Name: 3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer
Uploaded: 2018-09-13T14:45:00
Description: Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

Forfatterne vil gerne takke Dr. Howard Crawford (University of Michigan) laboratoriet for teknisk support og leverer materialer og udstyr til denne undersøgelse, og Alan Kelleher for teknisk support.
Dette arbejde blev finansieret af tilskud fra University of Michigan Rogel kræft Center kerne Grant CA046592-26S3, NIH K08 CA201335-01A1 (PLP), BKAN YIA (PLP), NIH R01 CA17483601A1 (DMS).

1. voksende kræft Spheroids
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Vokser fra cellelinjer
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<li>Kultur menneskelige blære cancerceller under konventionelle tilhænger celle kultur betingelser og vedligehold i et 37 ° C inkubator leveres med 5% CO2. Vedligeholde celler på < 90% confluency. Bemærk: kultur medier bruges er Dulbeccos modificerede minimum essential medier (DMEM) der indeholder 4,5 g/L D-glucose, L-glutamin, 110 mg/L natrium pyruvat, og leveres med 10% føtal bovint serum (FBS) i hele denne protoko...

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	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Society. , (2018).</li><li>Knowles, M. A., Hurst, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bladder+cancer:+new+insights+into+pathogenesis+and+clinical+diversity.">Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity.</a> Nature Reviews Cancer. 15 (1), 25-41 (2015).</li><li>DeGeorge, K. C., Holt, H. R., Hodges, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+Cancer:+Diagnosis+and+Treatment.+American+Family+Physician.">Bladder Cancer: Diagnosis and Treatment. American Family Physician.</a> American Family Physician. 96 (8), 507-514 (2017).</li><li>Repesh, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro.+assay+for+quantitating+tumor+cell+invasion.">A new in vitro. assay for quantitating tumor cell invasion.</a> Invasion Metastasis. 9 (3), 192-208 (1989).</li><li>Doillon, C. J., Gagnon, E., Paradis, R., Koutsilieris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+system+as+a+model+for+studying+cancer+cell+invasion+capacity+and+anticancer+drug+sensitivity.">Three-dimensional culture system as a model for studying cancer cell invasion capacity and anticancer drug sensitivity.</a> Anticancer Research. 24 (4), 2169-2177 (2004).</li><li>Duong, H. S., Le, A. D., Zhang, Q., Messadi, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+BBN+as+an+urothelial+carcinogen.">Review: BBN as an urothelial carcinogen.</a> In Vivo. 26 (4), 727-739 (2012).</li><li>Palmbos, P. L., 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=ATDC/TRIM29+Drives+Invasive+Bladder+Cancer+Formation+through+miRNA-Mediated+and+Epigenetic+Mechanisms.">ATDC/TRIM29 Drives Invasive Bladder Cancer Formation through miRNA-Mediated and Epigenetic Mechanisms.</a> Cancer Research. 75 (23), 5155-5166 (2015).</li><li>Wang, L., 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=ATDC+induces+an+invasive+switch+in+KRAS-induced+pancreatic+tumorigenesis.">ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis.</a> Genes &amp; Development. 29 (2), 171-183 (2015).</li><li>Fife, C. M., McCarroll, J. A., Kavallaris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movers+and+shakers:+cell+cytoskeleton+in+cancer+metastasis.">Movers and shakers: cell cytoskeleton in cancer metastasis.</a> British Journal of Pharmacology. 171 (24), 5507-5523 (2014).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microtubule+organization+and+dynamics:+two+ends+in+the+limelight.">Control of microtubule organization and dynamics: two ends in the limelight.</a> Nature Reviews Molecular Cell Biology. 16 (12), 711-726 (2015).</li><li>Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+invasion+in+breast+cancer+requires+a+conserved+basal+epithelial+program.">Collective invasion in breast cancer requires a conserved basal epithelial program.</a> Cell. 155 (7), 1639-1651 (2013).</li><li>Kidd, M. E., Shumaker, D. K., Ridge, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vimentin+intermediate+filaments+in+the+progression+of+lung+cancer.">The role of vimentin intermediate filaments in the progression of lung cancer.</a> American Journal of Respiratory Cell and Molecular Biology. 50 (1), 1-6 (2014).</li><li>Lowery, J., Kuczmarski, E. R., Herrmann, H., Goldman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+Filaments+Play+a+Pivotal+Role+in+Regulating+Cell+Architecture+and+Function.">Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function.</a> Journal of Biological Chemistry. 290 (28), 17145-17153 (2015).</li><li>Papafotiou, 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=KRT14+marks+a+subpopulation+of+bladder+basal+cells+with+pivotal+role+in+regeneration+and+tumorigenesis.">KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis.</a> Nature Communications. 7, 11914 (2016).</li><li>Sun, W., Lim, C. T., Kurniawan, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+adaptability+of+cancer+cells+strongly+affects+anti-migratory+drug+efficacy.">Mechanistic adaptability of cancer cells strongly affects anti-migratory drug efficacy.</a> Journal of the Royal Society Interface. 11 (99), (2014).</li><li>Goddette, D. W., Frieden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization.+The+mechanism+of+action+of+cytochalasin+D.">Actin polymerization. The mechanism of action of cytochalasin D.</a> Journal of Biological Chemistry. 261 (34), 15974-15980 (1986).</li><li>Berrier, A. L., Yamada, K. 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Her beskriver vi en 3D-tumor klumpformet model, der giver mulighed for real-time observation af blære kræft invasion, som er kritiske for kræft progression og metastaser. Dette system er indstillet til inddragelsen af forskellige stromale og cellulære komponenter at tillade efterforskere at bedre sammenfatte væv mikromiljø hvor blære cancer invasion finder sted. Blære cancer spheroids kan genereres fra forskellige kilder såsom cellelinjer (herunder genetisk modificerede cellelinjer nyttige for undersøgelsen af ...

