Name: extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment
Uploaded: 2016-06-12T14:00:00
Description: Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Dit onderzoek werd gesteund door het Ministerie van Defensie Breast Cancer Research Program onder award nummer (ASH, W81XWH-13-1-0010), NIH CA100324, PPG CA100324, en de geïntegreerde Imaging Program.

Alle beschreven procedures moeten worden uitgevoerd in overeenstemming met de richtlijnen en voorschriften voor het gebruik van gewervelde dieren, met inbegrip van de voorafgaande goedkeuring van het Albert Einstein College of Medicine Institutional Animal Care en gebruik Comite. 1. Productie fluorescent gelabelde tumoren en tumor-geassocieerde macrofagen <ol><li> Genereer fluorescent gelabelde tumorcellen door het oversteken van de spontane, autochtone, genetisch gemanipuleerde muizen borstkanker model waarbi...

<ol>
	<li>Gerlowski, L. E., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+permeability+of+normal+and+neoplastic+tissues.">Microvascular permeability of normal and neoplastic tissues.</a> Microvasc Res. 31 (3), 288-305 (1986).</li><li>Wang, H. -. L., Lai, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Evans+blue+quantitation+in+limited+rat+tissue+samples.">Optimization of Evans blue quantitation in limited rat tissue samples.</a> Sci Rep. 4, (2014).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rous-Whipple+Award+Lecture.+How+tumors+make+bad+blood+vessels+and+stroma.">Rous-Whipple Award Lecture. How tumors make bad blood vessels and stroma.</a> Am J Pathol. 162 (6), 1747-1757 (2003).</li><li>Nagy, J. A., 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=Permeability+properties+of+tumor+surrogate+blood+vessels+induced+by+VEGF-A.">Permeability properties of tumor surrogate blood vessels induced by VEGF-A.</a> Lab Invest. 86 (8), 767-780 (2006).</li><li>Egawa, G., 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=Intravital+analysis+of+vascular+permeability+in+mice+using+two-photon+microscopy.">Intravital analysis of vascular permeability in mice using two-photon microscopy.</a> Sci Rep. 3, (2013).</li><li>Yuan, F., 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=Vascular+permeability+in+a+human+tumor+xenograft:+molecular+size+dependence+and+cutoff+size.">Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size.</a> Cancer Res. 55 (17), 3752-3756 (1995).</li><li>Dellian, M., Yuan, F., Trubetskoy, V. S., Torchilin, V. P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+a+human+tumour+xenograft:+molecular+charge+dependence.">Vascular permeability in a human tumour xenograft: molecular charge dependence.</a> Br J Cancer. 82 (9), 1513-1518 (2000).</li><li>Entenberg, 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=Setup+and+use+of+a+two-laser+multiphoton+microscope+for+multichannel+intravital+fluorescence+imaging.">Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging.</a> Nat Protocols. 6 (10), 1500-1520 (2011).</li><li>Entenberg, 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=Imaging+tumor+cell+movement+in+vivo.">Imaging tumor cell movement in vivo.</a> Curr Protoc Cell Biol. Chapter. 19, (2013).</li><li>Patsialou, A., 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=Intravital+multiphoton+imaging+reveals+multicellular+streaming+as+a+crucial+component+of+in+vivo.+cell+migration+in+human+breast+tumors.">Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo. cell migration in human breast tumors.</a> Intravital. 2 (2), e25294 (2013).</li><li>Gligorijevic, B., Bergman, A., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+classification+links+tumor+microenvironments+with+tumor+cell+phenotype.">Multiparametric classification links tumor microenvironments with tumor cell phenotype.</a> PLoS Biol. 12 (11), e1001995 (2014).</li><li>Roussos, E. T., 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=Mena+invasive+(MenaINV)+promotes+multicellular+streaming+motility+and+transendothelial+migration+in+a+mouse+model+of+breast+cancer.">Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.</a> J Cell Sci. 13, 2120-2131 (2011).</li><li>Harney, A. 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=Real-Time+Imaging+Reveals+Local,+Transient+Vascular+Permeability,+and+Tumor+Cell+Intravasation+Stimulated+by+TIE2hi+Macrophage-Derived+VEGFA).">Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA).</a> Cancer Discov. 5 (9), 932-943 (2015).</li><li>Robinson, B. 