This work is supported by The Jackson Laboratory Cancer Center Pilot Grant and The Maine Cancer Foundation.

1. Protocol
<ol>
 <li>Anesthetize the mouse with ketamine and xylazine at a dose of 0.1 mg ketamine and 0.01 mg of xylazine per 1 g of body weight. Carprofen (0.005 mg per 1 g of body weight) is used for analgesia and is administered preoperatively.</li>
 <li>All of the surgical tools, including the dental drill bit, are steam sterilized in an autoclave. If batch surgeries are to be performed, the tips of surgical instruments must be re-sterilized with a glass bead sterilizer (Fine Science Tools FST 250) before each su...

<ol>
	<li>Paulino, A. C., Teh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+brain+tumors.">Treatment of brain tumors.</a> N. Engl. J. Med. 352, 2350-2353 (2005).</li><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> N. Engl. J. Med. 352, 987-996 (2005).</li><li>Singh, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Cheng, L., Bao, S., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+therapeutic+implications+of+cancer+stem+cells+in+glioblastoma.">Potential therapeutic implications of cancer stem cells in glioblastoma.</a> Biochem. Pharmacol. 80, 654-665 (2010).</li><li>Cheng, L., Ramesh, A. V., Flesken-Nikitin, A., Choi, J., Nikitin, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+cancer+stem+cell+research.">Mouse models for cancer stem cell research.</a> Toxicol. Pathol. 38, 62-71 (2010).</li><li>Dirks, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+stem+cells:+the+cancer+stem+cell+hypothesis+writ+large.">Brain tumor stem cells: the cancer stem cell hypothesis writ large.</a> Mol. Oncol. 4, 420-430 (2010).</li><li>Ebben, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+paradigm:+a+new+understanding+of+tumor+development+and+treatment.">The cancer stem cell paradigm: a new understanding of tumor development and treatment.</a> Expert Opin. Ther. Targets. 14, 621-632 (2010).</li><li>Germano, I., Swiss, V., Casaccia, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+brain+tumors,+neural+stem+cell,+and+brain+tumor+cancer+cells:+where+is+the+link.">Primary brain tumors, neural stem cell, and brain tumor cancer cells: where is the link.</a> Neuropharmacology. 58, 903-910 (2010).</li><li>Park, D. M., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+glioma+cancer+stem+cells.">Biology of glioma cancer stem cells.</a> Mol. Cells. 28, 7-12 (2009).</li><li>Bao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-like+glioma+cells+promote+tumor+angiogenesis+through+vascular+endothelial+growth+factor.">Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.</a> Cancer Res. 66, 7843-7848 (2006).</li><li>Calabrese, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perivascular+niche+for+brain+tumor+stem+cells.">A perivascular niche for brain tumor stem cells.</a> Cancer Cell. 11, 69-82 (2007).</li><li>Barami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+neural+stem+cells+with+their+vascular+niche:+implications+in+the+malignant+progression+of+gliomas.">Relationship of neural stem cells with their vascular niche: implications in the malignant progression of gliomas.</a> J. Clin. Neurosci. 15, 1193-1197 (2008).</li><li>Gilbertson, R. J., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+tumour's+bed:+glioblastoma+stem+cells+and+the+vascular+niche.">Making a tumour's bed: glioblastoma stem cells and the vascular niche.</a> Nat. Rev. Cancer. 7, 733-736 (2007).</li><li>Cho, R. W., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+cancer+stem+cells.">Recent advances in cancer stem cells.</a> Curr. Opin. Genet. Dev. 18, 48-53 (2008).</li><li>Drew, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nat. Methods. 7, 981-984 (2010).</li></ol>

The key to a successful PoRTS cranial window is the thinning and polishing. While the initial thinning can be performed quickly, care should be taken to ensure homogenous thinning of the skull over a large area. We typically apply a thin layer of saline to the skull, and then thin the skull one pass at a time, such that the saline solution evaporates shortly after the microdrill has passed over the skull once. This allows us to slowly but homogenously further thin the skull. A steady hand under the microscope is also key...

<table border="1">
 <tr>
 <td>Name of 			reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments 			(optional)</td>
 </tr>
 <tr>
 <td>DMEM/F12(1:1) 			with Sodium Pyruvate</td>
 <td>Thermo Sci 			Hyclone</td>
 <td>SH3026101</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 Serum 			Free Supplement (50x)</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>GlutaMax-1 			Supplement</td>
 <td>Invitrogen</td>
 <td>35050061</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin 			Solution</td>
 <td>Thermo Sci 			Hyclone</td>
 <td>SV30010</td>
 <td></td>
 </tr>
 <tr>
 <td>Accutase-Enzyme 			Cell Detachment Medium</td>
 <td>eBiosciences</td>
 <td>00-4555-56</td>
 <td></td>
 </tr>
 <tr>
 <td>T25 			flasks-vent cap green</td>
 <td>SARSTEDT</td>
 <td>83.1810.502</td>
 <td></td>
 </tr>
 <tr>
 <td>70% 			alcohol</td>
 <td>JAX LAHS</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>10% 			povidone-iodine topical solution</td>
 <td>JAX LAHS</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine 			HCl</td>
 <td>Butler 			Animal Health Supply</td>
 <td>NDC# 			11695-0550-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Akorn, Inc.</td>
 <td>NADA# 			139-236</td>
 <td></td>
 </tr>
 <tr>
 <td>Carprofen</td>
 <td>JAX LAHS</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ophthalmic 			ointment</td>
 <td>Dechra 			Veterinary Products</td>
 <td>17033-211-38</td>
 <td></td>
 </tr>
 <tr>
 <td>0.5% 			Lidocaine HCl</td>
 <td>REGENT, 			Inc.</td>
 <td>NDC 			0517-0625-25</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyanoacrylate 			glue</td>
 <td>Henkel 			Corp.</td>
 <td>46551</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile 			Swabs</td>
 <td>Fisher 			Scientific</td>
 <td>23-400-114</td>
 <td></td>
 </tr>
 <tr>
 <td>Diamond 			paste</td>
 <td>Widget 			Supply</td>
 <td>BBE60</td>
 <td></td>
 </tr>
 <tr>
 <td>Tin oxide</td>
 <td>LORTONE, 			Inc.</td>
 <td>591-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Liner Bond 			2V</td>
 <td>KURARAY 			Medical Inc.</td>
 <td>1921-KA</td>
 <td></td>
 </tr>
 <tr>
 <td>Clearfil 			AP-X</td>
 <td>KURARAY 			Medical Inc.</td>
 <td>1721-KA</td>
 <td></td>
 </tr>
 <tr>
 <td>Saline</td>
 <td>JAX LAHS</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Cover glass</td>
 <td>Warner 			Instruments</td>
 <td>64-0720</td>
 <td></td>
 </tr>
 <tr>
 <td>Hex Nut</td>
 <td>Small 			Parts, Inc.</td>
 <td>HNX-0090-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe 			Pump</td>
 <td>Syringepump.com</td>
 <td>NE-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Two-Photon 			imaging system</td>
 <td>Custom 			built (Any commercial system would work)</td>
 <td></td>
 <td></td>
 </tr>
</table>

imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Watch this Scientific Journal Video about Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window at JoVE.com

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

imaging-glioma-initiation-vivo-through-polished-reinforced-thin-skull

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JoVE Journal

Medicine

13.0K visualizações.  The Jackson Laboratory. Ao combinar um polido e reforçado crânio-fino (portas) craniana janela e injeção de células do glioblastoma (GBM), podemos observar glioma iniciação e crescimento de células GBM injetadas no cérebro de um rato vivo longitudinalmente.