Splenic hemangiosarcoma (HSA) is often called a “silent killer” because these tumors are usually not detected in dogs until the disease has advanced to the point to where it is no longer treatable. Currently, the field is missing the level of molecular detail needed to develop new, more effective, evidence-based diagnostic tools and therapies. We have carried out Chromatin- Run- On sequencing (ChRO-seq) analysis on HSA tumor and normal splenic tissues. ChRO-seq maps bound RNA polymerase in order to quantify nascent transcriptional activity throughout the genome. Our preliminary studies revealed that the genes associated with extracellular matrix remodeling (ECM) were significantly upregulated in HSA tissue. We hypothesize that factors within the ECM remodeling pathway are overrepresented in HSA and will likely function as novel biomarkers and therapeutic targets for this disease. In this proposal, we will test our hypothesis with 3 objectives: 1) Define the active gene regulatory elements in HSA tumor tissue samples using ChRO-seq. 2) Identify molecular biomarkers that can distinguish HSA from benign lesions using ChRO-seq, qPCR and immunohistochemistry. 3) Define the transcriptional profiles of specific histological regions within HSA tumors by performing Laser Capture Microdissection coupled with RNA-sequencing (LCM-Seq). This proposed research will provide a better understanding of the unique molecular drivers of HSA and will also facilitate in the development of HSA-specific biomarkers that could be used for rapidly identifying HSA in the clinics and in high risk dogs prior to metastatic spread.