Shoshana Bloom – Neil Forbes Lab
Targeting nuclear proteins for cancer therapy
Shoshana Bloom, Neil Forbes
Salmonella preferentially accumulate in tumors over other organs, providing a targeted vector for anti-cancer therapeutics and overcoming transport limitations of other anti-cancer therapies. Additionally, Salmonella invade cancerous cells and can lyse when specific promoters are activated. Following lysis, cytosolic contents of Salmonella flow into cancerous cells. This allows for the use of therapeutic agents that normally cannot penetrate eukaryotic cells to target intracellular signaling pathways and organelles, specifically the nucleus. Nuclear import is controlled by nuclear pore complexes (NPC) and importins, which recognize nuclear localization sequences (NLS). Nonpathogenic Salmonella were engineered to produce ovalbumin (OVA) or green fluorescent protein (GFP) with and without a NLS and to lyse intracellularly. OVA-NLS accumulated in the nucleus more frequently than OVA. However, this trend was not observed with GFP, which is a much smaller protein. This suggests that smaller proteins, even when delivered by Salmonella, are able to bypass NPC channels while larger proteins cannot. Additionally, in some instances, clear nuclei could be observed in cells invaded with Salmonella producing OVA-NLS, which did not occur for cells invaded with bacteria producing OVA. By delivering proteins with Salmonella, proteins tagged with a NLS can reach the nucleus, opening up the range of potential targets for anti-cancer therapies.
Aritra Nath Kundu – Shelly Peyton Lab
Engineering a synthetic 3D lung
Aritra Nath Kundu1, Lauren E Jansen1, Sualyneth Galarza1, Samar A Mahmoud2, Samuel R Polio1, Shelly R Peyton1, 2
1Department of Chemical Engineering, University of Massachusetts, Amherst
2Molecular and Cellular Biology Department, University of Massachusetts, Amherst
Lung is a viscoelastic and mechanically robust tissue capable of sustaining its structural and functional integrity over millions of pulmonary expansion and contraction cycles. Lung parenchyma, the area of the lung consisting of interstitium and alveolar wall and involved in gas exchange, derives its mechanical integrity from the extracellular matrix (ECM), which is primarily composed of elastin, laminins, and collagens. Besides providing structural integrity to the tissue, the ECM also regulates the diverse functions and phenotypes of contractile cells, including smooth muscle cells and fibroblasts by providing biochemical and biomechanical cues, primarily through ECM protein composition and tissue stiffness respectively . On the other hand, remodeling of the ECM by fibroblasts has been suggested to regulate cell function and phenotype, during injuries and chronic pathological conditions by creating fibrotic ECM . To better understand this feedback interaction between cells and ECM during tissue homeostasis and disease, an in vitro experimental model is needed to mimic the lung ECM in a controllable and reproducible manner, as currently existing in vitro lung tissue derived natural hydrogel platforms, such as decellularized lung ECM hydrogels mimic some aspects of the ECM architecture, but the compositions of these hydrogels are neither consistent nor controllable .
Toward achieving this goal, our lab has developed a maleimide-terminated poly(ethylene glycol) (PEG-MAL) based hydrogel system that can be crosslinked to mimic the stiffness of soft tissues and be coupled with peptides to mimic the ECM protein composition. To determine the mechanics for this material, we measured the Young’s modulus of lung tissue using multiple mechanical characterization techniques, including cavitation rheology (6.1±1.6 kPa), small amplitude oscillatory shear (3.3±0.5 kPa), micro-indentation (1.4±0.4 kPa), and uniaxial testing (3.4±0.4 kPa) . Moreover, we combined proteomics and bioinformatics to develop an approach for identifying the protein complexity of the lung ECM. We identified and quantified the ECM proteins of the lung using mass spectrometry data obtained from multiple healthy human lung samples, and bioinformatics data obtained from protein atlas , and identified 18 lung-specific cell-instructive peptides corresponding to previously identified proteins, that will be incorporated in the hydrogel to make a “lung-like” hydrogel cell culture environment. Separately, we are developing a new protocol to validate the specific location of these ECM proteins with imaging mass spectrometry (IMS). This method employs matrix assisted laser dissociation/ionization – time of flight (MALDI-TOF) mass spectrometry to identify the proteins, which first needs to be digested to ensure better identification. The preliminary IMS data will provide us with the spatial distribution of these proteins. In parallel, we are exploring which healthy cells of the lung produce these ECM proteins, by stimulating ECM production by lung fibroblasts, the most commonly found cell type in the alveolar wall of lung parenchyma. Our in vitro lung ECM model mimics several key aspects of the lung ECM including stiffness, and proteomic landscape, and can be further used to explore hypothesis-driven mechanistic studies important for regenerative medicine and disease.
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