Metabolomics and cytokine profiling of mesenchymal stromal cells identify markers predictive of T-cell suppression.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have shown great promise in the field of regenerative medicine, as many studies have shown that MSCs possess immunomodulatory function. Despite this promise, no MSC therapies have been licensed by the Food and Drug Administration. This lack of successful clinical translation is due in part to MSC heterogeneity and a lack of critical quality attributes. Although MSC indoleamine 2,3-dioxygnease (IDO) activity has been shown to correlate with MSC function, multiple predictive markers may be needed to better predict MSC function. METHODS: Three MSC lines (two bone marrow-derived, one induced pluripotent stem cell-derived) were expanded to three passages. At the time of harvest for each passage, cell pellets were collected for nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography mass spectrometry (MS), and media were collected for cytokine profiling. Harvested cells were also cryopreserved for assessing function using T-cell proliferation and IDO activity assays. Linear regression was performed on functional data against NMR, MS and cytokines to reduce the number of important features, and partial least squares regression (PLSR) was used to obtain predictive markers of T-cell suppression based on variable importance in projection scores. RESULTS: Significant functional heterogeneity (in terms of T-cell suppression and IDO activity) was observed between the three MSC lines, as were donor-dependent differences based on passage. Omics characterization revealed distinct differences between cell lines using principal component analysis. Cell lines separated along principal component one based on tissue source (bone marrow-derived versus induced pluripotent stem cell-derived) for NMR, MS and cytokine profiles. PLSR modeling of important features predicted MSC functional capacity with NMR (R2 = 0.86), MS (R2 = 0.83), cytokines (R2 = 0.70) and a combination of all features (R2 = 0.88). CONCLUSIONS: The work described here provides a platform for identifying markers for predicting MSC functional capacity using PLSR modeling that could be used as release criteria and guide future manufacturing strategies for MSCs and other cell therapies.

Dynamic Model of Protease State and Inhibitor Trafficking to Predict Protease Activity in Breast Cancer Cells.

INTRODUCTION: Cysteine cathepsins are implicated in breast cancer progression, produced by both transformed epithelial cells and infiltrated stromal cells in tumors, but to date, no cathepsin inhibitor has been approved for clinical use due to unexpected side effects. This study explores cellular feedback to cathepsin inhibitors that might yield non-intuitive responses, and uses computational models to determine underlying cathepsin-inhibitor dynamics. METHODS: MDA-MB-231 cells treated with E64 were tested by multiplex cathepsin zymography and immunoblotting to quantify total, active, and inactive cathepsins S and L. This data was used to parameterize mathematical models of intracellular free and inhibited cathepsins, and then applied to a dynamic model predicting cathepsin responses to other classes of cathepsin inhibitors that have also failed clinical trials. RESULTS: E64 treated cells exhibited increased amounts of active cathepsin S and reduced amount of active cathepsin L, although E64 binds tightly to both. This inhibitor response was not unique to cancer cells or any one cell type, suggesting an underlying fundamental mechanism of E64 preserving activity of cathepsin S, but not cathepsin L. Computational models were able to predict and differentiate between inhibitor-bound, active, and inactive cathepsin species and demonstrate how different classes of cathepsin inhibitors can have drastically divergent effects on active cathepsins located in different intracellular compartments. CONCLUSIONS: Together, this work has important implications for the development of mathematical model systems for protease inhibition in tissue destructive diseases, and consideration of preservation mechanisms by inhibitors that could alter perceived benefits of these treatment modalities.

Endothelial cells and cathepsins: Biochemical and biomechanical regulation.

Cathepsins are mechanosensitive proteases that are regulated not only by biochemical factors, but are also responsive to biomechanical forces in the cardiovascular system that regulate their expression and activity to participate in cardiovascular tissue remodeling. Their elastinolytic and collagenolytic activity have been implicated in atherosclerosis, abdominal aortic aneurysms, and in heart valve disease, all of which are lined by endothelial cells that are the mechanosensitive monolayer of cells that sense and respond to fluid shear stress as the blood flows across the surfaces of the arteries and valve leaflets. Inflammatory cytokine signaling is integrated with biomechanical signaling pathways by the endothelial cells to transcribe, translate, and activate either the cysteine cathepsins to remodel the tissue or to express their inhibitors to maintain healthy cardiovascular tissue structure. Other cardiovascular diseases should now be included in the study of the cysteine cathepsin activation because of the additional biochemical cues they provide that merges with the already existing hemodynamics driving cardiovascular disease. Sickle cell disease causes a chronic inflammation including elevated TNFα and increased numbers of circulating monocytes that alter the biochemical stimulation while the more viscous red blood cells due to the sickling of hemoglobin alters the hemodynamics and is associated with accelerated elastin remodeling causing pediatric strokes. HIV-mediated cardiovascular disease also occurs earlier in than the broader population and the influence of HIV-proteins and antiretrovirals on endothelial cells must be considered to understand these accelerated mechanisms in order to identify new therapeutic targets for prevention.