The drug development process is inefficient and expensive, in part because most therapeutics are initially screened with two-dimensional cell cultures. These cell cultures do not accurately reflect tissue structures in the human body. To improve drug screening, more accurate model systems should be used. Three- dimensional cell cultures, also known as spheroids, fill this gap. Similar to a tumor, colon cancer spheroids contain radially symmetric nutrient and oxygen gradients. The spheroid develops distinct cellular populations that reflect these chemical gradients. In this proposal, we are developing an analytical approach, called Spatial SILAC, to specifically label the divergent cellular populations present within spheroids using different isotopes.
In Aim 1, we will show that these isotopes can be readily distinguished by a mass spectrometer, thus providing an isotopic ?zip code? of the cell?s origin within a spheroid.
In Aim 2, we will combine imaging mass spectrometry drug distribution studies with an evaluation of proteomic changes in response to therapeutics, using the isotopic labels. We will evaluate irinotecan, 5-fluorouracil and Cetuximab as we have previously examined the distribution of these three drugs in spheroids by imaging mass spectrometry. We will compare the spatially localized proteomic changes in response to therapeutic treatment with the imaging distribution maps.
In Aim 3, we will apply our Spatial SILAC approach to screen newly developed cyclic penetrating peptides (CPPs), designed by Co-I Professor Dehua Pei. These promising cancer drug candidates require preclinical screening and Spatial SILAC in the spheroids provides an ideal testbed to evaluate their in vitro pharmacokinetics and pharmacodynamics. We will first perform imaging mass spectrometry studies to determine the distribution of the CPPs and their metabolites in the spheroids. We will then evaluate both the targeted and off-targeted proteomic changes caused by these drugs by Spatial SILAC. The results generated in this aim will be used to optimize the design of future CPPs. In summary, this proposal describes a novel mass spectrometric approach that will streamline the drug development process, while also providing rich in vitro pharmacokinetic and pharmacodynamic information on new therapeutic candidates.
The primary goal of this research is to develop Spatial SILAC?an approach to apply distinctive labels to the different cell populations found in tumor mimics. We can then use the tumor mimics to test possible drug candidates and infer the responses of the different types of cells using the specific labels. Our approach will let us evaluate both the drug and the tumor?s response to a drug at the same time and this valuable information will be used to improve drug design.
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