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Genomic Analysis of a Spontaneous Model of Breast Cancer Metastasis to Bone Reveals a Role for the Extracellular Matrix Genomic Analysis of a Spontaneous Model of Breast Cancer Metastasis to Bone Reveals a Role for the Extracellular Matrix Bedrich L. Eckhardt,1 Belinda S. Parker,1 Ryan K. van Laar,1 Christina M. Restall,1 Anthony L. Natoli,1 Michael D. Tavaria,1 Kym L. Stanley,1 Erica K. Sloan,1 Jane M. Moseley,2 and Robin L. Anderson1 1Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Melbourne, Victoria, Australia and 2Department of Medicine, University of Melbourne, St. Vincent’s Hospital, Fitzroy, Victoria, Australia Abstract involvement in several tissues, including bone, lung, lymph A clinically relevant model of spontaneous breast cancer node, and liver (1). The selective distribution of metastases is metastasis to multiple sites, including bone, was dictated by several factors, including the pattern of vascular characterized and used to identify genes involved in flow from the primary site, complementary adhesive contacts, metastatic progression. The metastatic potential of and molecular interactions between the tumor cell and the several genetically related tumor lines was assayed stroma at the secondary site (2). using a novel real-time quantitative RT-PCR assay of Breast cancer metastases have a strong avidity for bone tumor burden. Based on this assay, the tumor lines were (3, 4), leading to metastases that cause intractable pain, spinal categorized as nonmetastatic (67NR), weakly metastatic cord compression, bone fractures, and hypercalcemia (5, 6). to lymph node (168FARN) or lung (66cl4), or highly Current therapies, including cytotoxic chemotherapy and the metastatic to lymph node, lung, and bone (4T1.2 and administration of bisphosphonates, are rarely curative but can 4T1.13). In vitro assays that mimic stages of metastasis alleviate symptoms arising from bone metastases (7). An showed that highly metastatic tumors lines were more improved understanding of the biological and genetic regula- adhesive, invasive, and migratory than the less tion of breast cancer metastasis to bone is essential to identify metastatic lines. To identify metastasis-related genes novel and more effective molecular targets for therapy. in this model, each metastatic tumor was array profiled The advent of microarray technology has greatly enhanced against the nonmetastatic 67NR using 15,000 mouse the search for genetic regulators and markers of metastasis. cDNA arrays. A significant proportion of genes relating Indeed, arrays have been employed to identify genetic patterns to the extracellular matrix had elevated expression in that are predictive of metastatic relapse (8-10). They have also highly metastatic tumors. The role of one of these genes, been used in animal models of metastasis to determine POEM, was further investigated in the model. In situ molecular mechanisms that dictate metastatic spread (11-14). hybridization showed that POEM expression was Animal models provide an essential and powerful resource to specific to the tumor epithelium of highly metastatic investigate mechanisms of metastasis. However, breast cancer tumors. Decreased POEM expression in 4T1.2 tumors metastasis research is dominated by the use of xenograft models significantly inhibited spontaneous metastasis to the of experimental metastasis, typically involving the injection of lung, bone, and kidney. Taken together, our data human breast tumor cells directly into the circulatory system of support a role for the extracellular matrix in metastatic immunocompromised mice, resulting in metastases in either progression and describe, for the first time, a role for lung (following tail vein injection) or bone (following POEM in this process. (Mol Cancer Res 2005;3(1):1–13) intracardiac injection; refs. 14-17). These models have been valuable for studying the final stages of metastasis, and when Introduction coupled with microarray analysis, they can identify genes that Metastasis is the main cause of morbidity and mortality in regulate the colonization of specific tissues (13, 14). However, cancer patients. In late-stage breast cancer, patients have tumor these models of experimental metastasis involve human tumors in a mouse host and may lack some of the critical tumor-host interactions. In addition, they do not encompass the initial Received 6/1/04; revised 11/19/04; accepted 11/30/04. stages of primary tumor growth, invasion, and metastasis from Grant support: Department of Defense Breast Cancer Research Program grants an orthotopic site. Thus, array analysis using these experimental DAMD17-98-1-8144 (R.L. Anderson) and DAMD17-01-1-0371 (M.D. Tavaria), metastasis models provides little insight into the biology of Susan G. Komen Breast Cancer Foundation predoctoral fellowship (E.K. Sloan), and NIH/National Cancer Institute grant ROI CA90291 (R.L. Anderson). spontaneous metastasis. The costs of publication of this article were defrayed in part by the payment of A clinically relevant animal model of spontaneous breast page