Invasionen er et kritisk skridt i kræft progression, som er nødvendig for metastaser, og er forbundet med lavere overlevelse og dårlig prognose hos patienter. I menneskelige blærekræft, den mest almindelige malignitet i urinvejene, som forårsager omkring 165.000 dødsfald om året på verdensplan, er kræft stadie, behandling og prognose direkte relateret til tilstedeværelsen eller fraværet af invasion1. Omkring 75% af tilfældene af blærekræft er ikke-muskel invasive og er lykkedes med lokale resektion. Derimod muskel-invasive blære kræftformer (ca. 25% af alle tilfælde) er aggressive tumorer med høje metastatisk og behandles med aggressiv multimodalitet terapi2,3. Derfor er forstå de molekylære processer, der udløser invasion væsentligt bedre karakterisere risikoen for invasiv progression og udvikle terapeutiske indgreb, som kan forhindre invasive progression.
Tumor invasive progression opstår i et komplekst tredimensionale (3D) miljø og indebærer tumor celle interaktion med andre tumorceller, stroma, basalmembranen og andre typer af celler, herunder immun celler, fibroblaster, muskelceller og vaskulære endotelceller. Permeable støtte (f.eks.Transwell) assay systemer er ofte ansat til at kvantificere kræft celle invasion4, men disse systemer er begrænset, fordi de ikke tillader mikroskopiske overvågning af invasion processen i real-tid og den hentning af prøver til yderligere farvning og molekylær analyse er udfordrende. Udvikling af en 3D-blære tumor klumpformet system at studere invasion er ønskelig, fordi det giver mulighed for indarbejdning af definerede microenvironmental komponenter med bekvemmeligheden af in vitro- systemer.
I denne protokol, vi beskriver et system for at afhøre de invasive processer af menneskelige blære cancerceller ved hjælp af en 3D-klumpformet invasion assay indarbejde kollagen-baseret gel matricer og konfokal mikroskopi til at tillade efterforskere at overvåge celle motilitet og invasion i realtid (fig. 1A). Dette system er alsidige og kan ændres til at afhøre forskellige stromale/tumor indstillinger. Det kan optage de fleste blære cancer cellelinjer eller primære blære tumorer og yderligere stromale celler, såsom kræft forbundet fibroblaster og immunceller5,6,7. Denne protokol beskriver en matrix bestående af type-1 kollagen, men kan ændres til at omfatte andre molekyler såsom fibronektin, laminin eller andre kollagen proteiner. Invasive processer kan følges i 72 timer eller længere afhængigt af evnen til mikroskop og systemet bruges. Fiksering og immunfluorescens farvning af tumor indlejret i matrixen 3D-før, under og efter invasionen tillader afhøring af proteiner upregulated i invasive celler, hvilket giver afgørende oplysninger der normalt fraværende eller vanskeligt at samle ved hjælp af andre modeller, 3D-kultur. Dette system kan også udnyttes, at skærmen forbindelser som blokerer invasion og afgrænse signaling veje ramt af sådanne forbindelser.

