Rare steroid receptor-negative basal-like tumorigenic cells in luminal subtype human breast cancer xenografts

Abstract

There are two major subtypes of human breast cancers: the luminal, estrogen, and progesterone receptor-positive, cytokeratin 18-positive (ER(+)PR(+)CK18(+)) subtype, and the basal ER(-)PR(-)CK18(-)CK5(+) subtype. Tumor-initiating cells (CD44(+)) have been described for human breast cancers; whether these are common to the two subtypes is unknown. We have identified a rare population of cells that are both CD44(+) and ER(-)PR(-)CK5(+) in luminal-like ER(+)PR(+) T47D human breast tumor xenografts. The tumor-isolated CD44(+) cell fraction was highly enriched for clonogenic (in vitro culture) and tumorigenic (in vivo reimplantation) cells compared with the CD44(-) cell fraction. Rare ER(-)PR(-)CK5(+) cells were present within CD44(+)-derived colonies. Tumor-isolated cells placed in minimal media also contained rare ER(-)PR(-)CK5(+) cells at early time points (<10 cells); however, this population did not expand with increasing colony size. The number of ER(+)PR(+)CK5(-) cells, conversely, increased linearly with colony growth. Similary, tumors originating in vivo from CD44(+) cells contained a rare static ER(-)PR(-)CK5(+) population, an intermediate ER(-)PR(-)CK5(-) population, and an expanding ER(+)PR(+)CK5(-) population. Putative ER(+)PR(+)CK5(+) transitional cells could be seen only in colonies or tumors treated with a progestin. We propose that luminal ER(+)PR(+) breast tumors contain a minor ER(-)PR(-)CK5(+) population that has the capacity to generate the majority of ER(+)PR(+)CK18(+)CK5(-) cells. Luminal breast cancers are treated with endocrine therapies that target ER. The rare ER(-)PR(-)CK5(+) progenitor cells would escape such treatments and survive to repopulate the tumor.

Fig. 1.

Coexpression of CK5 and CD44 in human breast tumor xenografts. ER⁺PR⁺ T47D human breast tumor xenografts were grown in ovariectomized nude mice treated with estradiol alone (E2) or with E2 plus the progestin MPA (P). CK5⁺ and CK5⁻ cells, from E2 and E2+P sets were isolated by laser-capture microdissection of frozen tumor sections immunostained for CK5. Cells were subjected to gene expression profiling. (A) Volcano plot of genes positively correlated with CK5 expression by significance (P value) vs. fold change (CK5⁺/CK5⁻) from E2+P-treated tumors. Genes with the highest positive correlation are shown in red. Probes for CK5 (KRT5) and CD44 are indicated. (B) Gene expression profile of CD44 (relative expression, SEM) in CK5⁻ and CK5⁺ cells isolated from E2- and E2+P-treated tumors. (C) Immunohistochemistry for CD44 (red) and CK5 (green) of paraffin sections from T47D tumors grown with E2 or E2+P. Sections were counterstained for DAPI (blue). The arrow points to a rare CK5⁺CD44^−/low cell. (Scale bar, 10 μm.)

Fig. 2.

CK5⁺ cells in T47D tumors are mainly PR-negative. Sections of T47D tumors treated with estrogen (E2) or estrogen plus progestin (E2+P) were stained for nuclear PR (red) and cytoplasmic CK5 (green). The percentage of CK5⁺ cells that are also PR⁺ was 5.1% (E2) and 19.8% (E2+P). A rare CK5⁺PR⁺ cell in the E2+P set is marked with an asterisk. (Scale bar, 10 μm.)

Fig. 3.

Tumor-isolated CD44⁺ cells form heterogeneous colonies in 3D culture that contain CK5⁺PR⁻ cells. (A) Lin⁻ZsGreen⁺CD44⁺ cells were isolated from estrogen-treated ER⁺PR⁺ T47D tumors by FACS. For the depicted sort, 1.33% of ZsGreen⁺ tumor cells were CD44⁺ (upper box). Lin⁻ZsGreen⁺CD44⁻ cells were isolated from the lower boxed area. (B) Cells (2 × 10³ CD44⁺ and CD44⁻) were plated into eight-well chambers on Matrigel. Colonies were photographed after 3 weeks. (Scale bars, 100 μm.) (C) Sections containing CD44⁺ colonies were stained with antibodies against CD44, CK5, PR, and CD24. (Scale bar, 10 μM.) (D and E) Established CD44⁺ colonies were treated with vehicle (D) or 10 nM MPA (P) for 24 h (E) before fixation and paraffin embedding. Sections were stained by dual immunohistochemistry for CD44 (green)/PR (red), or CK5 (green)/PR (red). (Scale bars, 10 μm.) Arrows indicate CK5⁺PR⁻ cells (D Lower Center) and a rare CK5⁺PR⁺ cell (E Lower Right).

Fig. 4.

Relationship between CK5 positivity, PR expression, and colony size. Cells isolated from ER⁺PR⁺ T47D tumor xenografts were plated into minimal media in six-well dishes overlaid with glass coverslips and allowed to form colonies for 3–14 days. Cells were treated with either vehicle (control) or 10 nM MPA for 24 h before fixation and staining for CK5 and PR. (A) Relationship between colony size (log) and the number of cells positive for CK5 (green triangles) and PR (red triangles) in vehicle (control) or progestin-treated colonies. Lines represent linear regression for CK5 (m = −0.03 ± 0.01, r² = 0.18) and PR (m = 1.01 ± 0.02, r² = 0.98) in 49 control colonies and CK5 (m = 0.37 ± 0.04, r² = 0.64) and PR (m = 1.05 ± 0.03, r² = 0.97) in 45 MPA-treated colonies. (B) Representative CK5 (green)/PR (red) immunostaining of vehicle and MPA-treated small colonies (3 days, 10–15 cells, Left) and large colonies (7–14 days, >50 cells, Right). Slides were counterstained with DAPI (blue). (Scale bars, 10 μm.)

Fig. 5.

The CD44⁺ fraction of T47D xenografts is enriched for tumorigenic cells, which form tumors that acquire luminal-like properties over time. An equal number of CD44⁺ ESA⁺ZsGreen⁺ and CD44⁻ ESA ⁺ZsGreen⁺ cells (10⁴) were implanted into opposite abdominal mammary glands of recipient estrogen-treated ovariectomized nude mice. Tumor formation was monitored by fluorescence imaging, and tumors were excised from select mice at the indicated days postimplantation. (A) Fluorescent image of CD44⁻ and CD44⁺ tumors in one mouse dissected after necropsy at day 30 (Left) and in vivo fluorescent images of bilateral tumor pairs from representative mice bearing 28- and 45-day tumors (Left and Center). (B) Immunohistochemical analyses of CD44⁺ tumors grown in mice for 30 or 60 days. Paraffin sections were stained for CD44, CK5 (positive cells indicated by arrows), PR, and CK18. Select mice were injected with 1 mg of MPA (P) for 24 h before death. CK5 staining of these tumors is shown at days 45 and 70 (+P, far right). (Scale bars, 10 μm.)