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=Tumor+microenvironment+of+metastasis+in+human+breast+carcinoma:+a+potential+prognostic+marker+linked+to+hematogenous+dissemination.">Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination.</a> Clin Cancer Res. 15, 2433-2441 (2009).</li><li>Sasmono, R. T., 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+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Ovchinnikov, D. A., 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=Expression+of+Gal4-dependent+transgenes+in+cells+of+the+mononuclear+phagocyte+system+labeled+with+enhanced+cyan+fluorescent+protein+using+Csf1r-Gal4VP16/UAS-ECFP+double-transgenic+mice.">Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice.</a> J Leukcocyte Biol. 83 (2), 430-433 (2008).</li><li>Liu, H., 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=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).</li><li>Nakasone, E. S., Askautrud, H. A., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+drug+responses+in+the+tumor+microenvironment+in+mouse+models+of+breast+cancer.">Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer.</a> J Vis Exp. (73), (2013).</li><li>Weis, S., Cui, J., Barnes, L., Cheresh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+barrier+disruption+by+VEGF-mediated+Src+activity+potentiates+tumor+cell+extravasation+and+metastasis.">Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis.</a> J Cell Biol. 167 (2), 223-229 (2004).</li><li>Wyckoff, J., Gligorijevic, B., Entenberg, D., Segall, J., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Multiphoton+Imaging+of+Tumors.">High-Resolution Multiphoton Imaging of Tumors.</a> In Vivo. Cold Spring Harb Protoc. (10), (2011).</li><li>Lathia, 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=Direct+in+vivo.+evidence+for+tumor+propagation+by+glioblastoma+cancer+stem+cells.">Direct in vivo. evidence for tumor propagation by glioblastoma cancer stem cells.</a> PLoS One. 6 (9), e24807 (2011).</li><li>Nakasone, E. 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=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer Cell. 21 (4), 488-503 (2012).</li><li>Dreher, M. 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=Tumor+vascular+permeability,+accumulation,+and+penetration+of+macromolecular+drug+carriers.">Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers.</a> J Natl Cancer Inst. 98 (5), 335-344 (2006).</li><li>Wyckoff, J. B., 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=Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors.">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67 (6), 2649-2656 (2007).</li><li>Chittajallu, D. 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=In+vivo.+cell-cycle+profiling+in+xenograft+tumors+by+quantitative+intravital+microscopy.">In vivo. cell-cycle profiling in xenograft tumors by quantitative intravital microscopy.</a> Nat Methods. 12 (6), 577-585 (2015).</li><li>Kedrin, 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=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat Methods. 5 (12), 1019-1021 (2008).</li><li>Yano, 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=Spatial-temporal+FUCCI+imaging+of+each+cell+in+a+tumor+demonstrates+locational+dependence+of+cell+cycle+dynamics+and+chemoresponsiveness.">Spatial-temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness.</a> Cell Cycle. 13 (13), 2110-2119 (2014).</li><li>Yang, M., Jiang, P., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+subcellular+multicolor+imaging+of+tumor-host+interaction+and+drug+response+in+real+time.">Whole-body subcellular multicolor imaging of tumor-host interaction and drug response in real time.</a> Cancer Res. 67 (11), 5195-5200 (2007).</li><li>Manning, C. 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=Intravital+imaging+reveals+conversion+between+distinct+tumor+vascular+morphologies+and+localized+vascular+response+to+Sunitinib.">Intravital imaging reveals conversion between distinct tumor vascular morphologies and localized vascular response to Sunitinib.</a> Intravital. 2 (1), 24790 (2013).</li><li>Alexander, S., Koehl, G. E., Hirschberg, M., Geissler, E. K., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+cancer+growth+and+invasion:+a+modified+skin-fold+chamber+model.">Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model.</a> Histochem Cell Biol. 130 (6), 1147-1154 (2008).</li><li>Brown, E. B., 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=In+vivo.+measurement+of+gene+expression,+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy.">In vivo. measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nat Med. 7 (7), 864-868 (2001).</li></ol>

