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Figure 1. Establishment of the mouse model of human secondary lung cancer. (A) MDA-MB-231 subline 3475 cell line stably transduced with lentiviral constructs for expression of TdTomato-luciferase (pUltraChili-Luc) and pCT-CD63-GFP. Confocal imaging of a single clone co-expressing both TdTomato and GFP, sorted by FACS [See Supplementary Figure 1], with DAPI staining to locate cellular nuclei (Blue color, left panel) and Td-tomato red protein expression (Red colored cells, second panel from the left) and the CD63-GFP protein on extracellular vesicles (EVs, Green dots, third panel from the left), with a Merged image all staining and fluorescent imaging on the right panel. CD63-GFP detection shows that cells at different mitotic stages produced different amounts of EVs as observable by the differential detection of GFP protein. The central dividing cell displays the highest amount of observable EVs; (B) Bioluminescent in vivo imaging of tumor-bearing mice at weeks 0, 6, and 12 after inoculation of TdTomato-Luc+/CD63-GFP+ MDA-MB-231 subline 3475 cells. Bioluminescence intensity is indicated by means of radiant efficiency (photons/sec/cm²/sr) scale bars, with red being the most intense (See scale bar); (C) Representative formalin-fixed whole lung tissue images collected from two healthy mice (control; left) and two TdTomato-Luc+/CD63-GFP+ MDA-MB-231 subline 3475 inoculated lung tumor-bearing mice (case; right); (D) Representative images of Hematoxylin and Eosin (H&E) stained 5 mm tissue sections of lungs harvested from one control (left panels; 0 × and 20 × magnifications) and one lung tumor-bearing animal (right panels; 0 × and 20 × magnifications). The image right panel from one lung tumor-bearing animal shows extensive infiltration of metastatic carcinoma legions with a few rare immature lymphocytes seen interspersed.