The primary goal of this study was to address how luminal breast cancers disseminate and metastasize. Because luminal cancers predominantly metastasize to the skeleton [23
], and the resulting lesions represent a major source of morbidity, we used in vivo selection to isolate bone-tropic metastatic cell lines. First, we injected MCF-7 luminal breast carcinoma cells directly into the tibiae of nude mice, and monitored tumor formation by microCT. Secondary isolates of MCF-7 (MCF-7-5624) readily gave rise to tumor growth in the bone marrow microenvironment after reinjection into the tibia. Moreover, these tumor lesions retained ERα expression, and were characterized by a highly epithelioid phenotype, as demonstrated by expression of cytokeratins and membrane associated E-cadherin (Supplemental Fig. 1
). In addition, these lesions induced a strong osteoblastic response of the surrounding bone, as shown by orange G and phloxine positivity, a measure of new bone formation. On the other hand, there was little or no evidence of osteolytic activity (TRAP negativity). Comparison was made with the SCP2 bone-tropic subclone of the basal-like human breast cancer cell line, MDA-MB-231. When injected into the tibia, SCP2 cells also gave rise to bone lesions. However, the phenotype of these tumors was quite distinct from that of MCF-7-derived lesions in several key respects (Supplemental Fig. 1
). As expected, SCP2-derived tumors failed to expressed ERα or progesterone receptors (PR) (not shown). Secondly, SCP2-induced lesions were associated with significant osteolysis, as evidenced by TRAP positivity. Finally, SCP2-derived tumors clearly displayed mesenchymal features not seen in MCF-7-5624 derived lesions. Specifically, pan-cytokeratin expression in SCP2-derived tumors was significantly weaker than in MCF-7-5624 and E-cadherin was absent from the cell membrane (Supplemental Fig. 1
Strikingly, detailed histological analysis of the entire skeleton and other organs of mice that had been inoculated with MCF-7-5624 cells into one tibia revealed metastatic lesions at neighboring sites within the skeleton such as the fibula and femur. In addition, loco-regional lymphatic channels as well as retroperitoneal lymphatics and lymph nodes were filled with tumor (Supplemental Fig. 2
). To determine the frequency and time course of these locoregional metastases, second generation MCF-7-5624A bone-tropic cells were infected with a lentiviral vector so that they would constitutively express firefly luciferase to allow localization and quantification of tumor burden in vivo. Animals were inoculated with MCF-7-5624A-GF or MCF-7-ERE-Fluc cells in one tibia and monitored by serial bioluminescence imaging (BLI) in vivo. Metastases first became detectable 12 weeks after tumor cell inoculation and were seen in 5 of 15 mice (Supplemental Fig. 2A
). These lesions appeared in a predictable sequence, with the BLI signal first appearing in the iliac lymph nodes, followed by lumbar, and, eventually, high retroperitoneal lymph nodes. To further characterize this regional dissemination, the entire lymph node chain was dissected post mortem and examined histologically (Supplemental Fig. 2B, C
). We were able to confirm the presence of extensive tumor deposits throughout retroperitoneal lymphatic vessels and lymph nodes. In addition, in one case, we also found clusters of tumor cells in the right cardiac ventricle as well as in both lungs (Supplemental Fig. 2
). Most strikingly, in-transit metastases in retroperitoneal lymphatic vessels had a highly cohesive appearance, suggesting that entire cohorts of cells were disseminating collectively. Moreover, the tumor cells appeared to remain confined to the lymphatic system, as no extravasation was observed. These observations suggested that tumor cells might be metastasizing from the initial lesions in the tibia by collective migration rather than as individual mesenchymal cells. To test whether we were dealing with cohesive clusters of cells or with random aggregates of single cells, we performed immunostaining for E-cadherin. As can be seen in Supplemental Fig. 3
, in all of the metastatic lesions in the lymphatics, lymph nodes, heart and lungs, E-cadherin was strongly expressed at the cell membrane. Thus, we concluded that tumor cells disseminate regionally from initial lesions in the tibia as collective sheets or strands along lymphatic channels. Moreover, the process appears to be directional, as we never observed lymphatic metastases distal to the originating tibia lesion.
