Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231

Abstract

Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGFβ1. Knockdown of BSP or its inactivation by specific antibodies were found to reduce the metastatic potential of MDA-MB-231 triple-negative breast cancer cells in xenografts. While the role of BSP in bone metastasis was studied using such in vivo models, valid in vitro test systems to investigate BSP biology have been lacking since this protein is expressed at very low levels in classical 2D cell cultures and the frequently used breast cancer cell line MDA-MB-231 is difficult to grow in 3D. Here, we have developed a long-term 3D spheroid culture model using MDA-MB-231 cells in a sandwich approach using cell embedding between a non-adherent surface and basement membrane extracts. This allowed consistent growth of spheroids for more than 21 days. Also, co-culturing of MDA-MB-231 with CCD-1137Sk fibroblasts yielded stably growing spheroids, suggesting the importance of extracellular matrix (ECM) in this process. In addition, we have set up a novel and simple open source analysis tool to characterize protein expression in 2D cultures and spheroids by immunofluorescence. Using this approach in combination with Western blot analysis, the expression profile of BSP was analyzed. BSP was enriched at the rims of spheroids, both in mono- and co-cultures and its abundance in general correlated with that of TGFβ1 under different conditions, including spheroid maturation, cytostatic treatment, and fibroblast co-culture. Conversely, correlation of IGF-1 and BSP was limited to mono-culture time course profiles. In conclusion, we present novel tools to study the regulation of gene expression in combination with cell proliferation and apoptosis in a long-term 3D model of breast cancer and find dynamic abundance profiles of the metastasis-relevant protein BSP and its regulators.

Keywords: 3D cell culture; BSP; MDA-MB-231; apoptosis; bone sialoprotein; breast cancer; proliferation; spheroid.

Figure 1

Sandwich method using SPA and BME yields most consistent growth of MDA-MB-231 spheroids. (A) Representative phase-contrast microscopy images of MDA-MB-231 3D cultures. These were generated using four different methods: hanging drop (HD), inlay method (IM1; IM2; IM3), on top method (OM1; OM2), and sandwich method (SM1; SM2; SM3; SM4). From HD to SM3, equal initial seeding densities were used (10,000 cells/well), for SM4 5,000 cells/well were used. Spheroids were cultured until day 24 in vitro (DiV). Representative images from DiV 4, 8, 12, 16, 20, and 24 are shown. Scale bars, 200 μm. (B,C) Quantitative analysis of 3D culture growth or shrinkage. Graphs depict areas of 3D cultures as a function of DiV (mean values ± SD, n = 4 independent experiments with 5 replicates). Statistical analysis was performed using a two-way ANOVA with post-hoc Tukey's multiple comparison test to compare statistically significant differences across methods for each day. Statistical significance values were observed for 24 DiV: (SM1 vs. SM2, ^****P < 0.0001; SM2 vs., SM3 ^****P < 0.0001; SM3 vs. SM4, n.s.).

Figure 2

Co-culture of MDA-MB-231 and CCD-1137Sk fibroblast cells yields slowly growing spheroids and extensive outward cell movement. (A) Representative phase-contrast microscopy images of MDA-MB-231 plus CCD-1137Sk 3D co-cultures from 7, 14, 21, and 25. DiV. These were generated by co-seeding of 10,000 cells for each type in ultra-low attachment plates. Scale bar, 200 μm. (B) Quantitative analysis of 3D culture growth. Graphs depict average values of dark compact spheroid areas and excluding the outward spreading cell region, as a function of DiV (mean values ± SD, n = 96 spheroids). (C) Quantitative assessment of outward moving cells. Columns indicate the fraction of the total culture area that was occupied by the dark compact spheroid structures in percent and as a function of DiV (mean values ± SD, n = 96 spheroids; ^***P < 0.001). (D) Slices of co-culture spheroids at 7., 14., and 21. DiV were stained with DAPI for nuclei and anti-Collagen I antibody for ECM. Panels show single confocal sections of representative samples. Scale bar, 200 μm.

Figure 3

Mature and hypo-BSP protein levels are increased in 3D vs. 2D cultures at 7. DiV. (A) Wide field images of representative 7. DiV monolayer cells and 7.,14., and 21. DiV spheroids as used for biochemical analyses. Scale bars, 200 μm. (B,C) Equal amounts of protein of lysates from monolayer cells (2D) and spheroids (3D) were loaded on SDS-PAGE and Western blots were performed. Left panels depict quantitative analyses of Western blot bands as shown for representative cases in right panels. (B) Results upon incubation with human anti-recombinant human CD-BSP (aa 108–122) showing hypo-BSP expression. Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparisons test (n = 6, ^**P < 0.01, ^*P < 0.05, mean ± SD). (C) Results upon incubation with rat anti-recombinant human CD-BSP (aa 108–122) TGC-9 indicating expression of mature BSP. Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparisons test (n = 6, ^*P < 0.05, mean ± SD.). (D) Lysates of non-cancerogenous breast cell line MCF 10A were compared to those of MDA-MB-231. Graph shows quantitative analysis of Western blot band intensities as exemplified on the right side. Significant differences between groups were tested using an unpaired, two-tailed t-test (n = 3, ^**P < 0.01, mean ± SD).

Figure 4

Upregulation of BSP protein levels in young SM3 MDA-MB-231 spheroids is confirmed by immunofluorescence analysis. (A) 2D cell cultures were stained with DAPI (blue, cell nuclei) and hypo-BSP antibody (green). Ten micrometer thick cryosections of 7, 14, and 21. DiV spheroids were additionally labeled with Phalloidin-TRITC (red, actin cytoskeleton). Scale bars, 200 μm. Large panels depict optical sections, small panels show details from central and border regions of cultures to illustrate the BSP signal intensity and the enrichment of BSP at the spheroid rims. Detail pictures are taken from the dashed squared areas shown in the large panels. (B) Quantitative analysis of confocal images. The graph depicts antibody fluorescence signals as a function of DiV. Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparisons test (n = 3 for 2D and n = 4 for 3D; a total of 148 confocal images were analyzed; ^**P < 0.01, ^*P < 0.05, mean ± SD).

Figure 5

Cell proliferation decreases in MDA-MB-231 spheroids with increasing DiV. (A,B) Monolayer cell culture and 10 μm thick cryosections of 7., 14., and 21. DiV spheroids were stained with DAPI (blue) and immunolabeled for either Ki 67 (A, proliferation marker, green) or Cleaved Caspase 3 (B, apoptosis marker, green). Images show single confocal slices of representative samples. Scale bars, 200 μm. (C,D) Quantitative analysis of confocal images. Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparisons test. (C) Graphs depict mean ± SD (n = 3 for 2D and 3D; a total of 85 confocal images were analyzed; ‡ = Significant difference to all other values). (D) Graphs depict mean ± SD (n = 3 for 2D and 3D; a total of 81 confocal images were analyzed; ^**P < 0.01, ^*P < 0.05).

Figure 6

In untreated SM3 spheroids, expression profiles of TGFβ1, IGF-1, and RUNX2 are similar to that of BSP. Equal amounts of protein of lysates from monolayer cells (2D) as well as 7., 14., and 21. DiV spheroids (3D) were loaded on SDS-PAGE and Western blots were performed. (A) Representative Western blot profiles for TGFβ1, RUNX2, IGF-1, and GAPDH (loading control). (B-D) Quantitative analyses of Western blot bands. Statistical analysis was performed with one-way ANOVA followed by Tukey's multiple comparisons test (n = 4, ^****P ≤ 0.0001, ^***P < 0.001, ^**P < 0.01, ^*P < 0.05, mean ± SD).