Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: Elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases

Tumor-stroma interactions are of primary importance in determining the path ogenesis of metastasis. Here, we describe the application of sensitive comp etitive polymerase chain reaction (PCR) techniques for detection and quanti tation of human breast cancer cells (MDA-MB-231) in an in vivo mouse model of experimental metastasis. Human-specific oligonucleotide primers in compe titive PCR reactions were used to quantify the amount of MDA-MB-231 cells p er tissue per organ. Using this species-specific (semi)quantitative PCR app roach, gene expression patterns of (human) tumor cells or (mouse) stromal c ells in metastatic lesions in the skeleton or soft tissues were investigate d and compared. In all metastatic lesions, MDA-MB-231 cells express angioge nic factors (vascular endothelial growth factors [VEGFs]; VEGF-A, -B, and - C) and bone-acting cytokines (parathyroid hormone-related protein [PTHrP] a nd macrophage colony-stimulating factor [M-CSF]). In these metastases, PECA M-1-positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEG F receptors flt-1/flk-1/flk-4 and CD31/PECAM-1). Strikingly, steady-state m essenger RNA (mRNA) levels of VEGF-A and -B and the major bone resorption s timulators PTHrP and M-CSF by tumor cells were elevated significantly in bo ne versus soft tissues (p less than or equal to 0.05, p less than or equal to 0.0001, p less than or equal to 0.001, and p less than or equal to 0.05, respectively), indicating tissue-specific expression of these tumor progre ssion factors. In conclusion, MDA-MB-231 breast cancer cells express a vari ety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We h ypothesize that the observed up-regulated expression of angiogenic and bone -resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species-specific compet itive PCR-based assay in vivo can provide new information concerning the in volvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor-stroma interactions in cancer invasi on and metastasis. Key words: competitive polymerase chain reaction, breast cancer.