In order to determine whether MCF-7-5624- and MCF-7-ERE-Fluc-derived tibia lesions had retained their estrogen- dependence in vivo, 17β-estradiol (E2) pellets were removed from tumor-bearing animals (estrogen withdrawal, EWD) and bone lesions were monitored in vivo by microCT (). In control animals, ovariectomy by itself resulted in a moderate reduction in bone mass (). In E2-supplemented animals, tumor growth in the tibia was associated with a much more dramatic loss of bone mass (). However, in response to EWD, these tumors regressed and the tibiae progressively regained bone mass until it reached the same level as in the control animals (). As shown in parallel experiments using MCF-7-ERE-Fluc cells, EWD was associated with a decline in ERα signaling and regression of regional metastases (). To address the mechanism whereby estrogen might be driving locoregional dissemination, we examined the effects of 17β-estradiol on collective migration of luminal breast cancer cells in vitro. As can be seen in Supplemental , treatment with 17βestradiol significantly accelerated migration of luminal breast cancer cells in vitro. Thus, estrogen-driven cell migration may be contributing to dissemination of luminal breast cancer cells in vivo.
Fig 1 Bone-tropic luminal breast cancer cell lines retain estrogen-dependence in vivo. MCF-7-derived bone tropic MCF-7-5624 cells were injected into the tibiae of ovariectomized female nude mice supplemented with E2. a Tumor growth was monitored in vivo using (more ...)
Fig 4 Growth of MCF-7-5624A-GF breast cancer metastases is dependent on the level of circulating estrogen. MCF-7-5624A-GF cells were inoculated via the left cardiac ventricle into mice that had been either ovariectomized (low estrogen) (n = 6), ovariectomized (more ...)
Primary cultures were established from each of the tibial tumors (). These secondary cell lines were used to develop systemic metastasis models. Previous attempts at generating in vivo metastasis models of ERα-positive (luminal) breast cancer have used orthotopic or intracardiac (IC) injection to give rise to bone- or visceral metastases [24
]. These models have been of limited utility, largely because of the unpredictable and delayed tumor formation and the relatively insensitive in vivo imaging modalities available at the time. Therefore, we decided to test whether luciferase-expressing isolates from tibial tumors () had acquired a bone-tropic metastatic phenotype when injected systemically. Female mice that were at least 3 weeks old underwent bilateral ovariectomy. 17β-estradiol supplementation was provided in the form of slow-release E2 pellets. The two independently in vivo selected cell lines, MCF-7-5624A-GF and MCF-7-6012-ERE-FLuc cells, were then injected directly into the systemic arterial circulation via the left cardiac ventricle. Serial in vivo BLI confirmed that both cell lines gave rise to metastatic lesions (). Lesions were detectable by BLI in over half the mice by 7 days following IC injection, and in all mice by day 35 (). In both models, mice developed an average of five metastatic lesions each, suggesting that the frequency of metastasis-initiating cells is approximately 1:100,000 (). In addition, both MCF-7-5624A-GF and MCF-7-6012-ERE-FLuc cells gave rise to remarkably similar patterns of metastasis (). The predominant metastatic sites included the skeleton (upper- and lower extremities, spine and pelvic bones), the floor of the mouth and mandible, and the adrenal glands. Less common sites included the liver, lungs, brain and the mammary fat pad (MFP) (). Each of the sites detected by BLI was confirmed histologically (Supplementary ). Most strikingly, metastatic lesions that appeared following IC inoculation expanded very rapidly, with a doubling time of approximately 2 days. This was in sharp contrast with tumors that formed following local injection of the same cell lines into the tibia, which had a doubling time of approximately 10 days (). Moreover, the development of systemic metastases was associated with significant weight loss ().
