LICs are enriched within a small tumor subpopulation with active Wnt signaling. (A) Schematic diagram of the integrated fluorescent Wnt reporters, 7TGP and 7TGC, used to mark leukemia cells with active Wnt signaling. The reporter element is composed of 7 Tcf/Lef-binding sites upstream of a minimal promoter and GFP, followed by a separate SV40-puromycin (7TGP) or SV40-Cherry (7TGC) selection marker. The construct backbone is a self-inactivating lentiviral vector. (B) Schematic diagram of experimental approach. Primary mouse NOTCH1-ΔE leukemias were explanted and transduced with 7TGP or 7TGC lentivirus. 7TGP-transduced cells were selected with 1 μg/ml puromycin on MS5-DL1 puro feeder cells for 5 days beginning on day 2 posttransduction. 7TGC-transduced cells were FACS-sorted on day 2 posttransduction. Transduced cells were then transplanted into recipient mice, all of which subsequently developed leukemia. These secondary 7TGP/7TGC-transduced leukemias were then analyzed by flow cytometry for GFP expression. GFP⁺ and GFP⁻ subsets were then FACS sorted (also Cherry⁺ in 7TGC experiments) and transplanted at limiting dilution into tertiary recipients. (C) Summary of GFP⁺ cell abundance in Wnt reporter-transduced leukemias after passage in vivo. Freshly explanted BM from clinically morbid recipients of 7TGP or 7TGC-transduced primary leukemias was analyzed by flow cytometry for GFP expression within gated NGFR⁺ (7TGP) or NGFR⁺ Cherry⁺ (7TGC) leukemia cell populations (labeled “2° leukemia” in [B]). Results depicted are compiled from 10 independent primary leukemias (26 recipients in total; 11 × 7TGP and 15 × 7TGC). Each data point represents an individual mouse. Mean ± standard deviation (SD) values are indicated by horizontal lines. (D) Survival of recipient mice after transplantation with FACS-sorted Wnt active (GFP⁺) or Wnt inactive (GFP⁻) subsets from 7TGP and 7TGC-transduced secondary leukemias. Each of 4 recipient animals (n = 4) was injected with each of the cell doses as indicated in parentheses. Two of 3 transplant experiments using independent primary leukemias are depicted. ****P < .0001 (log-rank test). LTR, long terminal repeat; sinLTR, self-inactivating LTR.