Simultaneous detection of lymphocytes and tumor cells <i>in vivo</i> in response to STING-TLR9 immunotherapy with Raman active multiplexed gold nanostars

Immunotherapies show heterogeneous response in patients and identifying those likely to benefit from these therapies remains challenging. This is in part because histopathology, the current clinical standard, cannot accurately predict response. Dynamic changes occur in both tumor cells and immune cells <i>in vivo</i> during and after treatment which are not captured by histopathology or by single biomarker imaging. To address this urgent need, this study leverages multiplexed profiling of both CD8⁺ T cells and VEGFR2⁺ expressing tumor cells in 4T1 murine breast cancer tumors with surface-enhanced Raman spectroscopy (SERS) using multiplexed gold nanostars (MGNs). MGNs are conjugated with antibodies targeting each cell type and Raman labels to enable multiplexing. Real time SERS <i>in vivo</i> imaging enables detection of dynamic longitudinal changes in CD8 and VEGFR2 in response to STING + TLR9 (stimulator of interferon genes + toll like receptor 9) immunotherapies, a treatment that increases tumor immunogenicity through a type I interferon response. MGNs also distinguished nonresponders of immunotherapies where 4T1 tumors were treated with antiOX40 antibodies. <i>In vivo</i> endpoints were validated <i>ex vivo</i> with flow cytometry analysis of immune cell population, cytokine analysis, STING activation, and immunofluorescence (IF) imaging of key markers (CD8, VEGFR, CD31, Ki67, and STING). Further, high resolution SERS maps provided a spatial context of CD8 and VEGFR2 distribution that showed the molecular makeup of tumors in responder and nonresponder mice. Biomarker distribution in <i>ex vivo</i> SERS aligned with <i>in vivo</i> findings and showed moderate to strong correlations <i>via</i> a Pearson's correlation to quantification of IF markers in tumors.