Cellulaire interacties die spontaan in de tumor micro-omgeving kan leiden tot veranderingen in motiliteit en tumorcel intravasation. Hoge-resolutie intravitale beeldvorming van levend tumorweefsel maakt de visualisatie van meercellige dynamiek die zeer voorbijgaand 10,13,24 zijn. Eindpunt in vivo testen of time-lapse beelden die met discrete tijdstippen kan essentiële informatie verschaffen over de moleculaire mechanismen van processen in de tumor micro. Intravitale imaging studies zijn gebruikt om ...

Verspreiding van tumorcellen van primaire borsttumor is aangetoond dat niet alleen tumorcellen stromale cellen zoals macrofagen en endotheliale cellen omvatten, maar gastheer. Bovendien tumorvasculatuur met abnormaal verhoogde permeabiliteit 1. Aldus bepalen hoe tumorcellen, macrofagen en endotheelcellen interageren vasculaire permeabiliteit en tumorcel intravasation in de primaire tumor micro mediëren van belang voor het begrip metastase. Inzicht in de kinetiek van vasculaire permeabiliteit, tumorcel intravasation en de onderliggende signaleringsmechanisme van meercellige interacties in de tumor micro kan cruciale informatie bij het ontwikkelen en toediening van anti-kankertherapieën. Het belangrijkste middel van het bestuderen tumor vasculaire permeabiliteit in vivo meten van de extravasculaire kleurstoffen zijn zoals Evans blue 2, hoogmoleculaire dextranen molecuulgewicht (155 kDa)3 en ​​fluorofoor of tracer-geconjugeerde eiwitten (zoals albumine) 4 op vaste tijdstippen na injectie van de kleurstof. De vooruitgang in microscopie, hebben diermodellen en fluorescerende kleurstoffen de visualisatie van cellulaire processen en vasculaire permeabiliteit in levende dieren aanzetten met intravitale microscopie 5. Levende dieren beeldvorming met de overname van statische afbeeldingen of korte time-lapse sequenties over verschillende tijdstippen niet mogelijk is voor het volledig begrip van dynamische gebeurtenissen in de tumor micro 6,7. Inderdaad, statische beeldacquisitie de loop van 24 uur toonde dat de tumorvasculatuur lekt, maar de dynamiek van vasculaire permeabiliteit werd niet waargenomen 6. Aldus langere continue tijd-lapse imaging tot 12 uur vangt de kinetiek van dynamische gebeurtenissen in de tumor micro-omgeving. Dit protocol beschrijft het gebruik van verlengde time-lapse multiphotop intravitale microscopie dynamische meercellige gebeurtenissen in de tumor micro bestuderen. Meerdere celtypes in de tumor micro gelabeld met injecteerbare kleurstoffen of in transgene dieren tot expressie fluorescerende eiwitten. Via een staartader katheter vasculaire kleurstoffen of eiwitten kunnen worden geïnjecteerd na het begin van de beeldvorming kinetische gegevens van meercellige gebeurtenissen in de tumor micro verwerven. Voor live cell imaging de borsttumor wordt belicht door de chirurgische voorbereiding van een huid flap. Beelden werden tot 12 uur met een multifoton microscoop uitgerust met meerdere fotomultiplicatorbuizen (PMT) detectors 8. Door gebruikmaking van geschikte filters, een aftrekking algoritme stelt 4 PMT detectors 5 fluorescentiesignalen in de tumor micro gelijktijdig 9 verwerven. Hoge-resolutie multifoton intravitale microscopie vangt enkele cel resolutie afbeelding van tumor-stroma interacties in de tumor micro-omgeving, wat leidt tot een betere understanding van vasculaire permeabiliteit en tumorcel intravasation 10-13. Specifiek uitgebreid intravitale beeldvorming onthulde sterk gelokaliseerde, voorbijgaande vasculaire permeabiliteit gebeurtenissen die selectief plaatsvinden op plaatsen van interactie tussen een tumorcel, een macrofaag en een endotheelcel (gedefinieerd als de tumor micro van metastase, TMEM 14) 13. Bovendien tumorcel intravasation alleen optreedt bij TMEM en ruimtelijk en temporeel gecorreleerd met vasculaire permeabiliteit 13. Enkele cel resolutie van de dynamiek van deze gebeurtenissen is mogelijk gemaakt door het gebruik van verlengde time-lapse multifoton microscopie van fluorescent gelabelde cellen in de tumor micro-omgeving.

