(A) Immunocytochemical detection of calbindin in dissociated HARA or HARA 3D5 cell pellets (left). Scale bars: 100 μm. Insets are 10× magnified images of HARA cells. Percentage of calbindin⁺ cells in the same preparations (right). (B) Immunofluorescence detection of calbindin in HARA cell cultures (left). Scale bars: 20 μm. Percentage of calbindin⁺ cells in the same cultures (right). (C) Immunocytochemical detection of calbindin in HARA cells grown in 3D collagen matrices (left). Scale bars: 50 μm. Percentage of calbindin⁺ cells in the same cultures (right). In A–C, symbols are averages of independently acquired images. (D) H&E staining (left) and calbindin immunostaining (right) of sections of HARA cell tumors growing in the lungs of Rag2^–/–Il2rg^–/–Cd47^–/– recipients. Scale bars: 200 μm. (E) HERVH-CALB1 expression in TRACERx-100 LUSC patient samples (EGAD00001004591). Symbols represent individual tumor regions from each patient. Only patients with at least 2 regions sampled are shown. (F) HERVH-CALB1 expression according to the evolutionary history of each region from representative samples from E. Circles denote the positions of the cancer cell subpopulations sampled in each region from a given patient on the constructed phylogenetic tree (gray lines) for all regions in that patient. The areas enclosed by the circles represent the proportions of each cancer cell subpopulation in the sampled region. (G). Flow cytometric example of gating of HARA.LTR7-GFP cells, according to GFP expression, used from the purification of positive and negative subpopulations (left). Time course of GFP expression in cells that were initially LTR7-GFP^– and LTR7-GFP⁺ HARA.LTR7-GFP cells in 2 separate cultures (middle and right, respectively).