Detecting multiple molecular markers related to immune cells in vivo can revolutionize tumor phenotyping, monitor the effects of immunotherapy, and provide personalized treatment. Raman imaging with surface enhanced Raman scattering (SERS) nanoprobes is an optical imaging technique with high sensitivity and unparalleled multiplexing capacity. Here, we present a generalized methodology for the synthesis and visualization of SERS nanoprobes to detect multiple targets in vivo after intravenous administration in mouse models of cancer. A multiplexed imaging approach allows simultaneous scanning, unmixing, quantification, and visualization of the nanoprobes. We use this method to monitor the response of tumor-bearing mice (murine breast and colon cancer) to immune checkpoint blockade therapy. We visualize 8 immunological markers simultaneously, with subsequent immunohistological verification of each target. A spatially-aware multivariate approach is used for the segmentation of the acquired spectral images, which allowed their subsequent classification into responders or non-responders using machine learning.
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