Fig 2 Development of in vivo luminal breast cancer metastasis models. a In vivo selection and tagging of bone-tropic of MCF-7- and MCF-7-ERE-Fluc-derived cell lines. In most cases, we were able to re-establish cell lines from individual tibia tumors. Second (more ...)
Fig 5 TGF-β signaling is inactive in ERα-positive breast cancer cell lines because of transcriptional silencing of the TGFBR2 gene. a TGF-β treatment failed to induce Smad phosphorylation in ERαpositive human breast cancer cell (more ...)
Fig 3 Response of luminal breast cancer metastases to estrogen deprivation. Bone tropic MCF-7-5624A-GF tumor cells were injected into the tibia (n = 8) or into the left cardiac ventricle (n = 13) of viral antibody-free 4- to 5-week-old female athymic nude mice (more ...)
Even though a large body of experimental work supports the notion that growth of human ERα-positive breast cancer cell lines is dependent on estrogen both in vitro and as xenografts [26
], little is known of the role estrogen signaling plays in systemic metastatic dissemination. We addressed this question in several different ways. First, once systemic metastases had become detectable by BLI, approximately half the animals were treated with EWD by removing the E2 pellets. EWD immediately resulted in stabilization of the BLI signal, reflecting tumor growth arrest (). Moreover, EWD and clinical tumor response were associated with weight gain () and a significant prolongation in survival (). As expected, EWD was associated with a decrease in estrogen signaling as determined by ERE-Luc activity (). Thus, growth of established metastatic lesions is highly dependent on the levels of circulating estrogen. These findings are entirely consistent with the clinical experience of treating metastatic luminal breast cancer, indicating that our models phenocopy the human disease [27
In order to address the role of estrogen in homing to and colonization of secondary sites, we introduced MCF-7-5624A-GF cells via IC injection into 4–5 week old mice that had been either ovariectomized (low estrogen), ovariectomized followed by E2 pellet implantation (high estrogen), or were virgins (intermediate levels of circulating estrogen). As shown in , the average numbers of metastatic lesions detectable by BLI were significantly lower in ovariectomized mice than in either of the other two treatment groups. Moreover, in mice with the highest levels of circulating estrogen, metastatic lesions grew exponentially from the time they first became detectable, while, in the other two groups, lesions appeared to go through a lag phase prior to entering an exponential growth (). This finding suggests that metastasis-initiating cells require some time to adapt to different levels of circulating estrogen. Moreover, once metastatic lesions entered an exponential growth phase, the rates of growth were clearly dependent on the level of circulating estrogen (). In addition, the patterns of metastasis varied considerably as a function of circulating estrogen levels (). Specifically, the frequency of skeletal and floor of mouth metastases was highest in E2-supplemented animals, while ovariectomized mice developed metastatic lesions only in the adrenal glands and the MFP, two organs that produce endogenous estrogen [29
We went on to address the question whether or not homing and establishment of micrometastases were dependent on estrogen in two different ways: First, we introduced E2 pellets into the ovariectomized animals at 10 weeks following MCF-7-5624A-GF inoculation. Several new areas of metastasis appeared, indicating that tumor cells had seeded those areas after the initial IC injection but had remained dormant, presumably because of a lack of estrogen (Supplemental Fig. 6
). Similarly, we inoculated tumor cells into the tibiae of ovariectomized animals and introduced E2 pellets 12 weeks later. No tumor growth was observed by microCT over the ensuing 18 weeks. Nonetheless, we were able to isolate viable tumor cells from the tibia post mortem, and propagate these cells in vitro in estrogen-supplemented medium (MCF7-5624-6022) (). Thus, these tumorigenic cells had remained dormant but viable for a prolonged period of time, even in an estrogen-deficient bone marrow microenvironment. These results are entirely consistent with the clinical observation that micrometastases can remain dormant for many years during anti-estrogen adjuvant therapy, but become manifest as macrometastases once anti-estrogen therapy is discontinued [31
In order to begin to elucidate the molecular mechanisms that drive the ability of luminal breast cancer cells to metastasize, we characterized the metastatic clones by gene expression profiling using Affymetrix Human 1.0 ST Gene Arrays. One hundred and seventy genes were significantly downregulated by ≥2 fold (p
≤ 0.05) in MCF-7-5624A-GF cells compared to the parental cell line. Conversely, 166 genes were significantly upregulated by ≥2 fold (p
≤ 0.05) (Supplemental Table 1
). The first striking observation was that the metastatic cells failed to overexpress any mesenchymal markers or inducers of EMT. The second striking observation was that many of the genes that were upregulated in the metastatic cells are known to be involved in collective migration during development ().