charges. This article must therefore be hereby marked advertisement in cancer metastasis to multiple sites, including bone, is now accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Robin L. Anderson, Trescowthick Research Laboratories, available (18). Several syngeneic tumor lines with a spectrum of Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, metastatic phenotypes have been isolated from a spontaneous Victoria, Australia 8006. Phone: 61-3-9656-1284; Fax: 61-3-9656-1411. E-mail: [email protected] mammary tumor in a BALB/cfC3H mouse (19). When injected Copyright D 2005 American Association for Cancer Research. into the mammary gland of mice, these tumor lines are either Mol Cancer Res 2005;3(1). January 2005 1 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. 2 Eckhardt et al. nonmetastatic (67NR; ref. 20), produce spontaneous metastases ALN along with numerous metastatic foci in the lungs (Fig. 1C). to lymph node (168FARN; ref. 21), lung (66cl4; ref. 22), or both When analyzed by q-RT-PCR, high RTB signals were detected in (4T1; ref 20). 4T1.2 and 4T1.13 tumor lines are rare variants the lung (54% tumor tissue), ALN (29%), femur (17%), spine isolated from 4T1 cells that closely mimic the metastatic (10%), kidney (10%), and heart (8%; Fig. 1B). A similar distribution of human breast cancer (18). When grown as a metastatic pattern was observed for the 4T1.13 tumor line. Bone primary tumor in the mammary fat pad of a mouse, these tumor lysis was evident in bone metastases derived from 4T1.2 and lines develop overt metastases in bone, lung, and lymph nodes 4T1.13 tumors (data not shown), consistent with previous reports (18). Furthermore, mice bearing 4T1.2 and 4T1.13 tumors of these tumor lines (18, 24). Tumor signals were also detected in occasionally develop hind limb paralysis and have elevated the brain, liver, and spleen in mice harboring 4T1.2 and 4T1.13 plasma levels of calcium and parathyroid hormone-related tumors albeit to a minor extent (<1%). Thus, 4T1.2 and 4T1.13 protein (PTHrP), two pathologic hallmarks of the human disease can spontaneously metastasize to several organs, but preferential (18, 23). With a diverse range of metastatic capacities, these growth occurs in specific tissues (lung, ALN, and bone). The genetically related tumor lines provide a powerful model to presence of tumor within the ALN of mice bearing 4T1.2 and investigate the molecular events that dictate metastasis of breast 4T1.13 tumors suggests that these lines can also metastasize via cancer. a lymphogenous route. For more accurate analysis of metastasis, a real-time The extent of metastasis from 4T1.2 and 4T1.13 tumors to quantitative reverse transcription-PCR (q-RT-PCR) assay for ALN and lung was f20-fold higher than metastases produced the measurement of tumor burden within a tissue has been by 168FARN and 66cl4 tumors, respectively, indicating that developed. All tumor lines are tagged with a neomycin 4T1.2 and 4T1.13 have a greater metastatic capacity than resistance gene, allowing them to be detected by q-RT-PCR 168FARN and 66cl4. Furthermore, the metastatic distribution of genomic DNA. Using this model of spontaneous breast of 4T1.2 and 4T1.13 tumors is similar to that observed in cancer metastasis, critical metastasis-related genes can be advanced human breast cancer and, to our knowledge, provides identified. Gene expression profiles of metastatic tumors in the only syngeneic mouse model of spontaneous breast cancer this model were compared with the nonmetastatic 67NR using metastasis to bone. cDNA arrays consisting of 15,000 mouse gene elements. Array Representative sections of the kidney, heart, lung, spine, and analysis revealed altered expression of extracellular matrix femur provide further evidence of the difference in extent of (ECM) genes in highly metastatic tumors. One of these genes, metastatic dissemination of the 4T1.2 and 66cl4 tumors POEM, was shown to have a functional role in spontaneous (Fig. 1C). Metastatic lung nodules from 4T1.2 tumors were breast cancer metastasis in the model. larger and more numerous compared with those in the lungs of mice with 66cl4 tumors. Overt metastases in the spine and femur were detected in 4T1.2 and 4T1.13 tumors but not from Results 66cl4 tumors (Fig. 1C; data not shown). These histologic data Characterization of the Metastatic Dissemination of agree with the RTB values determined by q-RT-PCR. Several Syngeneic Mammary Tumor Lines The spontaneous metastatic distribution of each tumor line Increased Adhesion, Migration, and Invasion, but Not was profiled using a sensitive q-RT-PCR assay for tumor Angiogenesis and Proliferation, Correlate with Enhanced burden (Fig. 1A). Tumor cells were inoculated into the fourth Metastatic Capacity inguinal mammary glands of mice. Mice were culled and The processes of angiogenesis, proliferation, cell motility, tissues were excised for genomic DNA extraction when the invasion, and adhesion are critical for the formation of distant average primary tumor weight of each group was f1.5 g or if metastases.
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