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In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.

ABSTRACT: Tumor hypoxia is known to activate angiogenesis, anaerobic glycolysis, invasion, and metastasis. However, a comparative analysis of the potentially distinct functions of hypoxia in primary tumor growth and organ-specific metastasis has not been reported. Here, we show distinct hypoxia kinetics in tumors generated by the MDA-MB-231 breast cancer sublines with characteristically different primary tumor growth rates and organotropic metastasis potentials. Hypoxia-induced angiogenesis promotes both primary tumor growth and lung metastasis but is nonessential for bone metastasis. Microarray profiling revealed that hypoxia enhances the expression of a significant number of genes in the lung metastasis signature, but only activates a few bone metastasis genes, among which DUSP1 was functionally validated in this study. Despite the different mechanisms by which hypoxia promotes organ-specific metastasis, inhibition of HIF-1alpha with a dominant-negative form of HIF-1alpha or 2-methoxyestradiol reduced metastasis to both lung and bone. Consistent with the extensive functional overlap of hypoxia in promoting primary tumor growth and lung metastasis, a 45-gene hypoxia response signature efficiently stratifies breast cancer patients with low or high risks of lung metastasis, but not for bone metastasis. Our study shows distinct functions of hypoxia in regulating angiogenesis and metastasis in different organ microenvironments and establishes HIF-1alpha as a promising target for controlling organotropic metastasis of breast cancer.

SUBMITTER: Lu X

PROVIDER: S-EPMC2872139 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.

Lu Xin X Yan Carol H CH Yuan Min M Wei Yong Y Hu Guohong G Kang Yibin Y

Cancer research 20100504 10

Tumor hypoxia is known to activate angiogenesis, anaerobic glycolysis, invasion, and metastasis. However, a comparative analysis of the potentially distinct functions of hypoxia in primary tumor growth and organ-specific metastasis has not been reported. Here, we show distinct hypoxia kinetics in tumors generated by the MDA-MB-231 breast cancer sublines with characteristically different primary tumor growth rates and organotropic metastasis potentials. Hypoxia-induced angiogenesis promotes both ...[more]

PMID: 20442288

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Project description:It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1? (HIF-1?) to drive transcription of various genes. Therefore, we have used a HIF-1? reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1? bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O?) for 24 hr before BLI, while the cells in normoxia (21% O?) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1? binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1? bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

Project description:BACKGROUND: The study of breast cancer metastasis depends on the use of established breast cancer cell lines that do not accurately represent the heterogeneity and complexity of human breast tumors. A tumor model was developed using primary breast tumor-initiating cells isolated from patient core biopsies that would more accurately reflect human breast cancer metastasis. METHODS: Tumorspheres were isolated under serum-free culture conditions from core biopsies collected from five patients with clinical diagnosis of invasive ductal carcinoma (IDC). Isolated tumorspheres were transplanted into the mammary fat pad of NUDE mice to establish tumorigenicity in vivo. Tumors and metastatic lesions were analyzed by hematoxylin and eosin (H+E) staining and immunohistochemistry (IHC). RESULTS: Tumorspheres were successfully isolated from all patient core biopsies, independent of the estrogen receptor ? (ER?)/progesterone receptor (PR)/Her2/neu status or tumor grade. Each tumorsphere was estimated to contain 50-100 cells. Transplantation of 50 tumorspheres (1-5 × 103 cells) in combination with Matrigel into the mammary fat pad of NUDE mice resulted in small, palpable tumors that were sustained up to 12 months post-injection. Tumors were serially transplanted three times by re-isolation of tumorspheres from the tumors and injection into the mammary fat pad of NUDE mice. At 3 months post-injection, micrometastases to the lung, liver, kidneys, brain and femur were detected by measuring content of human chromosome 17. Visible macrometastases were detected in the lung, liver and kidneys by 6 months post-injection. Primary tumors variably expressed cytokeratins, Her2/neu, cytoplasmic E-cadherin, nuclear ? catenin and fibronectin but were negative for ER? and vimentin. In lung and liver metastases, variable redistribution of E-cadherin and ? catenin to the membrane of tumor cells was observed. ER? was re-expressed in lung metastatic cells in two of five samples. CONCLUSIONS: Tumorspheres isolated under defined culture conditions from patient core biopsies were tumorigenic when transplanted into the mammary fat pad of NUDE mice, and metastasized to multiple mouse organs. Micrometastases in mouse organs demonstrated a dormancy period prior to outgrowth of macrometastases. The development of macrometastases with organ-specific phenotypic distinctions provides a superior model for the investigation of organ-specific effects on metastatic cancer cell survival and growth.

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In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.

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In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.

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