Two-plex <i>in vivo</i> molecular imaging in the second near-infrared window for immunotherapeutic response

Tumor-infiltrating CD8⁺ T cells and programmed death-1 (PD1) levels are critical indicators for tumor immunophenotyping and therapeutic decision-making. Noninvasive optical imaging in the second near infrared window (NIR-II) is particularly well-suited for investigating the biological processes within tumors in live mammals, thanks to its deep-tissue penetration and superior spatiotemporal resolution. However, <i>in vivo</i> NIR-II imaging has primarily been restricted to a single probe at a time. <b>Methods:</b> Herein, we developed a two-plex NIR-II molecular imaging method utilizing the non-overlapping fluorescence emission of indocyanine green (ICG) in the NIR-IIa window (1000-1200 nm) and PbS/CdS core-shell quantum dots (QDs) in the NIR-IIb window (1500-1700 nm). By integrating PD1 aptamer-labeled ICG (ICG-Apt-PD1, targeting PD1) and CD8 aptamer-labeled QDs (QDs@Apt-CD8, targeting CD8⁺ T cells), our two-plex NIR-II molecular imaging enabled simultaneous and noninvasive monitoring of the number of CD8⁺ T cells and PD1 levels in tumors. <b>Results:</b> QDs@Apt-CD8 demonstrated the excellent ability for <i>in vivo</i> imaging of tumor infiltrating CD8⁺ T cells, owing to its strong NIR-IIb luminescence and the high selectivity and specificity. This two-plex <i>in vivo</i> molecular imaging allowed for dynamic monitoring for PD1 levels and the number of CD8⁺ T cells in tumors. We observed the heterogeneous bio-distributions of PD1 and CD8⁺ T cells across different tumor types and revealed the tumor immunophenotypes. Moreover, our findings indicated that the low PD1 and high CD8⁺ T cells levels in tumors predicted a better anti-tumor effect. <b>Conclusions:</b> Such <i>in vivo</i> noninvasive NIR-II molecular imaging would complement <i>ex vivo</i> biopsy-based diagnostic techniques, and it could contribute to developing an <i>in vivo</i> tumor immune-scoring algorithm to offer a more precise prediction for immunotherapeutic response.