Monitoring Response to Combinatorial Immunotherapies by Tracking both T Cells and Natural Killer Cells <i>In Vivo</i> via ImmunoPET-Raman Multimodal Gold Nanostars

Real-time imaging of multiple immunomarkers <i>in vivo</i> is crucial to predict treatment response and to understand the complex interplay of immune cells such as CD8⁺ T cells and natural killer (NK) cells in the tumor microenvironment (TME). Since dynamic changes occur in these immune cells during combinatorial immunotherapies, these changes cannot be captured by single biomarker tracking or by <i>ex vivo</i> measurement of receptors at the endpoint of treatment. There is an unmet need for imaging probes that cohesively integrate the merits of multiple imaging modalities while surmounting the challenges of each modality. Here, we have developed multimodal gold nanostars (MGNs) functionalized with Raman reporters, immune-targeting antibodies, and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated ⁶⁴Cu radiolabels. These MGNs integrate the high sensitivity and whole-body imaging of immunoPET (positron emission tomography) with high spatial resolution and multiplexing of Raman spectroscopy for real-time <i>in vivo</i> detection of recruited CD8⁺ T cells and NKp46⁺ NK cells in the TME. MGNs predict treatment response to antiPD-L1 + antiCD47 combination immunotherapies in both immunologically "hot" CT26 murine tumors that are responders of therapy and immunologically "cold" 4T1 murine tumors that are poor responders. <i>In vivo</i> endpoints were validated with conventional assays (immunofluorescence, ELISA, and flow cytometry) to confirm immune cell recruitment. Our results show that MGNs enable imaging capabilities that cannot be achieved with either modality alone, allowing early detection of immunomarkers to improve therapeutic outcomes for responders and accelerate clinical decisions for poor responders.