Bu çalışma Hariri Ailesi Vakfı ve TJ Martell Vakfı tarafından desteklenmiştir.

1.. Hasat ve Cerrahi Örneklerinin İnsan Prostat Kanseri Doku İşleme <ol><li> Roswell Park Memorial Institute (RPMI),% 10 fetal sığır serumu (FBS) ve% 1 penisilin / streptomisin ile takviye edilmiş 1640 kültür ortam maddesi içinde 15 ml içeren iki 50 ml konik polistiren tüpler hazırlayın. </li><li> Patoloji servis personeli protokolü onaylanan Kurumsal Değerlendirme Kurulu altında sırasıyla prostatektomi ve palyatif cerrahi örneklerinden primer ve metastatik prostat kanseri örnekleri, hasat. Metastatik örnekleri birincil, ameliyat sırasında lenf düğümü kesitlerden elde edilebilir, diğerleri arasında, bir metastatik lezyonların teşhis çıkarılması esnasında palyatif cerrahi gibi omurilik dekompresyon gibi işlemler, ve akciğer sırasında omur. <ol><li> Aşama 1.1 ilk 50 ml konik bir tüp içine polistiren Klinik teşhis için gerekli olmayan fazla toplu doku hasat ve 4 ° C &#39;de hemen depolamak Hasat edilird doku, en azından bir bölgede 4-5 cm 2 ve kalınlığı 1-2 cm boyutlarında olmalı. </li></ol></li><li> Laboratuvar personeli hücre süspansiyonları histolojik inceleme ve nesil için hazırlık patoloji servisinden alınan toplu doku işlemek. <ol><li> Kütle dokuların işlenmesi maksimum hücre canlılığı garanti etmek amacıyla cerrahi rezeksiyon 24 saat geçmemelidir. </li><li> Steril bir neşter ve forseps ile bir biyogüvenlik kabini içinde çalışmak, makroskopik tümör nodüller 2-4 mm kalınlığında yatay doku bölümleri alır. Hemen aşama 1.1 saniye 50 ml konik bir tüp içinde polistiren bölümleri saklayın. </li><li> Makroskopik nodüller prostatektomi örneklerinde görülmediği zaman, arka lob ve geçiş bölgesinden rastgele 2-4 mm kalınlığında yatay doku bölümleri toplamak. </li></ol></li><li> Steril bir bisturi bıçağını ve forsepsler kullanılarak cerrahi dokusunun bir kısmı ayrılır ve önce cinse% 10 formalin içinde tamirHücre süspansiyonları tion. Arşiv ve prostat kanseri dokusunun varlığını doğrulamak için bu malzeme histolojik analiz. </li></ol> 2. Doku bölümleri gelen hücre süspansiyonları Üretimi <ol><li> Steril bir biyo-güvenlik kabini içinde çalışan, steril 1 x fosfat tamponlu tuzlu su (PBS) içinde 1 ml ihtiva eden bir 60 x 15 mm kültür çanağı için bir doku bölümü aktarın. <ol><li> Mekanik bir steril neşter ve forseps kullanarak örnek çiğnemek. </li><li> Hayır enzimatik sindirimler bu protokol vardır. </li></ol></li><li> Bir steril 50 ml konik bir tüp içine 1 ml süspansiyon aktarın. <ol><li> Kültür çanak 1 ml ekleyin ve doku bölümü tamamen genellikle 3-4x, ayrışmış kadar ezildikten adımı tekrarlayın. </li><li> Tüm doku bölümleri ayrışmış kadar seri olarak bu işlemi tekrarlayın. </li></ol></li><li> 5 ml&#39;lik bir serolojik pi ile 50 ml polistiren konik tüp içeriğini yeniden süspansepette ve 1 dakika boyunca maksimum hızda (yaklaşık 3,200 rpm) vorteks. </li><li> 35 mikron hücre süzgecinden elde edilen süspansiyon filtre edilir ve ikinci bir steril 50 ml konik tüp içine polistiren toplar. </li><li> 10 dakika boyunca 450 x g&#39;de santrifüj ile pelet oluşturur. </li><li> , Süpernatant atılır alyuvar lisiz tamponu, 5 ml pelet tekrar süspansiyon ve oda sıcaklığında 5 dakika için çözelti inkübe edin. Oda sıcaklığında 450 x g&#39;de 5 dakika boyunca santrifüj ile kırmızı kan hücre parçalama tamponunu çıkarın. </li><li> , Süpernatant atılır,% 5 FBS ile desteklenmiş 1 x PBS içinde tekrar süspansiyon pelet ve bir hemasitometre veya otomatik hücre sayacı kullanılarak uygulanabilir (tripan mavi-negatif) hücrelerin sayısını ölçmek. Bir 1x10 6 hücre / ml süspansiyon oluşturmak ve artık 1 saat daha buz üzerinde saklayın. <ol><li> Canlılık ve hücre verim tümör örnekleri arasında önemli ölçüde değişebilir. Prostatektomi ortamda, canlılığı 45-70 ve% fi arasında değişebilirBir makroskopik tümör nodül kalın yatay doku kesitleri yaklaşık 3x10 6 canlı hücreler elde bekleniyor olabilir 2-4 mm ettik. </li></ol></li></ol> 3. FACS için Hücrelerinin antikor boyama <ol><li> Aşağıda gösterildiği gibi beş 15 ml polistiren konik tüpler etiketleyin. Sırasıyla, hematopoetik ve endotelial elemanları hariç CD45 ve CD31 antijeni etiketleme kullanın. <ol><li> Lekesiz </li><li> IgG 2a κ-PE + IgG 1 κ-FITC </li><li> HLAI-PE + IgG 1 κ-FITC </li><li> IgG 2a κ-PE + CD31 + CD45-FITC-FITC </li><li> HLAI-PE + CD45 + CD31-FITC-FITC </li></ol></li><li> Adım 3.1 &#39;de beş tüplere önceki bölümde (adım 2.7) den sayısallaştırılmış hücre süspansiyonu dağıtın. 1:250 seyreltide antikor ekleyin ve 30 dakika boyunca buz üzerinde inkübe hücre süspansiyonları. </li><li> 4 ° C&#39;de 3 dakika boyunca 450 x g&#39;de santrifüj hücreleri ve süpernatantlar atın. </li&gt; <li> Steril 1x PBS ile her bir topağın tekrar askıda bırakılmasıyla hücreleri yıkanır, 4 ° C&#39;de 3 dakika boyunca 450 x g&#39;de santrifüj ile ve ardından,% 10 FBS ile takviye edilmiş </li><li> 10 ug / ml &#39;lik bir konsantrasyonda 4 1x PBS ihtiva eden bir çözelti, 1 ml&#39; ,6-diamidino-2-fenilindol (DAPI) &#39;de tekrar süspansiyon hücreleri. </li><li> 12 mm x 75 mm polistiren tüpler içine 35 um süzgeç kapaklar aracılığıyla nihai çözüm Filtre. </li></ol> 4. FACS ile Prostat CSCS izolasyonu <ol><li> Hücre sıralama için bir flowsitometri yararlanın. </li><li> Tüplerden # 1, # 2, # 3 hücreleri kullanılarak tazminat kontrolleri ayarlayın, ve 4.. </li><li> Ileri ve yan dağılım parametreleri kullanarak enkaz ve hücre kümeleri hariç kapıları oluşturun. </li><li> Sırasıyla, tüp # 3 ve # boru 4 süspansiyonlar kullanılarak FITC ve PE kapıları oluşturulması. </li><li> Pasifik mavi-A kanalı kullanılarak kapı canlı (DAPI-negatif) hücreler. </li><li> Tüp # 5 ıskarta CD31-pozitif ve CD45-pozitif hücreler kullanılarak veRPMI içinde 2 ml% 10 FBS ile takviye içeren steril 15 ml konik polistiren tüpler içine de HLAI-negatif ve pozitif HLAI popülasyonları toplar. </li><li> Santrifüj 5 dakika boyunca 450 x g&#39;de hücre süspansiyonları sıralanmış ve daha sonra% 10 FBS ile takviye edilmiş RPMI içinde 200-500 ul pelet tekrar süspansiyon. </li><li> Hemasitometre ya da otomatik hücre sayıcı kullanarak canlı (trypan mavi-negatif) hücrelerini ölçmek. </li></ol>

<ol>
	<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>Magee, J. A., Piskounova, E., Morrison, S. J. <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:+impact,+heterogeneity,+and+uncertainty.">Cancer stem cells: impact, heterogeneity, and uncertainty.</a> Cancer Cell. 21, 283-296 (2012).</li><li>Nguyen, L. V., Vanner, R., Dirks, P., Eaves, C. J. <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:+an+evolving+concept.">Cancer stem cells: an evolving concept.</a> Nat. Rev. Cancer. 12, 133-143 (2012).</li><li>Valent, P., 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+cell+definitions+and+terminology:+the+devil+is+in+the+details.">Cancer stem cell definitions and terminology: the devil is in the details.</a> Nat. Rev. Cancer. 12, 767-775 (2012).</li><li>Visvader, J. E., Lindeman, G. J. <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:+current+status+and+evolving+complexities.">Cancer stem cells: current status and evolving complexities.</a> Cell Stem Cell. 10, 717-728 (2012).</li><li>Lapidot, 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+cell+initiating+human+acute+myeloid+leukaemia+after+transplantation+into+SCID+mice.">A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.</a> Nature. 367, 645-648 (1994).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., 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=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 3983-3988 (2003).</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>Collins, A. T., Berry, P. A., Hyde, C., Stower, M. J., Maitland, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Res. 65, 10946-10951 (2005).</li><li>Domingo-Domenech, 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=Suppression+of+acquired+docetaxel+resistance+in+prostate+cancer+through+depletion+of+notch-+and+hedgehog-dependent+tumor-initiating+cells.">Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.</a> Cancer Cell. 22, 373-388 (2012).</li><li>Patrawala, L., Calhoun-Davis, T., Schneider-Broussard, R., Tang, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+organization+of+prostate+cancer+cells+in+xenograft+tumors:+the+CD44+alpha2beta1++cell+population+is+enriched+in+tumor-initiating+cells.">Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+alpha2beta1+ cell population is enriched in tumor-initiating cells.</a> Cancer Res. 67, 6796-6805 (2007).</li><li>Qin, 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=The+PSA(-/lo)+prostate+cancer+cell+population+harbors+self-renewing+long-term+tumor-propagating+cells+that+resist+castration.">The PSA(-/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration.</a> Cell Stem Cell. 10, 556-569 (2012).</li><li>Siegel, R., Naishadham, D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2013.">Cancer statistics, 2013.</a> CA Cancer J. Clin. 63, 11-30 (2013).</li><li>Patrawala, 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=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li></ol>

Çıkar çatışması ilan etti.

Bu protokol, taze insan prostat kanseri dokulardan CSCS izolasyonunu tarif eder. Birçok önemli hususlar bu protokolün başarılı sonucunu etkiler. Uygun bir prostat kanseri hücrelerinin yüksek sayıda geri kazanım Cerrahi numunelerin dikkat brüt değerlendirmesine bağlıdır. Makroskopik tümör nodüller gözlendi ve 10 işlendiğinde bizim deneyim, tümörijenik prostat hücrelerinin başarılı izolasyonu iyi sağlanır. Bununla birlikte, p...

