Project Summary The -omics era has made it possible to identify several molecular markers involved in predicting survival and response to therapies. We currently lack an easy way to obtain high content molecular information while providing high resolution spatial profiling across a patient’s tissue. This proposal aims to provide physicians with an entirely new multiplexed molecular imaging technology that has the potential to offer both high content molecular expression and spatial profiling in a single histology image. Raman spectroscopy in conjunction with surface enhanced Raman scattering (SERS) nanoparticles (NPs) is an optical imaging technique that can offer unsurpassed sensitivity and multiplexing capabilities to the field of histology imaging with the potential to provide rich molecular details on the microscopic level. Clinicians will be able to utilize the imaging strategy on the same tissue sections prepared for histology. Incorporating it into the pathology workflow could enable physicians to better understand the patient’s molecular profile and stratify patients to receive the most effective therapeutic regimen possible. This unique histology imaging strategy also has the potential to identify new molecular trends in patient’s tissue samples that could be used to predict how aggressive their disease is or how well the patient is likely to respond to given therapies. This innovative ex-vivo diagnostic strategy has a high likelihood for clinical translation, offering rapid whole tissue section imaging for multiple molecular biomarkers simultaneously. Our approach begins by developing a new set of sensitive SERS NP batches, each designed with a unique spectral barcode to enable simultaneous molecular interrogation of an entire tissue sample within a single image. After fabrication and characterization of our newly developed multiplexed SERS NPs, we will test their multiplexed imaging capabilities and targeting efficiency on various biomarkers in cell culture and on de- identified human tissue sections. We will first test our new multiplexed imaging technology to target immune cells. Recently, studies have shown that the immune system plays a key role in cancer development, and thus the density, location, and type of immune cells found across a patient’s tumor can predict therapeutic response. Failure to fully understand the immune profile and tumor heterogeneity across a patient’s tumor can lead to administration of ineffective therapies that increase patient morbidity. Our NPs will actively target multiple immune receptors through chemically conjugated antibodies. We will assess the targeting efficiency of our newly developed NPs with microscopic Raman imaging tools and compare with gold standard immunohistochemistry (IHC) staining. These results will be an important step in the clinical translation of this new multiplexed Raman imaging approach; to provide rapid spatial molecular profiling while enabling improved personalized therapy....