Established breast cancer stem cell markers do not correlate with in vivo tumorigenicity of tumor-initiating cells

Abstract

The tumor-initiating capacity of primary human breast cancer cells is maintained in vitro by culturing these cells as spheres/aggregates. Inoculation of small cell numbers derived from these non-adherent cultures leads to rapid xenograft tumor formation in mice. Accordingly, injection of more differentiated monolayer cells derived from spheres results in significantly decelerated tumor growth. For our study, two breast cancer cell lines were generated from primary tumors and cultured as mammospheres or as their adherent counterparts. We examined the in vivo tumorigenicity of these cells by injecting serial dilutions into immunodeficient mice. Inoculation of 106 cells per mouse led to rapid tumor formation, irrespective of cell line or culture conditions. However, after injection of only 103 cells, solely sphere cells were highly tumorigenic. In vitro, we investigated differentiation markers, established breast CSC markers and conducted mRNA profiling. Cytokeratin 5 and 18 were increased in both monolayer cell types, indicating a more differentiated phenotype. All cell lines were CD24(-)/CD44(+) and did not express CD133, CD326 or E-cadherin. ALDH1 activity was not detectable in any cell line. A verapamil‑sensitive Hoechst side population was present in sphere cells, but there was no correlation with tumorigenicity in vivo. mRNA profiling did not reveal upregulation of relevant transcription factors. In vitro cell cycle kinetics and in vivo tumor doubling times displayed no difference between sphere and monolayer cultures. Our data indicate that intrinsic genetic and functional markers investigated are not indicative of the in vivo tumori-genicity of putative breast tumor-initiating cells.

Figure 1

Tumor xenograft growth characteristics of S2N and S2 breast cancer sphere and monolayer cells at different cell number inoculations. Subcutaneous injection of sphere cells into the right flank (solid lines) and monolayer cells into the left flank (broken lines), respectively. Tumor size values represent mean ± SEM (10 mice per group). The numbers adjacent to the lines indicate the mean ± SEM of the population doubling times calculated from the xenograft growth curves (S2 sphere 10³ group: tumor growth only in 1/10 mice).

Figure 2

Morphology and cell cycle kinetics of monolayer and sphere S2N and S2 breast cancer cells cultivated under serum-free FGF/EGF and 10% FBS cell culture conditions. Bright field microscopic images of more spindle-like monolayer and aggregated sphere cells [Axiovert 135 microscope (Zeiss), CoolSnap K4 camera (Visitron), bar length 100 μm]. The BrdU/Hoechst FACS cell cycle kinetic analysis was done after a 48 h BrdU labeling period (1st cell cycle, G1, S, G2M; 2nd cell cycle, G1’, S’, G2M’; 3rd cell cycle G1”).

Figure 3

Western blot analysis of intermediate filament expression in highly tumorigenic S2N sphere cells compared to weakly tumorigenic, more differentiated S2N monolayer cells, S2 sphere and S2 monolayer cells. Breast cancer cell differentiation markers: cytokeratin 5 (basal breast cancer) and cytokeratin 18 (luminal breast cancer), vimentin (mesenchymal marker) and GAPDH loading control.

Figure 4

FACS expression analysis of putative CSC/TIC cell surface receptors of highly tumorigenic S2N sphere cells compared to more differentiated, weakly tumorigenic S2N monolayer cells, S2 sphere and S2 monolayer cells. CD324 marker, E-cadherin; CD326 marker, EpCAM. FACS receptor analysis was done by gating on viable, non-aggregated single cells. IgG isotype control (broken line) and receptor specific antibody (solid line).

Figure 5

Aldehyde dehydrogenase 1 (ALDH1) and Hoechst side population (SP) activity in S2N and S2 sphere and monolayer cells. (A) ALDH1 activity in absence or presence of the ALDH1 inhibitor DEAB. Cells within framed region represent ALDH1 positive cells. (B) Multidrug resistance transporter activity in absence or presence of MDR transporter inhibitor verapamil. The framed region indicates cells of the Hoechst 33342 side population (cellular fluorochrome exclusion). FACS gating in both assays was on viable, non-aggregated single cells.

Figure 6

Comparison of mRNA gene expression pattern of highly tumorigenic S2N sphere cells, more differentiated weakly tumorigenic S2N monolayer cells and weakly tumorigenic S2 sphere and monolayer cells. The comparison is based on 183 probe sets representing 136 genes which are up- or downregulated differently by at least a factor of 2.

Figure 7

Genes exclusively expressed in highly tumorigenic or weakly tumorigenic cells. (A) Genes expressed in the highly tumorigenic S2N spheroids only but not in weakly tumorigenic S2 spheroid/monolayer and S2N monolayer cells. (B) Genes expressed in weakly tumorigenic S2 spheroid/monolayer and S2N monolayer cells but not expressed in highly tumorigenic S2N spheroids (filled bars, S2 spheroids; dotted bars, S2N monolayer; hatched bars, S2 monolayer).