Tümör içine heterojenlik kanser 1 ayırt edici bir özelliği olarak kabul edilir. Gerçekten de, tümör heterojenite çeşitli mekanizmalar genetik mutasyon ve mikro etkileşimler de dahil olmak üzere tarif edilmiştir. Buna ek olarak, bazı kanser türleri seri nakli deneylerinde 2-5 tümör başlatıcı kapasitesi ve kendini yenileme özelliklerini sergileyen kanser kök hücreleri (rulmanları) bir alt popülasyonunun bir hücresel hiyerarşisi içerebilir. Başlangıçta hematolojik 6, meme 7, 8 ve beyin habis de tarif CSCS aynı zamanda prostat kanseri 9-12 hem de diğer tümör tipleri 2-5 incelenmiştir. CSCS genellikle oluşan heterojen bir topluluk içinde 2-5 hücresel fraksiyon olarak kabul edilir. Bu nedenle, CSCS fonksiyonel ve moleküler karakterizasyonu toplu nüfus onların zenginleşmesine şarta. Buna göre, son iki yılda çeşitli buluştuCSC zenginleştirme hodologies tipik olarak akış sitometrisi ile işaretlenmiş popülasyonlarının ayrılmasını içerir ki tasarlanmıştır. Buna ek olarak, CSCS çalışmada önemli bir husustur insan dokularının hiyerarşik organizasyonu, kültür veya bağışıklık yetersizliği olan farelerde seri pasaj olarak deneysel manipülasyonlar tarafından kesintiye edilebilir olmasıdır. Sonuç olarak, insan dokularından CSCS doğrudan izolasyon CSC alanda önemli bir yöntem olarak ortaya çıkmıştır. Prostat kanseri kanser morbidite ve Amerika Birleşik Devletleri&#39;nde ve dünya çapında 13 ölüm önde gelen bir nedenidir. Bu nedenle, prostat CSCS izolasyonu ve biyolojik karakterizasyonu önemli ilgi konusudur. Prostat CSCS önce hücre hatları, hasta türetilmiş ksenograftlarının ve düşük geçiş hasta süspansiyon kültürlerinden türetilmiş 9,10,12,14 ikinci zenginleştirilmiştir. Biz son zamanlarda direkt olarak insan cerrahi s prostat CSCS izolasyonuna bildirdiOnların HLAI-negatif hücre yüzey fenotip 10 sayesinde depas&#39;lar. Burada ayrıntılı bu hücrelerin izolasyonu için uygulanan prosedürler WE. Prostat tümörler cerrahi örneklerden hasat edildi ve hücre süspansiyonları içine yapılır. Hücreler daha sonra HLAI karşı antikorlar hem de stromal ve canlılığı belirteçleri kullanılarak lekelenmektedir, CSCS ve floresan aktive hücre sıralama (FACS) ile izole edilir. İzole CSCS sonra yaşayabilir hücreler gerektiren deneyleri için de kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">11875-093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Penicillin Streptomycin</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:195px;">Corning cell gro</td>
			<td style="width:187px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">60 mm-15 mm cell culture dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:187px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">35 &#181;m Cell Strainer</td>
			<td style="width:195px;">BD Falcon</td>
			<td style="width:187px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">50 ml conial tube</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">15 ml conical tune</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Red blood cell lysing buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:187px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HLA class I (W6/32) PE antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab43545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD31 FITC antibody</td>
			<td style="width:195px;">ebioscience</td>
			<td style="width:187px;">11-0319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD45 FITC antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab27287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG2a&#954; PE antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">555574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG1&#954; FITC antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">551954</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DAPI</td>
			<td style="width:195px;">Invitrogen&#160;</td>
			<td style="width:187px;">d3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">12 x 75 mm polystyrene tubes with cell strainer cap</td>
			<td style="width:195px;">BD Falcon&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:187px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vortex Mixer</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">CG-BV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">10% Neutral Buffer Formalin</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:187px;">RBBP-0480</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cancer stem cells from human prostate cancer samples

