Summary Project 2– Defining the relationship between tumor composition, spatial heterogeneity, drug delivery, and drug efficacy The ultimate goal of this project is to determine the physical factors regulating therapeutic distribution and therapeutic efficacy in brain tumors. To this end, we have developed a highly innovative integrated strategy to quantitatively map therapeutic distribution with spatially registered characterization of the tumor architecture and therapeutic efficacy, all within a given tumor specimen. Specifically, in this approach we will combine MALDI-MSI to quantify drug distribution, stimulated Raman scattering imaging for label free analysis of tumor architecture with optical imaging resolution, immunohistochemistry to determine the tumor cell state and target distribution, proximity ligation assays for cellular spatial resolution of signaling response to therapy, and laser- capture microdissection RNASeq to quantify spatially resolved transcriptional response to therapy. All of these approaches will be performed in serial sections from individual tumors, thereby enabling the integration of spatially registered data. Together with mass spectrometry based phosphoproteomics and RNASeq analysis to quantify the dynamic signaling and transcriptional network response to a spectrum of defined drug concentrations in additional tumor specimens, the data generated in this project will (1) map spatially heterogeneous drug distribution and drug efficacy and (2) enable the computational modeling of the physical factors governing distribution and regulating the cellular and molecular response to different local drug concentrations. The novel integration of cutting-edge imaging and systems biology approaches applied to different sections of the same tumor, combined with computational models to identify the physical factors governing drug distribution and tumor cell response, will provide unprecedented insight into the complex dynamic behavior of tumor cells in vivo in response to spatially heterogeneous levels of therapeutics.