Fig. 4.

Functional validation of the computationally predicted differentiation states in BC. In vivo validation of three phenotypically distinct subtypes of bladder cancer (BC) according to their differentiation states: (A) basal, (C) intermediate, and (E) differentiated as defined by surface marker profiles (CD90, CD44, and CD49f). BC cells were purified by FACS and xenotransplanted intradermally into immunodeficient mice in limited dilution (10³ and 10⁴). (B, D, and F) The immunophenotypes of xenograft tumors derived from each BC subtype were reanalyzed by FACS postengraftment. (A) The basal BC subtype is composed of all differentiation states [CD90⁺CD44⁺CD49f⁺ (red box) → CD90⁻CD44⁺CD49f⁺ (blue box) → CD90⁻CD44⁻CD49f⁺ (green box) → CD90⁻CD44⁻CD49f⁻ (light blue box)]. (B) Only the most upstream (CD90⁺CD44⁺CD49f⁺) population forms xenograft tumors and recapitulates all downstream differentiation states (CD90⁻CD44⁺CD49f⁺ → CD90⁻CD44⁻CD49f⁺ → CD90⁻CD44⁻CD49f⁻). (C) The intermediate BC subtype lacks the basal differentiation state (CD90⁺CD44⁺CD49f⁺). (D) Only the most upstream differentiation state (CD90⁻CD44⁺CD49f⁺) forms xenograft tumors and can reconstitute all downstream states (CD90⁻CD44⁻CD49f⁺ → CD90⁻CD44⁻CD49f⁻). (E) In differentiated BCs that lack both the basal (CD90⁺CD44⁺CD49f⁺) and the intermediate (CD90⁻CD44⁺CD49f⁺) differentiation states, (F) only the existing differentiation state (CD90⁻CD44⁻CD49f⁺) forms xenograft tumors and recapitulates the terminally differentiated (CD90⁻CD44⁻CD49f⁻) downstream state. (A, C, and E) The terminally differentiated subpopulations (CD90⁻CD44⁻CD49f⁻) never give rise to tumors. (G) Frequency of tumor formation of all transplanted cell populations described in A, C, and E.