Examples of genes upregulated in metastatic MCF-7-5624A-GF cells
Interestingly, one of the most highly expressed genes was TGF-β2 (). Given the preeminent role TGF-β plays in driving metastasis of basal-like breast carcinoma cell lines in vivo [8
], this suggested that this cytokine might play a similar role in luminal breast cancer metastasis. To our surprise, while treatment with TGF-β induced brisk phosphorylation of Smad2 and −3 in ERαnegative human breast cancer cell lines, ERα-positive cell lines display this response either weakly or not at all (). Consistent with their inability to respond to TGF-β, luminal breast cancer cell lines do not express the TGF-β response gene signature (TBRS), while this is clearly represented within the gene expression profiles of ERα-negative basal-like and HER-2-positive cell lines (). The inability of the luminal cell lines to activate Smads was apparently due to transcriptional silencing of the TGF-β type II receptor gene (TGFBR2
), as we were unable to detect any TGFBR2
mRNA in most of the ERαpositive cell lines, while it was abundantly expressed in each of the ERα-negative cell lines (). To validate this observation, we examined published gene expression profiles of three large series of human breast cancer cell lines [33
] (). Strikingly, human breast cancer cell lines appear to express either ESR1
mRNA but rarely or ever both. Moreover, Charafe-Jaufret et al. [36
] identified TGFBR2
as one of the 10 genes most strongly differentially expressed between basal-like and luminal breast cancer lines. Thus, by and large, expression of ESR1
mRNA appear to be inversely correlated.
MCF-7-5624A-GF cells produced much higher amounts of TGF-β2 than parental MCF-7 cells in culture, while production of TGF-β1 was similar in both cell lines (). In addition, MCF-7-5624A-GF cells strongly induced TGF-β-responsive luciferase activity in co-cultivated mink lung epithelial reporter cells, indicating that the TGF-β2 produced by MCF7-5624A-GF cells is biologically active. Moreover, treatment with the pan-TGF-β neutralizing antibody, 1D11, completely abolished luciferase activation (). Even though the metastatic MCF7-5624 cells were intrinsically unresponsive to TGF-β because they lack TβR-II receptor expression (), it was possible that TGF-β2 might promote metastasis in a paracrine manner. To address this question, mice were treated with the pan-TGF-β neutralizing antibody 1D11, an isotype control antibody (13C4) or vehicle only and then inoculated systemically with MCF-7-5624A-GF cells. Treatment with 1D11 failed to affect the rate of appearance and growth of metastases (). Moreover, neither the number of metastases () nor the survival of the mice () was affected. These experiments demonstrate that the high levels of active TGF-β2 produced by the tumor cells do not contribute significantly to metastasis of MCF-7-5624A-GF cells. Thus, in contrast to basal-like breast cancer models, in which both tumor cell autonomous TGF-β signaling and paracrine effects of tumor cell-associated TGF-β play major roles in driving dissemination and metastasis [8
], luminal breast cancer cells appear to disseminate independently of auto- or paracrine TGF-β signaling.
Fig 6 Luminal breast cancer metastasis does not require TGF-β signaling. a Conditioned medium of MCF-7-5624A-GF cells (red circles) produced much higher amounts of TGF-β2 than parental MCF-7 cells (blue squares), while production of TGF-β1 (more ...)