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">155 kDa dextran-tetramethylrhodamine isothiocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">T1287</td>
			<td style="width:167px;">reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">70 kDa dextran-Texas Red</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">D-1830</td>
			<td>reconstitute at 10 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">10 kDa dextran-fluorescein isothyocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">FD10S</td>
			<td>reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Qdot 705 ITK Amino (PEG) Quantum Dots</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">Q21561MP</td>
			<td>Dilute 25 uL in 175 uL of 1 X PBS for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MMTV-PyMT mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">2374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">26051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-EGFP mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">1 x PBS</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Isoethesia (isoflurane)</td>
			<td style="width:95px;">Henry Schein Animal Health</td>
			<td align="right" style="width:128px;">50033</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen</td>
			<td style="width:95px;">AirTech</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">1 mL syringe, tuberculin slip tip</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">30G x 1 (0.3 mm X 25 mm) needle</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">305128</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Polyethylene micro medical tubing&#160;</td>
			<td style="width:95px;">Scientific Commodities Inc</td>
			<td style="width:128px;">BB31695-PE/1</td>
			<td>0.28 mm I.D. X 0.64 mm O.D.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope coverglass</td>
			<td style="width:95px;">Corning</td>
			<td style="width:128px;">2980-225</td>
			<td>thickness 1.5, 22 X 50 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MouseOx oximeter, software and sensors</td>
			<td style="width:95px;">STARR Life Sciences</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Laboratory tape</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:128px;">159015R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">soft rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8514K62</td>
			<td>Ultra-Soft Polyurethane Film, 3/16&#8221; Thick, 12&#34; x 12&#34;, 40 Oo Durometer, Plain Back</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">hard rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8568K615</td>
			<td>High-Strength Neoprene Rubber Sheet 1/4&#34; Thick, 12&#34; X 12&#34;, 50A Durometer,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope</td>
			<td style="width:95px;">Olympus</td>
			<td style="width:128px;"></td>
			<td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">7-Punch set</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td style="width:128px;">3429A12</td>
			<td>, 1/4&#34; to 1&#34; Hole Diameter, for Hammer-Driven Hole Punch,&#160;</td>
		</tr>
	</tbody>
</table>

extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

Uitgebreide time-lapse intravitale microscopie maakt enkele cel resolutie beeldvorming van meercellige processen in de tumor micro. Door fluorescent labeling tumorcellen, macrofagen, het vaatsysteem, en visualiseren van de collageenvezelnetwerk met de frequentieverdubbeling signaal worden meerdere compartimenten in de tumor micro gelijktijdig gevolgd tijdens de beeldvorming. Tumorcellen gelabeld met fluorescente proteïnen kunnen worden geproduceerd in transgene muizen zoals in MMTV-PyMT...

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Dit protocol beschrijft het gebruik van multiphoton microscopie tot uitgebreide time-lapse beeldvorming van meercellige interacties uit te voeren in real time, in vivo bij enkele resolutie cel.

Extended Time-lapse intravitale Imaging van Real-time Meercellige Dynamiek in de tumor micro

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Research

JoVE Journal

Medicine

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