A Cell-based Computational Modeling Approach for Developing Site-Directed Molecular Probes

Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration

Combined expression of caveolin-1 and an activated AKT/mTOR pathway predicts reduced disease-free survival in clinically confined renal cell carcinoma

We previously reported that tumour-associated caveolin-1 is a potential biomarker in renal cell carcinoma (RCC), whose overexpression predicts metastasis following surgical resection for clinically confined disease. Much attention has recently focused on the AKT/mTOR pathway in a number of malignancies, including RCC. Since caveolin-1 and the AKT/mTOR signalling cascade are independently shown to be important regulators of tumour angiogenesis, we hypothesised that caveolin-1 interacts with the AKT/mTOR pathway to drive disease progression and metastasis in RCC. The aims of this study were to determine (i) the expression status of the activated AKT/mTOR pathway components (phosphorylated forms) in RCC and (ii) their prognostic value when combined with caveolin-1. Immunohistochemistry for caveolin-1, pAKT, pmTOR, pS6 and p4E-BP1 was performed on tissue microarrays from 174 clinically confined RCCs. Significantly decreased mean disease-free survival was observed when caveolin-1 was coexpressed with either pAKT (2.95 vs 6.14 years), pmTOR (3.17 vs 6.28 years), pS6 (1.45 vs 6.62 years) or p4E-BP1 (2.07 vs 6.09 years) than when neither or any one single biomarker was expressed alone. On multivariate analysis, the covariate of ‘caveolin-1/AKT' (neither alone were influential covariates) was a significant influential indicator of poor disease-free survival with a hazard ratio of 2.13 (95% CI: 1.15–3.92), higher than that for vascular invasion. Tumours that coexpressed caveolin-1 and activated mTOR components were more likely to be larger, higher grade and to show vascular invasion. Our results provide the first clinical evidence that caveolin-1 cooperates with an activated AKT/mTOR pathway in cancer and may play an important role in disease progression. We conclude that evaluation of the ‘caveolin-1/AKT/mTOR axis' in primary kidney tumours will identify subsets of RCC patients who require greater postoperative surveillance and more intensive treatment

The neurogenic potential of astrocytes is regulated by inflammatory signals

Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-β1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes