Characterization of a metastasizing mammary adenocarcinoma model system in the Berlin-Druckrey IV rat

Vita.An animal model of metastatic breast cancer was developed using ENU1564, a cell line established from an ethylnitrosourea-induced mammary tumor in a Berlin-Druckrey IV (BD-IV) rat. Two sublines selected and cloned from brain metastases (Br7- C5) or from a slowly growing colony in vitro (FP2-A11) demonstrated differing malignant behaviors. Br7-C5 grew more rapidly in vitro and formed larger tumors in mammary fat pad (m.f.p.) inoculation sites than FP2-A11. Br7-C5, but not FP2-A11, spontaneously metastasized to lymph nodes and lung after orthotopic m.f.p. inoculation. After left cardiac ventricle (LV) inoculation, Br7-C5 metastasized widely to sites including brain, bone and lung. In contrast, FP2-A11 colonized primarily brain and bone, with minimal lung involvement Histologic lesions produced by FP2-A11 were pleomorphic, with epithelial and spindled cells, while Br7-C5 produced well-differentiated adenocarcinomas. The selected cells were examined for mutational activation of the c-Ha-ras, c-neu, and p53 oncogenes. Diagnostic restriction fragment length polymorphism (RFLP) analysis and oligonucleotide hybridization showed that no c-Ha-ras codon 12 point mutation is present. RFLP and single-strand conformational polymorphism analyses ruled out codon 664 mutation of c-neu. No p53 protein overexpression or mutation was revealed by immunoprecipitation and Western immunostaining. Cultured tumor cells from both clones grew slowly in 1% serum and survived poorly in serum-free medium. Estrogen supplementation did not stimulate mitogenesis. The cells secrete transforming growth factor alpha (TGFα), but tumor conditioned medium (TCM) did not stimulate tumor cell EGF-receptor tyrosine phosphorylation or DNA synthesis. TCM supported slow net population growth, possibly by decreasing cell loss rather than by stimulating DNA synthesis. TGFα apparently does not mediate autocrine growth, but could stimulate paracrine responses in vivo..