Characterizing Bone Tropism of Human ER+ Breast Cancer Cell Lines in a Murine Bone Metastasis Model

Breast cancer patients with estrogen receptor‐positive (ER+) tumors have the highest rate of developing clinically evident osteolytic bone metastasis. However, pre‐clinical models of ER+ bone metastasis are currently lacking; therefore we sought to characterize 3 ER+ breast cancer cell lines—MCF‐7, T47D, and ZR‐75‐1—in terms of their in vivo ability to form osteolytic bone metastasis and in vitro expression of cell surface receptors and signaling pathways thought to be important for bone metastases progression. Methods Female nude mice implanted with 0.72 mg 60 day extended‐release 17β‐estradiol pellets were inoculated with 1×10 5 ER+ human breast cancer cells (MCF‐7, T47D or ZR‐75‐1; ATCC) via the left cardiac ventricle. Radiographs of hind femurs and tibias were obtained weekly to assess osteolytic lesion formation and bone marrow was harvested from hind limbs with osteolytic lesions for tumor cell isolation and propagation. Cells (control and/or TGFβ1 stimulated) were analyzed for expression of ER, TGFβR and phosphorylated Smad2 by Western. Results The incidence of osteolytic bone metastases in mice injected 35 days prior with MCF‐7 (n = 11), T47D (n =12), or ZR‐75‐1 (n = 4) ER+ cells was 82%, 56%, and 25%, respectively. After 4 additional mice were injected with twice the number of ZR‐75‐1 cells, total incidence in ZR‐75‐1‐inoculated mice (n=8) increased to 38%, a rate that remained significantly lower than that seen with MCF‐7 cells (p ≤ 0.05). Osteolytic lesion area in the hind limbs of mice inoculated with MCF‐7, T47D, and ZR‐75‐1 cells were 6.5±1.5 mm 2 , 5.7±1.9 mm 2 , and 1.0±0.6 mm 2 , respectively, with MCF‐7 and T47D osteolytic lesion area significantly exceeding that of ZR‐75‐1 cells (p < 0.05). Phenotypically, T47D and ZR‐75‐1 formed small osteolytic lesions along the entire length of the long bones, while MCF‐7 formed larger lesions that were primarily located in the epiphyseal/metaphyseal areas. ERα, ERβ, TGFβRI, and TGFβRII protein levels were similar among the 3 cell lines and unchanged in MCF‐7, T47D, and ZR‐75‐1 cells isolated from osteolytic lesions and propagated in vitro . Evidence of Smad‐mediated TGFβ signaling (TGFβ induced Smad2 phosphorylation) was demonstrated in ATCC MCF‐7 cells but not in ATCC T47D or ZR‐75‐1 cells. Conclusion While all 3 ATCC cell lines formed visually detectable osteolytic lesions, MCF‐7 cells formed the most aggressive osteolytic bone lesions as assessed by incidence and osteolytic lesion size, while ZR‐75‐1 cells were the least aggressive. Although all 3 ATCC cell lines expressed similar levels of ERα, ERβ, TGFβRI, and TGFβRII, only MCF‐7 cells showed increased levels of phosphorylated Smad2 upon TGFβ1 stimulation. Because TGFβ‐mediated Smad signaling has been demonstrated to enhance the progression of ER− bone metastases, activation of this pathway in ER+ MCF‐7 cells may contribute to its more prominent ability to form osteolytic lesions in vivo. Retention of ER expression in cells isolated from osteolytic bone lesions recapitulated the phenotype most frequently seen in women with ER+ breast cancer bone metastases, which remain concordant for ER expression in the majority of cases. Support or Funding Information R01 CA174926‐01; R03 CA181893‐01