Expression profiling of primary tumors and matched lymphatic and lung metastases in a xenogeneic breast cancer model

Abstract

Using a purpose-designed experimental model, we have defined new, statistically significant, differences in gene expression between heavily and weakly metastatic human breast cancer cell populations, in vivo and in vitro. The differences increased under selection pressures designed to increase metastatic proficiency. Conversely, the expression signatures of primary tumors generated by more aggressive variants, and their matched metastases in the lungs and lymph nodes, all tended to converge. However, the few persisting differences among these selectively enriched malignant growths in the breast, lungs, and lymph nodes were highly statistically significant, implying potential mechanistic involvement of the corresponding genes. The evidence that has emerged from the current work indicates that selective enhancement of metastatic proficiency by serial transplantation co-purifies a subliminal gene expression pattern within the tumor cell population. This signature most likely includes genes participating in metastasis pathogenesis, and we document manageable numbers of candidates for this role. The findings also suggest that metastasis to at least two different organs occurs through closely similar genetic mechanisms.

Figure 1

Visualization of GFP-labeled tumors and metastases in our xenogeneic breast cancer metastasis model. Left: Increasing metastatic loads in the five pulmonary lobes of mice bearing 2-cm primary tumors developed after orthotopic inoculation of isogenic clonal cell lines with progressively enriched metastatic capabilities, as indicated. Right: A survey view of the heavy metastatic spread of a CL16 primary tumor in the whole animal. Numerous tumor cells leaving the primary tumor (P) distended the afferent lymphatic vessels (arrowheads). Pulmonary and lymphatic metastases are also indicated. An asterisk indicates the heart.

Figure 2

Protein validations of candidates selected on microarray-based differential expression between the nonmetastatic and the metastatic tumors in BalbC mice. Changes in mRNA expressions estimated by microarray analyses and quantified by real-time PCR (middle columns) were validated at the protein level by quantitative (ELISA, third column) or semiquantitative methods (Western blotting, left; immunohistochemistry, right) as described in Materials and Methods. Both the “Affy” and the “Q-PCR” fold changes came from the Q-PCR validation table provided in the supplemental material. This explains why, for some genes, the microarray data indicated in this figure may not exactly match those in Table 1, which are based on SCID comparisons. All of the stained tumor sections were observed at 400× magnification, and a scale bar was inserted in the NM-2C5 tumor section stained with the anti-Pmel-17 antibody. Arrows in the upper left show examples of positive signals of MITF localization in the nuclei of nonmetastatic tumor cells.

Figure 3

Schematic diagram of the “horizontal” (black arrows) and “vertical” (white arrows) comparisons performed in the microarray data analyses. dChip software was used to compare global expression datasets obtained with the HG-U133A array hybridized with the nonmetastatic (NM-2C5) and metastatic (CL16) primary tumors and the spontaneous metastatic deposits developing in the lung (Lung Mets) or in the thoracic lymph nodes (LN Mets) in SCID mice.

Figure 4

Signature gene clustering and sample classification. Hierarchical cluster analyses of the differentially expressed genes from in vivo and in vitro comparisons. A: Candidate genes differentially expressed (with P < 0.005 and fc >1.5) at least in one of the six in vivo comparisons (illustrated in Figure 3) were clustered according to their expression levels in the NM-2C5 and CL16 lines in vitro and in the primary and secondary tumors they generated in SCID mice. B: Candidate genes differentially expressed (with fc >3) at least in one of the three in vitro comparisons of the cell lines (Table 7) were clustered according to their expression levels in NM-2C5, LM3, and CL16 lines. The vertical bars on right of this panel indicate the candidate genes gradually overexpressed (white bar) or down-regulated (gray bar) in regard with the progressive acquirement of high metastatic capabilities. As shown in the color bar, red indicates high expression; green, low expression; and black, intermediate expression. The dendogram on the left indicates the pairing of genes, and the branch length is proportional to the distances between the clusters. C: Linear discriminant analysis using NM-2C5 (blue striped squares) and CL16 (red striped squares) primary tumors in SCID mice as the training set to classify unknown samples (test set) according to the combined candidate gene list used in A for the clustering analysis. Plain blue squares: Samples classified into the nonmetastatic group; plain red squares: samples classified into the metastatic group. The unknown samples include primary tumors (NM-2C5 and LM3), metastases in the lung (Lg) or lymph nodes (LN), and cell lines (underlined). The asterisk shows misclassified samples.