Kanser kök hücre (CSC) modeli ölçüde son iki yılda yeniden gözden geçirilmiştir. Bu süre boyunca CSCS tanımlanır ve doğrudan insan dokularından izole edilmiş ve seri olarak, tipik olarak akış sitometrisi ile hücrelerin ve fraksiyonlama alt grupları antikor etiketleme yoluyla, bağışıklık yetersizliği olan farelerde yayılır edilmiştir. Bununla birlikte, prostat kanseri benzersiz klinik özellikleri önemli ölçüde taze insan tümör örneklerinden prostat CSCS olan çalışma sınırlıdır. Yakın zamanda, HLA sınıf I (HLAI)-negatif fenotipi nedeniyle, doğrudan insan dokularından prostat CSCS izolasyonunu bildirdi. Prostat kanser hücreleri cerrahi örneklerden hasat edilir ve mekanik olarak ayrıştırılırlar. Bir hücre süspansiyonu üretilir ve floresan konjuge HLAI ve stromal antikorlar ile etiketlenir. HLAI-negatif hücrelerin alt grubu son olarak bir akış sitometrisi kullanılarak izole edilmiştir. Bu protokolün başlıca sınırlaması sık mikroskobik ve multifokal doğaprostatektomi örneklerinde primer kanser. Bununla birlikte, izole edilmiş canlı prostat CSCS bağışıklık yetersizliği olan farelerde nakli ile moleküler karakterizasyonu ve fonksiyonel doğrulama için uygundur.

<img alt="Şekil 1" fo:content-width="5in" fo:src="/files/ftp_upload/51332/51332fig1highres.jpg" src="/files/ftp_upload/51332/51332fig1.jpg" /> Şekil 1. İnsan prostat kanseri tümörü hasat ve bir insan prostat kanseri belirli popülasyonlarının arsa görüntüleyen akış sitometrisi. (A) Tümör nodülleri bir hücre süspansiyonu üretmek için hasat edilir. Işlenmiş örnek Tümör içeriği, geleneksel hematoksilen ve eozin boyama ile teyit edilir. D...

Watch this Scientific Journal Video about Isolation of Cancer Stem Cells From Human Prostate Cancer Samples at JoVE.com

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Doğrudan insan dokularından kanser kök hücreler (rulmanları) izolasyonu biyolojik karakterizasyonu için bir koşuldur. Ayrıca zor sorun giderme adımları konusunda ipuçları sağlarken Bu makale, insan dokularından prostat CSCS izolasyonu için bir yöntem anlatılmaktadır.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

isolation-of-cancer-stem-cells-from-human-prostate-cancer-samples

Research

JoVE Journal

Medicine

14.0K Görüntüleme.  Icahn School of Medicine at Mount Sinai.  Doğrudan insan dokularından kanser kök hücreler (rulmanları) izolasyonu biyolojik karakterizasyonu için bir koşuldur. Ayrıca zor sorun giderme adımları konusunda ipuçları sağlarken Bu makale, insan dokularından prostat CSCS izolasyonu için bir yöntem anlatılmaktadır. 

Video: Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital Görüntüleme Gerçek Zamanlı tümörler Nanopartikül Alımı Değerlendirme

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Tıp

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Birincil Preneoplastic Meme Epitel Hücrelerinin Time-lapse görüntüleme Meme Kanseri Genetik Fare Modelleri türetilen

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Kemirgen Ürogenital Sinüs mezenşimin ve İnsan Kök Hücre Doku Rekombinasyon kullanarak İnsan Prostat epiteli oluşumu

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

İnsan Prostat Kanser metastaz Ortotopik fare modeli

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

İnsan Hücre Lines Kemorezistans Yumurtalık Kanser Kök Hücreleri Zenginleştirme

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Fare Kanser Modeli Tümör Hücreleri Sirkülasyon İzolasyonu ve Yayılım

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).