In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Summary

Bioluminescence imaging of hypoxia inducible factor-1α activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Abstract

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-1α (HIF-1α) to drive transcription of various genes. Therefore, we have used a HIF-1α reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1α bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O₂) for 24 hr before BLI, while the cells in normoxia (21% O₂) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1α 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-1α 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.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center.

1. In vitro HIF-1α bioluminescence assay

1. Materials and Methods:
   • Human metastatic breast cancer cell line MDA-MB231 transfected with a novel HIF-1-dependent reporter gene, 5HRE-ODD-luc was generated by Dr. Harada.
   • In hypoxic condition, the enhanced expression of oxygen-dependent degradation domain (ODD)-Lucif.......

Discussion

Breast cancer brain metastasis occurs in 30% of breast cancer patients at stage IV. It is associated with high morbidity and mortality and has a median survival of 13 months⁶. There is a need to have appropriate animal models to mimic this clinically devastating disease in order to facilitate our understanding of its intracranial initiation and progression as well as pathophysiological profiles. Here, we have developed an orthotopic breast cancer brain metastasis model by injecting human breast cancer cells, d.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
D-luciferin	Gold Biotechnology	L-123	120 mg/kg in PBS in a total volume of 80 μl for in vivo study
Isoflurane	Baxter International Inc.	1001936060
Matrigel	BD Biosciences	354234
Hamilton syringe	Hamilton Company	1701
32G Hamilton needle	Hamilton Company	7803-04
Hypoxia chamber	Billups-Rothenberg, Inc.	MIC-101
Bioluminescence imaging system	Caliper Life Sciences	IVIS Spectrum system
G418	Fisher scientific	SV3006901