Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state

Abstract

Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.

Fig. 1.

Enrichment of a rare population of floating cells from cultured mammary epithelial cells. (A) Schematic illustrating the derivation of HME-flopcs and single-cell clones (SCCs), as well as the antigen profiles of the major cell subpopulations described in this paper. (B) Phase contrast micrographs (20×) of HME and HME-flopc populations in 2D culture. Immunofluorescence of HME and HME-flopc cells is shown, for E-cadherin (green), β-catenin (red), and ZO-1 [nuclei stained blue with 4,6-diamidino-2-phenylindole (DAPI)]. (C) Immunoblot with antibodies to cytokeratins 14, 18 and 19, vimentin, E-cadherin and β-catenin (total and active forms). (D) Flow cytometry plots for CD44, CD24, and ESA on bulk HME and HME-flopc cells. Gating is set to unstained control cells.

Fig. 2.

Normal and transformed HME-flopc-CD44^lo cells spontaneously convert into CD44^hi cells. (A) Population dynamics modeled by a simple growth model in which CD44^lo cells either divide (k_lo) or spontaneously switch (k_s) to a CD44^hi state. CD44^hi cells divide (k_hi) but cannot switch to the CD44^lo state. Cell populations are quantified by flow cytometry. (B) Growth rates of purified CD44^lo and CD44^hi cell populations analyzed in C, n = 6, Promega CellTiter 96 AQueous Assay. (C) Quantification of the spontaneous dedifferentiation of purified CD44^lo cells isolated from bulk HME cells, HME-flopcs, and HME-flopcs expressing SV40 early region only (HMLE-flopc), as well as fully transformed HME cells (HMLER) and HME-flopc cells (HMLER-flopcs) expressing the SV40 early region and Ras oncoproteins (n = 3–6, results are mean ± SEM). (D) Switching rate (k_s) of various CD44^lo cell types per population doubling time. (E) HME-flopc-CD44^lo cells purified from two independent single-cell clones (no color) were mixed with fluorescent (pLV-Tomato) HME-flopc-CD44^hi (CD44^hi-Tom) cells to recreate a heterogeneous population. The percentages of CD44^hi-Tom cells (red bars) and of de novo-derived CD44^hi cells arising from HME-flopc-CD44^lo cells expressing no fluorescent protein (gray bars) were measured over a time course of 12 d. Results are mean ± SEM.

Fig. 3.

CD44^hi cells display stem-like properties in vitro. (A) Mammosphere-forming ability of bulk HME or HME-flopc cells, HME-flopc-CD44^lo single-cell clones (SCCs, n = 5), HME-flopc-CD44^hi SCCs (n = 4), and CD44^hi and CD44^lo cells purified by FACS from two independent HME-flopc SCCs (FL1 and FL2). Results are mean ± SEM. (B) Representative flow cytometry plots for the markers CD44, CD24, and ESA of cells isolated from dissociated mammospheres from de novo-derived CD44^hi cells (FL1-CD44^hi and FL2-CD44^hi) and two stable HME-flopc-CD44^hi SCCs (FH1 and FH2). (C) Quantification of total CD44^lo cells, CD44^loESA⁻ and CD44^loESA⁺ populations [from (B) box P7; n = 3]. Results are mean ± SEM. (D) Frozen sections of mammospheres (5 μm) stained by immunofluorescence for myoepithelial [CD10, vimentin, and cytokeratin-14 (K14)] and luminal (K14 and MUC1) differentiation markers.

Fig. 4.

CD44^loCD24⁺ESA^lo cells isolated from primary HMECs convert into the CD44^hiCD24^loESA^lo mammary stem-like state. (A) Single-cell suspensions of (i) uncultured primary human mammary epithelial cells (HMECs), (ii) bulk HME cells (expressing hTERT), (iii) FH1 [preexisting HME-flopc CD44^hi single-cell clone (SCC)], and (iv) spontaneously arising CD44^hi cells (purified from HME-flopc CD44^lo SCC-FL1) were injected into the humanized mouse mammary fat pad. After 12 wk, mammary glands were sectioned and stained by immunofluorescence for basal (keratin 14, green) and luminal (pan-cytokeratin, red) markers. (B) Quantification of subpopulations of uncultured HMECs following direct isolation from primary tissue (SI Materials and Methods). (C) Flow cytometry plot illustrating the gating strategy used to isolate subpopulations of HMECs. P1, proposed mammary stem cell-enriched fraction; P2, CD44^loCD24⁺ESA^lo basal cells; P3, CD44^loCD24⁺ESA⁺ luminal fraction with phase contrast micrographs (20×) of purified P2 and P3 cell populations. (D) Purified (via flow cytometry) primary CD44^loCD24⁺ESA^lo and CD44^loCD24⁺ESA⁺ HMECs were monitored for 12 d in 2D culture for their ability to spontaneously convert into the CD44^hiCD24⁺ESA^lo cell state. Results are mean ± SEM. Flow cytometry plots of the purified CD44^loCD24⁺ESA^lo and CD44^loCD24⁺ESA⁺ cell populations at day 12 are shown.

Fig. 5.

CSCs are created de novo in vivo. (A) Representative FACS profiles of tumor cell populations before injection into NOD/SCID mice and of digested tumor cell populations following 8–10 wk in vivo. (B) Quantitation of in vivo de novo-derived CD44^hi cells isolated from various tumor populations, including tumor incidence, tumor weight, and a FACS analysis of immune cells recruited to the different tumor types (n = 6–9 animals/group, P < 0.001, two-way ANOVA).