functional in synthetic urine or blood serum. Signal enhancement through the incorporation of platinum nanoparticle film in the scheme offers additional design control within the sensing scheme. The presented sensing strategy has the potential to be applied to any disease that has a related biomolecule and corresponding oxidase enzyme and represents rare, adaptable, sensing capabilities.

Functionalized carbon nanotube adsorption interfaces for electron transfer studies of galactose oxidase.

Modified electrodes featuring specific adsorption platforms able to access the electrochemistry of the copper containing enzyme galactose oxidase (GaOx) were explored, including interfaces featuring nanomaterials such as nanoparticles and carbon nanotubes (CNTs). Electrodes modified with various self-assembled monolayers (SAMs) including those with attached nanoparticles or amide-coupled functionalized CNTs were examined for their ability to effectively immobilize GaOx and study the redox activity related to its copper core. While stable GaOx electrochemistry has been notoriously difficult to achieve at modified electrodes, strategically designed functionalized CNT-based interfaces, cysteamine SAM-modified electrode subsequently amide-coupled to carboxylic acid functionalized single wall CNTs, were significantly more effective with high GaOx surface adsorption along with well-defined, more reversible, stable (≥ 8 days) voltammetry and an average ET rate constant of 0.74 s-1 in spite of increased ET distance - a result attributed to effective electronic coupling at the GaOx active site. Both amperometric and fluorescence assay results suggest embedded GaOx remains active. Fundamental ET properties of GaOx may be relevant to biosensor development targeting galactosemia while the use functionalized CNT platforms for adsorption/electrochemistry of electroactive enzymes/proteins may present an approach for fundamental protein electrochemistry and their future use in both direct and indirect biosensor schemes.

First Generation Amperometric Biosensing of Galactose with Xerogel-Carbon Nanotube Layer-By-Layer Assemblies.

A first-generation amperometric galactose biosensor has been systematically developed utilizing layer-by-layer (LbL) construction of xerogels, polymers, and carbon nanotubes toward a greater fundamental understanding of sensor design with these materials and the potential development of a more efficient galactosemia diagnostic tool for clinical application. The effect of several parameters (xerogel silane precursor, buffer pH, enzyme concentration, drying time and the inclusion of a polyurethane (PU) outer layer) on galactose sensitivity were investigated with the critical nature of xerogel selection being demonstrated. Xerogels formed from silanes with medium, aliphatic side chains were shown to exhibit significant enhancements in sensitivity with the addition of PU due to decreased enzyme leaching. Semi-permeable membranes of diaminobenzene and resorcinol copolymer and Nafion were used for selective discrimination against interferent species and the accompanying loss of sensitivity with adding layers was countered using functionalized, single-walled carbon nanotubes (CNTs). Optimized sensor performance included effective galactose sensitivity (0.037 µA/mM) across a useful diagnostic concentration range (0.5 mM to 7 mM), fast response time (~30 s), and low limits of detection (~80 µM) comparable to literature reports on galactose sensors. Additional modification with anionic polymer layers and/or nanoparticles allowed for galactose detection in blood serum samples and additional selectivity effectiveness.