Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids.

Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

Relative Risk Assessment for Substandard Antibiotics Along the Manufacturing and Supply Chain: A Proof-of-Concept Study.

BACKGROUND: Ensuring good quality of antibiotics is essential for desired health outcomes. Risk assessment of products for quality issues arising along the manufacturing and supply chain can thus have an important role in surveillance and management of interventions designed to reduce the burden of substandard antibiotics. Demonstrated and validated risk assessments are currently limited. OBJECTIVES: The objective of this study was to investigate whether a comparative risk assessment framework, which adapts the WHO criteria for estimating risks for quality issues posed by individual medicines, is applicable and can identify antibiotics with a higher relative risk of substandard prevalence. METHODS: For a proof-of-concept study, a set of antibiotics from the WHO essential medicines list was selected. Quantitative and qualitative data were extracted for each risk assessment criteria pertaining to severity and probability. A final risk matrix was then compared to field data for validation. RESULTS: Antibiotic products were classified by relative risk. Of all the antibiotic products assessed (n = 28), 32% were categorized as highest risk, 46% as high risk, 18% as medium risk, and 4% as lowest risk. The comparison of the risk scores and incidence of quality failure from the USP Medicines Quality Database showed significant correlation. CONCLUSION: The framework and extracted data sets appear applicable to determine relative risk for substandard antibiotics. Results of the risk matrix may be valuable for guiding pharmacovigilance, surveillance strategies, standardizing risk-based approaches, and mitigation efforts. Refinement with increased data availability may improve results.