कैंसर मेटास्टेसिस के मात्रात्मक विश्लेषण एक एवियन भ्रूण मॉडल का उपयोग
Summary
मात्रात्मक पीसीआर का उपयोग करके, हम प्रदर्शन कैसे अच्छी तरह से स्थापित लड़की सांचा मॉडल मात्रात्मक दूर के अंगों को मानव ट्यूमर कोशिकाओं की मेटास्टेसिस का विश्लेषण के लिए इस्तेमाल किया जा सकता है.
Abstract
During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.
Protocol
Discussion
लड़की सीएएम कैंसर जीव विज्ञान और metastatic प्रगति के विभिन्न पहलुओं का अध्ययन करने के लिए एक मॉडल प्रणाली के रूप में दशकों के लिए इस्तेमाल किया गया है. Metastatic क्षमताओं के सिद्धांत मूल्यांकन निद्रा का विश्लेषण…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम उदारता से निषेचित अंडे प्रदान करने के लिए टायसन फूड्स इंक पहचान करना चाहते हैं. Shanna अर्नोल्ड इस प्रोटोकॉल के फिल्मांकन के दौरान महत्वपूर्ण भूमिका निभाई थी. Trenis पामर रुथ एल Kirschstein राष्ट्रीय अनुसंधान सेवा पुरस्कार (F31 राष्ट्रीय कैंसर संस्थान) के तहत स्वास्थ्य के राष्ट्रीय संस्थान द्वारा समर्थित किया गया. इस काम आंशिक रूप से CCSRI # 700537 JDL और NIH / NCI अनुदान # CA120711-01A1-Z CA120711-01A1 अनुदान द्वारा समर्थित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| 1X Dulbecco’s Modified Eagle Medium (DMEM) | Invitrogen | 11995073 | |
| Dulbecco’s Phosphate-Buffered Saline (D-PBS) (1X), liquid | Invitrogen | 14190250 | pH 7.4 |
| Trypsin, 0.05% (1X) with EDTA 4Na, liquid | Invitrogen | 25300054 | |
| Sportsman hatcher | Berry Hill | 1550HA | These incubators are some of the most readily available. However, the video will show different equipment. |
| Sportsman incubator | Berry Hill | 1502EA | |
| Dremel rotary tool | Dremel | ||
| Dremel cutoff wheels no. 36 | Dremel | 409 | |
| Portable Pipette Aid | Fisher | 1368115E | |
| Cotton Tipped Applicators | Fisher | 23-400-001) | |
| Fertilized Eggs | Various | Many different vendor can be used. The eggs should be approximately 35-55 grams. The hens that lays them should be between 36 and 55 weeks of age. | |
| Hemocytometer | Hausser Scientific, VWR | 15170-090 | |
| Fiber-optic microscope illuminator | Amscope | HL250-AY | 150W |
| 25 gauge needle | |||
| Sybr Green Extract–N-Amp Tissue PCR Kit | Sigma-Aldrich | XNATRG |
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