A human breast cell model of preinvasive to invasive transition

Abstract

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur "spontaneously" in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA-based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context.

Figure 1

Partial loss of tissue polarity and preinvasive phenotypes of S3 cells. A, steps involved in the isolation of the HMT-3522 series of cell lines. B, 3DlrBM cultures of HMT-3522-S2 cells and small (S3-A), medium (S3-B), and large (S3-C) colonies isolated from the parent culture of S2 on day 10. C, confocal images of immunostained colonies in 3DlrBM, day 10. D, invasion assays: two experiments, triplicate samples. Black columns, untreated cells; gray columns, cells treated with T4-2 conditioned medium (CM).

Figure 2

S3 cells display low tumorigenicity and squamous metaplastic phenotype in nude mice. A, percent injection sites that produced tumors sustained for the duration of the experiment. Top, s.c. injections (three experiments); 14 S1, 22 S2, 20 S3-A, 16 S3-B, 22 S3-C, 30 T4-2, and 3 media controls. Middle, s.c. + Matrigel (laminin-rich basement membrane, lrBM) injections (two experiments); 18 S1, 18 S2, 20 S3-A, 22 S3-B, 18 S3-C, and 18 T4-2 injections. Bottom, mammary fat pad injections; 15 S1, 16 S2, 16 S3-A, 16 S3-B, 15 S3-C, 24 T4-2, and 4 media controls. B, mean tumor volume (in mm³) excised from sacrificed animals. C, P values for the volume comparisons indicated; P < 0.05 in bold. Top, s.c. injections; middle, s.c. + Matrigel injections; bottom, mammary fat pad injections. D, H&E images of S1, S2, and S3-B s.c. + Matrigel tumors and of S3-A, S3-C, and T4-2 fat pad tumors. Bar, 50 μm. S1, low-grade adenosquamous carcinoma-like phenotype at the injection site; S2, squamous differentiation with whorls and bridges, transition from cuboidal cells to larger squamous cells in the center; S3-A, well-differentiated area with a small cyst, less well differentiated area with calcification; S3-B, cords of cells surrounding areas of extracellular matrix showing solid tumor areas with proliferation and squamous differentiation and displaying pleomorphic adenoma phenotype; S3-C, squamous differentiation, foamy squamous carcinoma cells, abundant stromal reaction; T4-2, squamous carcinoma invading the skeletal muscle.

Figure 3

CGH profile of S3 cells compared with other HMT-3522 cells and with primary tumors (for details of CGH data, see Supplementary File 2). A, chromosomal amplifications (red) and deletions (green) for each cell type examined at the indicated passage number (e.g., S1_27: S1 cells at passage 27). B, chromosomal aberrations common to all cell lines shown on the left (e.g., “S1” means common to all S1 lineages shown in A). C, hierarchical clustering of 119 significantly amplified or deleted regions for the indicated cell lines. D, chromosomal aberrations commonly found in primary tumors, as described in each study cited; shown in bold if shared with HMT-3522 cell lines; complete list of aberrations shared between the cell lines and primary tumors listed at the bottom (TOTAL).

Figure 4

Candidate MMPs from microarrays are differentially expressed between S3-C and T4-2 cells. A, quantitative RT-PCR for MMP13, normalized to GAPDH internal control, for S3-C and T4-2 in 3DlrBM (P < 0.05). B and C, cell-surface expression of MMP15 and MMP17; four independent experiments (P < 0.05). D, zymogram detecting gelatinolytic activity in cell lines, compared with p-aminophenylmercuric acetate–activated recombinant MMP2 control.

Figure 5

Candidate MMPs function in invasion through laminin-rich basement membrane. A, RT-PCR for MMP9, MMP13, MMP15, and MMP17 in cells transfected with siRNAs to the respective MMPs or a scrambled control. B, invasion assays for T4-2 cells treated with the indicated MMP inhibitor or control; two experiments, duplicate samples. C, invasion assays for T4-2 cells transfected with MMP9, MMP13, MMP15, or MMP17 siRNAs versus scrambled control; P values compared with scrambled control. D, invasion assays for S3-C cells; number of invading cells normalized to S3-C cells treated with T4-2 conditioned medium. Posttreated, conditioned medium was added to the S3-C medium after the indicated MMP inhibitor or control (40 μmol/L); Pretreated, conditioned medium was pretreated with the MMP inhibitor or control (40 μmol/L) for 48 h before being added to the S3-C medium in the invasion assays; three experiments.