Figure 3.

Tumorigenic properties of myoepithelial-like (CD49f^high/KIT^neg) and ductal-like (CD49f^low/KIT⁺) cells. A) Predicted outcomes of cell transplantation experiments under a clonal model, whereby different cell types give rise to distinct progenies, each retaining the phenotypic properties of the parent cells. B) Predicted outcomes of cell transplantation experiments under a differentiation model, whereby 1 or more cell types can serve as a progenitor of others, in a plastic and dynamic fashion. C) Experimental workflow of prospective xenotransplantation experiments aimed at comparing the tumor-initiating capacity of CD49f^high/KIT^neg and CD49f^low/KIT⁺ cells. The 2 populations were purified in parallel by FACS, starting from the same tumor lesion, double sorted to achieve high purity (>95%) and injected subcutaneously, side by side, into the opposite flanks of the same animal. D-E) Extreme limiting dilution analysis (ELDA) of xenotransplantation experiments using paired sets of CD49f^high/KIT^neg and CD49f^low/KIT⁺ cells sorted from ACCX5M1 (D) and SGTX6 (E) PDX lines. In both models, the frequency of tumor-initiating cells was higher in CD49f^high/KIT^neg as compared with CD49f^low/KIT⁺ cells. F-Q) Analysis by FACS and IHC of the cell composition of tumors originated from the xenotransplantation of purified preparations of either CD49f^high/KIT^neg or CD49f^low/KIT⁺ cells, sorted from either ACCX5M1 (F-K) or SGTX6 (L-Q) PDX lines. Analysis by FACS (F-G, I-J, L-M, O-P) showed that tumors originated from sorted cells contained both CD49f^high/KIT^neg and CD49f^low/KIT⁺ populations, irrespective of the original phenotype of sorted cells. In tumors originated from CD49f^high/KIT^neg cells, the percentage of CD49f^high/KIT^neg cells did not appear increased as compared with that observed in parent tumors but was lower than that observed in the purified preparations (G, M), indicating spontaneous in vivo differentiation (n.s. = not statistically significant; *P < .05; Mann–Whitney U test, 1-tailed). A symmetric scenario was observed in tumors originated from CD49f^low/KIT⁺ cells (J, P). Analysis by IHC of tumors originated from sorted cells (H, K, N, Q) confirmed the reconstitution of a cribriform histology and of a biphenotypic cell composition, defined by the coexistence of 2 distinct subsets of cancer cells with mutually exclusive expression of myoepithelial-specific (TP63) and ductal-specific (KIT) biomarkers. Scale bars: 50 µm. APC = allophycocyanin; CI = confidence interval; FACS = fluorescence-activated cell sorting; IHC = immunohistochemistry; PDX = patient-derived xenograft; PE = phycoerythrin.