PROJECT SUMMARY/ABSTRACT Despite the unprecedented effects of immunotherapy in treating cancer, over 50–80% of patients will not respond to treatment and will endure severe adverse and life-threatening events due to treatment. Approaches to sensitize tumors to immunotherapy are urgently being explored. One very promising approach is the use of radiation therapy due to known immunomodulating effects. Another promising approach is to target tumor endothelial cells with pharmaceuticals to minimize their immunosuppressive properties. In normal tissues, endothelial cells act as a regulating gateway for the immune system. In cancer, endothelial cells are highly abnormal and immunosuppressive. We propose to explore novel approaches to enhance tumor immunogenicity by mechanical targeting of tumor endothelial cells. This goes beyond pharmaceutical targeting localizing the treatment and minimizing systemic effects or adverse events. Our previous research demonstrated that ultrasound-stimulated microbubbles (USMB) can activate mechanotransduction pathways in tumor endothelial cells through mechano-acoustic forces, which in turn significantly enhance radiotherapy. Given that endothelial immunogenicity is known to be directly affected by mechanical forces, and that radiation is a known immunomodulator, we posit that USMB treatments can enhance the effects of immunotherapeutic strategies that may include radiation. Our proposed research includes: i) characterization of tumor endothelium immunogenicity resulting from mechanical forces, and ii) pre-clinical assessment of combined treatment regimens that include USMB, immunotherapy and/or radiation. Through this work, we will also gain a deeper understanding of the role of endothelial cells in immunotherapy, and whether mechanical forces can alter endothelial surface marker expression and general immunogenicity. Equipped with this research, our goal will be to initiate a larger research program designed to introduce mechano-acoustic forces for immunomodulation in cancer and in other immune-based diseases, and to study these effects in more advanced and clinically- relevant biological models. past year. In addition, as