A: ROI analyses of in vivo radiotracer uptake (tumor-to-muscle ratio, mean±SD) at two early time points (1 and 4 hours post-injection) in the CT26/hPD-L1-nblk, CT26/hPD-L1-blk, and Raji-nblk mouse groups (n=3–5/group). The 64Cu-FN3hPD-L1 tracer uptake value (expressed as %ID/g) in the CT26/hPD-L1-nblk group (5.6±0.9) was significantly greater than in the CT26/hPD-L1-blk group (2.1±0.2, p<0.001) and in the Raji-nblk group (2.2±0.2, p<0.001) at 1 hour. B: Histogram of 64Cu-FN3hPD-L1 tracer biodistribution in the three groups (n=3/group). All mice were injected with 3.7 MBq of 64Cu-FN3hPD-L1 tracer via tail vein and euthanized at 24 hours post-injection. Organs were then isolated, and uptake of tracer dose was measured by gamma counter (decay-corrected mean %ID/g ±SD,). Pre-blocked mice (blk) received 10 mg/kg of non-radioactive FN3hPD-L1 via tail vein 2 hours before 64Cu-FN3hPD-L1 radiotracer injection whereas non-blocked mice (nblk) did not.