Identification of differentially expressed genes in canine mammary tumor cell lines using a newly developed canine-specific cDNA microarray

Background

Tumors of the mammary gland belong to the most common tumors in the female dog. Ovarian hormones and signaling cascades involving them play an important role in mammary tumor formation and progression in the female dog, as early ovariectomy may reduce the incidence of mammary carcinoma from 30% to less than 2%. The phenotypic expression of canine mammary tumors may vary, even within one affected animal. In the past, growth factor-independent canine mammary cell lines have been isolated [1]. Molecular characterization of these cell lines and correlation of their molecular signatures to biological behavior may lead to better understanding of signaling pathways involved in mammary cancer and classification of spontaneous mammary tumors.

Methods

Three canine mammary tumor cell lines (CMT) originating from primary mammary spindle cell tumor (CMTU309), primary mammary osteosarcoma (CMTU335) and primary mammary anaplastic carcinoma (P114) were compared directly with each other in this study. Cell lines were tested for in vitro invasion using a Transwell assay. Total RNA was isolated from cells grown to near confluence. In vitro transcription followed by labeling and hybridization to a cDNA microarray was carried out according to published protocols [2]. In a loop design of hybridization, labeled cRNA from cell lines were hybridized against each other on a dog-specific cDNA microarray containing 20,160 independent genes, which was developed and spotted in our laboratory. Statistical analysis of microarray data was carried out using significance analysis of microarrays [3]. Further analysis of microarray data was done using GeneSpring.

Results

The Transwell invasion assay revealed a clear difference in in vitro invasiveness between canine mammary tumor cell lines. P114 showed a highly invasive phenotype whereas CMTU309 was the least invasive cell line. The cell lines under investigation showed a significant difference in doubling time but no difference in growth factor dependence. Microarray data analysis yielded a total of 451 differentially expressed genes. Among them, about 111 genes were differentially expressed in P114 compared with CMTU335, 110 genes were differentially expressed in P114 against CMTU309 and 230 genes were significantly regulated in CMTU309 against CMTU335. GeneSpring analysis of the microarray data revealed genes unique to each cell line, which were differentially expressed (twofold) in one cell line against the other two cell lines. Unique gene lists containing 19 genes for CMTU309, 62 genes for CMTU335 and 33 genes for P114 were obtained.

Conclusion

Our study yielded a novel set of genes unique for each canine mammary cell line in this study. Next, the contributions of these genes, among which some 50% are not annotated towards phenotypic differences between these cell lines, are under investigation.