Our purpose in TR&D1 is to develop and validate novel translatable molecular imaging and theranostic agents to detect and promote an immune reactive tumor microenvironment (TME) that will be integrated into the research of multiple collaborative and service projects. Immune checkpoint inhibitors have provided some of the most exciting treatment outcomes for some cancers, but for nearly 80% of cancers the promise of cancer immunotherapy (CIT) has not been realized. Cancers have complex TMEs with an altered extracellular matrix, hypoxia and acidic extracelluar pH (pHe) that present a formidable barrier to immune cell infiltration and cancer cell killing. Non-invasive translatable imaging methods that provide spatio-temporal information on mechanisms that create barriers to CIT and provide novel theranostic strategies to improve the outcome of CIT are urgently needed to accelerate progress in this field. During the previous funding period we have made significant advances in multimodal imaging of the TME and siRNA theranostics that we have built upon and combined with our expertise in PET/MR imaging, chemical exchange saturation transfer (CEST), theranostics and CIT to design three aims that will identify a TME hostile to CIT, and provide novel theranostic approaches to overcome such an environment to facilitate CIT. We have focused on breast and pancreatic preclinical cancer models in these studies as these cancers have limited response to CIT. In Aim 1 we will develop novel PET and CEST MRI probes based on decorin to expand our understand of the spatio-temporal dynamics between collagen 1, decorin, hypoxia, pHe, and immune checkpoint PD-L1/PD-1 expression in syngeneic models of pancreatic and breast cancer using multimodal imaging. In Aim 2 we will develop and validate multi-modality imaging/multiplexed siRNA theranostics of myeloid derived suppressor cells (MDSCs) in the spleen and tumor to create an immune reactive TME in these tumor models. In Aim 3 we will develop hypoxia targeted NPs to deliver siRNA or PX-478 to downregulate HIF-1 in tumors to understand the effects of HIF-1 downregulation on PD-L1/PD-1 expression. The reagents and resources developed in the three aims will be incorporated in a push pull mechanism by the CPs and by the SPs to collectively advance progress in CIT.