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			<td height="21" style="height:21px;width:527px;">Human bladder cancer cell lines UM-UC9, UM-UC13, UM-UC14, UM-UC18, 253J</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td style="width:612px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">DMEM cell culture medium</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">A3803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS), pH 7.4&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Ultral-low attachment 6-well cluster&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">3471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional inverted microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">491206-0001-000</td>
			<td>General use for cell culture and checking spheroids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen type 1 from rat tail, high concentration&#160;</td>
			<td>Corning</td>
			<td align="right" style="width:169px;">354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;">LSM800</td>
			<td>A confocal miscoscope with climate chamber, multi-location imaging, and Z-stack scanning function&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat micromtome</td>
			<td>Leica Biosystems</td>
			<td style="width:169px;">CM3050 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zen 2 Image processing software&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:169px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td align="right" style="width:169px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImmEdge Hydrophobic Barrier PAP Pen</td>
			<td>Vector Laboratories&#160;</td>
			<td style="width:169px;">H4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">O.C.T compound&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">23730571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 solution&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right" style="width:169px;">62249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-ATDC (Trim29) antibody</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:169px;">HPA020053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Cytokeratin 14 antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab7800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:527px;">Anti-Vimentin antibody</td>
			<td style="width:203px;">Abcam</td>
			<td style="width:169px;">ab24525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:169px;"></td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

3-d cell culture system for studying invasion and evaluating therapeutics in bladder cancer

Blærekræft er et væsentligt sundhedsproblem. Det anslås, at mere end 16.000 mennesker vil dø i år i USA fra blærekræft. Mens 75% af blære kræft er non-invasiv og usandsynligt at metastaserer, ca. 25% gå videre til en invasiv vækstmønster. Op mod halvdelen af patienter med invasiv kræft vil udvikle dødbringende metastatisk tilbagefald. Forstå mekanisme af invasive progression i blærekræft er således afgørende at forudsige patientens resultater og forhindre dødbringende metastaser. I denne artikel præsenterer vi en tre-dimensionelle kræft invasion model, som giver mulighed for indarbejdelsen af tumorceller og stromale komponenter til at efterligne i vivo betingelser, der forekommer i blæren tumor mikromiljø. Denne model giver mulighed for at observere den invasive proces i realtid ved hjælp af time-lapse imaging, afhøre den molekylære veje involveret ved hjælp af Konfokal immunfluorescent imaging og skærmen forbindelser med potentiale til at blokere invasion. Mens denne protokol fokuserer på blærekræft, er det sandsynligt, at lignende metoder kan bruges til at undersøge invasion og motilitet i andre tumortyper.

Vellykket skabelse af invasive blære cancer tumor klumpformet kræver dannelsen af passende størrelse tumor spheroids fra cellelinjer eller primære tumorer. Figur 2 A viser korrekt størrelse spheroids udviklet fra fire menneskelige blære cancer cellelinjer (UM-UC9, UM-UC13, UM-UC14, 253J og UM-UC18). Figur 2 B viser en tumor klumpformet fra en BBN-genereret mus blære tumor indlejret i kolla...

Watch this Scientific Journal Video about 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer at JoVE.com

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

3D-celle kultur System for at studere Invasion og evaluere Therapeutics i blærekræft

Processer for blære cancer invasion repræsenterer muligheder for biomarkør og terapeutisk udvikling. Her præsenterer vi en blære kræft invasion model, der indeholder 3D-kultur af tumor spheroids, time-lapse billedbehandling og konfokal mikroskopi. Denne teknik er nyttig til at definere funktioner af den invasive proces og for screening terapeutiske agenter.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

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Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

Metoder til evaluering af c-Fos rolle og Dusp1 i onkogen afhængighed

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

En tredimensionel Thymisk kultur system til at generere murine induceret pluripotente stamcelle-afledte tumor antigen-specifikke Thymisk emigranter

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

En 3D Spheroid model som et mere fysiologisk system for kræft-associeret fibroblaster differentiering og invasion in vitro undersøgelser

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...