Isolation and characterization of a highly metastatic hybrid cell line generated between estrogen receptor (ER) negative and ER positive breast cancer cells in mouse bone marrow.

Abstract #2053 Background: Breast cancer cells in a patient are heterogeneous. How the presence of these heterogeneous cancer cells may contribute to cancer progression is not clear. Experimental evidence indicates that soluble factors produced by aggressive cancer cells may generate metastasis niches for less aggressive cancer cells. In this study, we sought to determine how a growing orthotopic tumor formed by an aggressive ER negative breast cancer cell line might affect the metastatic potential of a less aggressive ER positive breast cancer cell line. Methods and Results: Five-week-old female nude mice were injected orthotopically with metastasis-competent ER negative human breast cancer MDA-MB-231/GFP/Neo cells. After three weeks, metastasis-incompetent human breast cancer ER positive ZR-75-1/GFP/puro cells were inoculated into these mice via intra-cardiac (IC) route and puromycin resistant metastatic ZR-75-1/GFP/puro cells were flushed out of the bone marrow of one mouse and established as a variant cell line called B6. B6 cells have both estrogen-dependent and -independent cells when they were orthotopically injected into female nude mice with or without estrogen supplementation. The cells isolated from an estrogen-dependent tumor were called B6TE and the cells isolated from an estrogen-independent tumor were called B6TC. Further characterization revealed that B6TC cell was more tumorigenic without estrogen supplementation and has a unique metastatic property. More interestingly, B6TC cell was resistant to both puromycin and G418 suggesting it was derived from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. When compared with parental MDA-MB-231/GFP/Neo, the B6TC cells were found to be more metastatic to lung and bone after IC inoculation as detected by GFP imaging, with significant development of paraplegia in the hind limbs after four weeks. More significantly, B6TC mice also developed metastases to brain, which was not observed in the MDA-MB-231/GFP/Neo group. B6TC cell has a low expression of ER alpha and CD24, and high expression of p-ERK, CD44, ALDH. It also expresses vimentin, CXCR4 and Integrin-β1. Its growth is refractory to the anti-estrogens, raloxifene and tamoxifen. Conclusion: Our study indicates that B6 cells generated in the bone marrow were a heterogeneous population of ER positive and ER negative cells with marked differences in their proliferative potential and malignancy. The low ER-expressing B6TC is a novel hybrid cell line generated spontaneously in a metastatic site, which has propensity to metastasize to brain in addition to lung and bone. The expression of CD44 and ALDH indicates that it has stem cell-like features. Further characterization and molecular profiling is underway to reveal the novel biomarkers. This cell line should be a useful model for the investigation of the molecular mechanism of brain metastasis and the therapeutic strategies for the treatment of metastatic breast cancer resistant to anti-estrogens. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2053.