Bioluminescence can be used to distinguish patterns and timing of organ involvement for both human and mouse leukemia. (A) Prone, supine, and organ images of an NSG mouse injected with 1 × 10⁶ ffluc⁺ primary ALL-1 cells. Outlined 2D regions of interest correspond to organ involvement validated at day 3 of engraftment by isolated imaging. DLIT image of mouse internal anatomy, with skin and gut removed for clarity, reconstructed from images is shown (right panel). Green voxels represent point sources of light within putative target organs, verified by ex vivo imaging and flow cytometry. (B) Day 3 intensity maps of NSG mice injected with 1 × 10⁶ ffluc⁺ leukemia cells as indicated. These representative mice show the differing patterns of organ involvement at this early time point. Human ALL cells establish within the liver and bone marrow (primary ALL, RS4-11, and Nalm-6) or bone marrow only (380), whereas both mouse cell lines studied rapidly home to the spleen and bone marrow (t309 and 289). (C) Time course of leukemia engraftment reveals consistent bioluminescent correlation with appearance of peripheral blasts and maximal moribund disease burden. Nalm-6 (20-22 days), primary ALL-1 (40-50 days), primary ALL-2 (80-100 days), and primary ALL-3 (35-40 days) all become moribund at 2 × 10¹¹ photons/s/cm². Appearance of peripheral blasts (> 1%) happens consistently at systemic bioluminescent tumor burdens of > 1 × 10¹⁰ photons/s/cm². In ALL-2, the stable bioluminescent burden from 46 to 75 days corresponds with stable but high peripheral blast burden (30%-50%) before a progression to end stage between 80 and 100 days. Results are presented as mean ± SE (n = 5 for primary ALL samples, n = 10 for Nalm-6).