| The lymphatic system is a common avenue for the spread of breast cancer cells. If primary tumors are estrogen (ER) and/or progesterone (PR) receptor positive, then the likelihood that lymph node (LN) metastases express receptors exceeds 80%. However, little is known about the role of hormones in breast cancer spread or the effects of the LN microenvironment on hormone responsiveness. We developed ER+ metastasis models using ZsGreen labeled ER+ human breast cancer cells. Tumors and metastases were tracked in living mice by fluorescent whole-body and intravital imaging. Tumor growth was estrogen dependent and required for intratumoral lymphangiogenesis. Seventy-five percent of estrogen treated tumors generate LN metastases. "Triads" of primary tumors, tumor-filled draining lymphatic vessels, and tumor-filled LNs from the same mouse showed that: (a) proliferation was higher in the LN than in the primary tumor. (b) High ER levels were extensively downregulated by estradiol in primary tumors. However, there was partial failure of ER downregulation in LNs associated with (c) reduced PR expression. (d) The hyaluronan receptor, CD44 was sparsely expressed in primary tumor cells but homogeneously overexpressed in cells transiting the lymphatics and populating LNs, suggesting that CD44 targets tumor cells for transport to LNs. To follow up on the observations that ER have reduced function in the LN microenvironment we developed a fluorescent xenograft model in which estradiol was withdrawn after the development of ER+ LN metastasis. Estradiol-withdrawal combined with laser-capture microdissection, gene expression profiling, and immunohistochemistry was used to assess estrogen responsiveness of primary tumor xenografts and matched LN metastases. Genes regulated by the tumor cell microenvironment, by hormones, or both were identified. Gene expression data confirmed the estrogen insensitivity of ER+ LN metastases. Fewer genes were estradiol regulated in breast cancer cells at the metastatic site. Interestingly, seventy-five of the genes were estradiol regulated in the opposite direction in tumors and LN metastases. We speculate that current estrogen withdrawal or antiestrogen therapies may differentially affect breast cancer cells growing within the breast and in the LNs, and that the LN microenvironment renders cancer cells relatively resistant to estrogen suppression therapies. |
