It has been indicated previously that spontaneous cell fusion between tumor cells or between tumor cells and bone marrow derived stem cells might play an important role in the acquisition of malignant phenotypes including aneuploidy, drug resistance, and cancer stem cell features 
. For example, fusion of two cell lines, which were resistant to two different drugs, produced a new cell line that was not only resistant to the two drugs, but also resistant to an additional drug 
. A recent study from Yibin Kang's group showed spontaneous cell fusion in vitro or in vivo between two sublines of the MDA-MB-231 breast cancer cell line with different metastasis organotropisms resulting in the formation of a stable new cell line with dual metastasis organotropisms 
. In this study, we have generated an estrogen independent highly malignant B6TC hybrid cell line, spontaneously formed between an ERα positive and ERα negative cell, and isolated from a bone marrow metastatic site. Since B6TC cells are much more malignant than its prarental cells with a number of stem cell characteristics, we speculate that it might be formed by the fusion of at least one cell with stem cell features.
Our study was initiated to investigate whether the presence of primary tumors formed by highly metastatic MDA-MB-231 or MDA-MB-435 cells can drive less metastatic ZR-75-1 cell homing to bone. In the process of characterizing bone metastatic ZR-75-1 cells, we identified the hybrid cell line, B6TC. Whether a specific metastasis-permissive niche in the bone established by the primary tumors might be involved in the generation of this hybrid cell line would require further investigation. The B6TC cells originated from B6 cells, which were generated in the bone marrow, and it was composed of a mixed population of ER high and low cells with marked differences in their proliferative potential and malignancy. Among them, the low ER expressing B6TC cells are highly metastatic. B6TC cell contains the identical p53R280K mutation as the MDA-MB-231 cell and in large part resembles MDA-MB-231 cell with respect to its growth property and response to estrogen. However, its brain metastatic property and ability to form mammospheres appears to resemble ZR-75-1 cell rather than MDA-MB-231 cell. Gene expression profiling indicates that B6TC cell is less different from MDA-MB-231 cell than from ZR-75-1 cell. We are currently in the process of identifying genes that contribute to the bone and brain metastatic property of B6TC cell.
The precise cellular and molecular mechanisms that lead to metastases of a tumor to a pre-determined location are not known. To specifically identify genes that mediate metastasis, animal models have been used to select in vivo for highly metastatic and organ specific derivatives of human cancer cell lines 
. In this study, the B6TC cells are highly metastatic to the bone as well as to the brain. Bone is the most common site for cancer metastases. Breast cancer metastasizes to bone in more than 80% of patients with advanced disease 
. Existing evidences suggest that enhanced formation and activation of osteoclasts induced by colonization of cancer cells are critical to the establishment of bone metastases. B6TC cells consistently caused osteolytic bone metastases in nude mice after intracardiac inoculation. This model provides opportunity to study pathogenesis and molecular mechanism of tumor cell-bone interactions in breast cancer metastases. Our results demonstrate B6TC tumor causes increased bone resorption, not counterbalanced by bone formation. Future investigation is needed to define the role of different tumor producing osteoclastogenic factors, which stimulates the differentiation and activation of the osteoclasts, leading to the progression of bone destruction in the steps of bone metastatic cascade of B6TC cells. This model can be used in search of effective treatment for osteoclastic bone metastasis.
The RNA microarray study for identifying genes that are unique and overexpressed in B6TC cells compared to the parental cell lines MDA-MB-231 and ZR-75-1 and characterization of those genes with respect to their biological function will certainly have great impact on our current understanding of metastasis. Brain metastases are the most feared complication in breast cancer. Nearly 20% of patients with advanced breast cancer are eventually diagnosed with brain lesions, making breast tumors the main source of metastatic brain disease in women 
. Because of the availability of many advanced cancer therapeutics, the life span of breast cancer patients has been increased. However, presently available treatment regimens are not effective in treating breast cancer brain metastases. Limited rodent model systems have been reported for brain metastasis in breast carcinoma 
. Therefore, there is a need of efficient model systems that can be utilized for our understanding of different factors from both the host and the tumor contributing to brain metastasis. Overexpression of the genes related to brain colonization in our B6TC model may reveal novel genes contributing to the brain metastasis by comparing the signal pathways involved. The brain tissues are well-protected by blood brain barrier 
. Intravasation of such complex barrier occurs at a very low rate compared to the intravasation in a lung capillary. Also, the unique microenvironment present in the brain is relatively poorly understood, but seems to be different from other organs and may influence brain metastases 
. There are reports that CXCR4 may act as a signature gene in contributing to the invasive and metastatic behavior of the breast cancer cells to the brain 
. Our gene profiling data also implicates CXCR4 in brain metastasis as it is upregulated in B6TC in comparison to its parental cells. The brain permeability is very important for drug development 
. The understanding of brain metastasis in our B6TC model will provide insights into the development of potential novel therapies for breast cancer-induced brain metastasis, especially for the search of efficacious small inhibitors that can cross the blood brain barrier.
Many solid tumor types, including breast cancer, exhibit a functional hierarchy of cancer cells of which only a small subpopulation of stem-like cells can give rise to the differentiated cells that comprise the bulk tumor 
. The cancer stem cell hypothesis is very important concept in cancer biology and also for the detection, prognosis and prevention of cancer. The development of cancer therapeutics based on tumor regression may have produced agents which kill differentiated tumor cells while sparing the small cancer stem cell population 
. Therefore development of successful cancer therapeutic regimen requires targeting this cancer stem cell population. Such cells represent a subpopulation of cancer cells that, by one mechanism or another, have the capacity to act as tumor-propagating cells 
. In our study, we show that B6TC cells express a high level of CD44+ and a low level of CD24−, a phenotype similar to the cancer stem cells. One candidate stem cell marker with conserved stem and progenitor cell functions is aldehyde dehydrogenase 1 (ALDH1), a detoxifying enzyme responsible for the oxidation of intracellular aldehydes 
. It has been shown that murine and human hematopoietic and neural stem and progenitor cells have a high ALDH activity 
. The B6TC cell populations show high expression of ALDH. Since it has been shown that cells with high ALDH activity contain the tumorigenic cell fraction, are able to self-renew, and recapitulate the heterogeneity of the parental tumor 
, B6TC cells likely have a population of cancer stem cells. The formation of mammospheres by B6TC cells confirms that it has breast cancer stem cells as mammospheres generated from normal mammary epithelium are enriched in stem/progenitor cells 
. Therefore, it will be of great interest to determine whether these CD44+/CD24−/ALDH+ cells are directly targeted by alternative therapies, such as novel small molecule inhibitors. Based on our results, using B6TC model to target CD44+/CD24−/ALDH+ cells for drug discovery offers a highly promising and reproducible means to identify therapies that prevent self-renewal of cancer stem cells in a tumor microenvironment.
In summary, our study shows that tumors formed by aggressive ER-negative breast cancer cells can enhance metastatic potential of less aggressive ER-positive breast cancer cell. Cell fusion may be a unique mechanism of tumor heterogeneity. The estrogen-independent highly malignant B6TC hybrid cell line with stem cell-like properties, spontaneously formed between an ERα positive and ERα negative cell and isolated from a bone marrow metastatic site, will be useful for the investigation of the molecular mechanism of brain metastasis and for the therapeutic targeting of breast cancer cells resistant to anti-estrogens.