स्तन कैंसर मस्तिष्क मेटास्टेसिस के ट्यूमर Hypoxia गतिशीलता के vivo bioluminescence इमेजिंग में एक माउस मॉडल में
Summary
Hypoxia inducible कारक 1α गतिविधि के bioluminescence इमेजिंग के लिए एक स्तन कैंसर मस्तिष्क मेटास्टेसिस माउस मॉडल में intracranial ट्यूमर hypoxia के विकास पर नजर रखने के लिए लागू किया जाता है.
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
Discussion
स्तन कैंसर मस्तिष्क मेटास्टेसिस के चतुर्थ चरण में स्तन कैंसर के रोगियों के 30% में होता है. यह उच्च रुग्णता और मृत्यु दर के साथ जुड़ा हुआ है और 13 6 महीने के एक औसत अस्तित्व है . वहाँ एक के लिए उपयुक्त पशु …
Divulgations
The authors have nothing to disclose.
Acknowledgements
इस अध्ययन में भाग DOD स्तन कैंसर आईडिया पुरस्कार द्वारा समर्थित है W81XWH-08-1-0583 और NIH / CA141348 01A1 NCI (DZ) और FAMRI नैदानिक वैज्ञानिक पुरस्कार (डी एस). इमेजिंग बुनियादी ढांचे पश्चिमी छोटे पशु इमेजिंग अनुसंधान CA126608 U24 और सीमन्स कैंसर केंद्र (CA142543 p30) और NIH 1S10RR024757 01 द्वारा भाग में समर्थित प्रोग्राम द्वारा प्रदान की गई है.
Materials
| 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 |
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