Physical Science in Cancer Research Pilot Program Grants Program (NBIC), 2011
Molecular Force Sensor
Department of Bioengineering
The ability of cancers to proliferate and invade has recently been shown to depend on integrin- mediated adhesion and mechanical tension generated between cells and their surrounding ECM. However, much of our understanding of when and where these cellular forces are generated are based on measurements on highly specialized deformable substrates. Here, we propose to develop a nanoscale force sensor that could be used in a wide variety of settings (2D and 3D) that currently are not amenable to force measurement. This tool would address a critical shortcoming in the field to quantify both the temporal and spatial cell tractions exerted by cancer cells and provide a quantitative window into this important normal and pathologic cellular physiology. We will develop a multicolored library of these mechanosensors that can spatially and temporally report specific cell traction amplitudes.
Real-time observation of phosphorylation dynamics. (A) A cell expressing a chromatin-targeted, FRET-based (for fluorescence resonance energy transfer) phosphorylation sensor for Aurora B was treated with a kinesin-5 inhibitor to create monopolar spindles; centromeres (labeled in red) are oriented towards the middle( chromosomes in green). (B) Cells were imaged live, and FRET changes at the indicated times were color coded. Scale bar 5 µm, time relative to kinase activation. Here, we proposed to reconstitute these reactions by using nanoparticles islands to mimic kinetochore, and by attaching FRET sensor to the coverslip.
Understanding cell division is important for developing new anti-cancer therapeutics. One of the most crucial regulators and possible targets for anti-cancer drugs is Aurora B kinase. During mitotic division Aurora B kinase orchestrates numerous events and is crucial for resolving erroneous kinetochore-microtubule interactions, thereby ensuring high accuracy of chromosome segregation. Aurora B kinase activity and intracellular localization are exquisitely regulated. Work towards understanding the underlying regulatory mechanisms is important, because it may lead to new strategies to diagnose and treat cancer. In this pilot grant, we will reconstitute key aspects of this regulation by using purified components and nano-engineered surfaces to assemble Aurora B-containing kinetochore complexes on the patterned nanoparticle surfaces. We build upon prior experience with nano-engineered surfaces to create targeted areas with high kinase activity, thereby mimicking the centremere region of the chromosomal. The spatio-temporal propagation of phosphorylation gradients will be dissected using our unique FRET biosensor method. A similar strategy will be used to create local islands of a crucial Aurora B substrate, Hec1 (Highly Expressed in Cancer), and interactions between differentially phosphorylated Hec1 proteins and the ends of microtubule polymers will be examined in real time. This challenging and highly novel approach at the interface of physics and biology is possible only because of ongoing rapid advancement in biochemical analysis of kinetochore composition and developments in nano-biotechnology. These findings will have significant impact by advancing our understanding of the basic principles of Aurora B-dependent regulation of kinetochore-microtubule interactions, as well as of how the fidelity of chromosome segregation is achieved.
ENHANCING TUMOR BLOOD: BRAIN BARRIER PENETRATION BY DRUG-LOADED NANOCARRIER WITH TARGETED RADIATION THERAPY
Illustration of the strategy of Targeted Radiation Therapy to enhance the permeabilization and retention of Filomicelle drug-loaded Nanocarrier through the Brain Tumor blood:brain barrier
The effectiveness of anticancer treatment for brain tumors is limited by the inability of standard anticancer agents to penetrate the blood-brain-barrier (BBB) into the tumor. One of the main methods of treating brain tumors is radiation therapy (RT). RT has been reported to increase BBB permeability, but modulation of the BBB by targeted RT for therapeutic gain has surprisingly not been extensively studied. We therefore propose to investigate the efficacy in animal models of RT-induced modulation of the tumor BBB coupled with novel filomicelle drug-loaded nanocarriers (DLNs) that feature enhanced half-life in the systemic circulation. Our hypothesis is that radiation-induced modulation of the tumor BBB will facilitate the enhanced permeation and retention (EPR) of paclitaxel-loaded nanocarriers in brain tumors, resulting in increased efficacy.
DENDRITICALLY-STABILIZED UP-CONVERTING NANOPARTICLES - IMAGING PLATFORM FOR QUANTIFICATION OF TUMOR MICROENVIRONMENT
Cancers are complex, evolving, multiscale systems that are characterized by high spatial and temporal heterogeneity. Quantitative dynamic information about the tumor microenvironment is the key to the ability to manipulate tumor physiological status in order to enhance the susceptibility of tumors to existing treatment modalities and to develop new anti-cancer protocols. Nevertheless, characterizing the local microenvironment of tumors on a microscopic scale is a challenging problem, and there is a need to established imaging technology to provide that insight. Here, we propose to develop a comprehensive imaging platform based on dendritically-stabilized up-converting lanthanide nanoparticle phosphors (UCNPs) for real-time quantification of tumor physiological parameters with high spatial and temporal resolution. These dendronized UNPC systems can be excited by deep penetrating near infrared radiation and internal upconversion that locally initiates shorter wavelength photodynamics with in the tumors, probing the local chemical environment. In this project, we focus specifically on imaging oxygen and pH – the two key variables of the tumor microenvironment.
RATIONAL DESIGN AND FACILE SYNTHESIS OF A LIBRARY OF POLYMERIC HOLLOW NANOPARTICLES AS MRNA CARRIERS FOR SAFE AND EFFICIENT CANCER THERAPEUTICS
(A) Amphiphilic polymer nanoparticles as mRNA carrriers and their SEM image and (B) schematics of three possible associations of NPs to the cell membrane: suspended in solution, partially wrapped with different degrees of wrapping, and endocytosed.
Recent advances in molecular biology, nanotechnology and polymer science open up new exciting possibilities in gene therapeutics, where polymeric nanoparticles (NPs) are used as drug and/or gene carriers for highly efficient and safe cancer therapeutics. We propose to develop a transient gene therapy that both delivers immune adjuvants and proteins that will lead to tumor necrosis. By combining these genes and transiently delivering them by complexation of modified messenger RNA (mRNA) with polymeric NPs, we hope to develop a safe and effective approach to inducing potent and curative immune responses without the need to obtain tumor cells for ex vivo manipulation. This will avoid current approaches to cancer vaccination that must first obtain tumor cells to either isolate RNA or proteins or modify with new genes.