Gene expression profiling of the tumor microenvironment during breast cancer progression

Abstract

Introduction: The importance of the tumor microenvironment in breast cancer has been increasingly recognized. Critical molecular changes in the tumor stroma accompanying cancer progression, however, remain largely unknown. We conducted a comparative analysis of global gene expression changes in the stromal and epithelial compartments during breast cancer progression from normal to preinvasive to invasive ductal carcinoma.

Methods: We combined laser capture microdissection and gene expression microarrays to analyze 14 patient-matched normal epithelium, normal stroma, tumor epithelium and tumor-associated stroma specimens. Differential gene expression and gene ontology analyses were performed.

Results: Tumor-associated stroma undergoes extensive gene expression changes during cancer progression, to a similar extent as that seen in the malignant epithelium. Highly upregulated genes in the tumor-associated stroma include constituents of the extracellular matrix and matrix metalloproteases, and cell-cycle-related genes. Decreased expression of cytoplasmic ribosomal proteins and increased expression of mitochondrial ribosomal proteins were observed in both the tumor epithelium and the stroma. The transition from preinvasive to invasive growth was accompanied by increased expression of several matrix metalloproteases (MMP2, MMP11 and MMP14). Furthermore, as observed in malignant epithelium, a gene expression signature of histological tumor grade also exists in the stroma, with high-grade tumors associated with increased expression of genes involved in immune response.

Conclusions: Our results suggest that the tumor microenvironment participates in tumorigenesis even before tumor cells invade into stroma, and that it may play important roles in the transition from preinvasive to invasive growth. The immune cells in the tumor stroma may be exploited by the malignant epithelial cells in high-grade tumors for aggressive invasive growth.

Figure 1

Laser capture microdissection experimental design. Example of the tumor microenvironment compartments targeted by laser capture microdissection: epithelial (white asterisk) and stromal (black outlined areas with black asterisk) compartments of the normal terminal ductal lobular unit, of ductal carcinoma in situ (DCIS) and of invasive ductal carcinoma (IDC).

Figure 2

Comparative analysis of gene expression changes in tumor and stroma. Gene expression changes in normal breast epithelium (N), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), normal stromal compartment (N-S), ductal carcinoma in situ-associated stroma (DCIS-S) and invasive ductal carcinoma-associated stroma (IDC-S). ↑, upregulated genes; ↓, downregulated genes.

Figure 3

Heatmap of expression patterns of ductal carcinoma in situ-associated and invasive ductal carcinoma-associated genes. (a) Heatmap of 849 genes with >3-fold differential expression in either ductal carcinoma in situ (DCIS) versus normal breast or invasive ductal carcinoma (IDC) versus normal breast in the epithelium. (b) Heatmap of 557 genes with >3-fold differential expression in either ductal carcinoma in situ-associated stroma (DCIS-S) versus normal stromal compartment or invasive ductal carcinoma-associated stroma (IDC-S) versus normal stromal compartment. Data shown are log₂(fold change) relative to the average expression in normal controls (normal breast epithelium or normal stromal compartment). In each heatmap, genes (rows) are hierarchically clustered using 1 – Pearson correlation as the distance metric. IS, ductal carcinoma in situ; INV, invasive ductal carcinoma; ISS, ductal carcinoma in situ-associated stroma; INVS, invasive ductal carcinoma-associated stroma.

Figure 4

Heatmap of differential expression of ribosomal protein genes in the malignant epithelium and tumor stroma. Differential expression of ribosomal protein genes in ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), ductal carcinoma in situ-associated stroma (DCIS-S) and invasive ductal carcinoma-associated stroma (IDC-S). Data shown are log₂(fold change) relative to the average expression level in the normal controls (normal breast epithelium or normal stromal compartment). Expression measurements for multiple probe sets representing the same gene were collapsed to the single representative probe set with the largest differential gene expression. All genes shown were significant at adjusted P < 0.05. IS, ductal carcinoma in situ; INV, invasive ductal carcinoma; ISS, ductal carcinoma in situ-associated stroma; INVS, invasive ductal carcinoma-associated stroma.

Figure 5

Heatmap of gene expression signature correlated with tumor grade in the stroma. Comparison of grade III tumors with grade I tumors identified 526 upregulated genes and 94 downregulated genes in grade III stroma. Data shown are log₂(fold change) relative to the median expression level across all samples. Genes in rows were hierarchically clustered, and samples in columns were arranged by sample type. E, epithelium; S, stroma.

Figure 6

Validation of selected genes. (a) to (d) Boxplots of relative gene expression by quantitative real-time PCR in ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), ductal carcinoma in situ-associated stroma (DCIS-S) and invasive ductal carcinoma-associated stroma (IDC-S). (a) and (b) Reference groups were the normal components (N, normal breast epithelium; N-S, normal stromal compartment). (c) and (d) Reference groups were grade I (EI, epithelium; SI, stroma). y axis, cycling threshold values relative to the median value for the entire series. Statistically significant differences by Wilcoxon rank sum test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.(e) Immunostaining of an estrogen-receptor-positive breast cancer. Arrows point to positive staining in stromal fibroblasts.