Yazarlar, Ulusal Doğa Bilimleri Vakfı Çin (31371390), devlet yüksek teknoloji geliştirme projesi (2014AA020702) programı ve programın Guangdong bilim ve teknoloji (2016B030231001) araştırma hibe destek kabul etmiş oluyorsunuz.

Tüm hayvan iş hayvan refahı Yasası, kurumsal yönergelere uygun olarak gerçekleştirilen ve kurumsal hayvan bakım ve kullanım Komitesi, Sun Yat-Sen'in Üniversitesi tarafından onaylanmıştır.
1. TEM hazırlanması
<ol><li>Fareler tarafından servikal çıkığı yürütmek ve steril cerrahi makas ve cımbız kullanarak dilleri kaldırın.</li>
	<li>Tongues yılında % 75 etanol 3 min için bırakın, sonra her dil bir 1,5 mL koymak Eppendorf (EP) tüp ile 1 mL 10 mM steril fosfat tamponlu çözüm (PBS). Not: PBS konsantrasyonu aşağıdaki adımlarda bu adımda konsantrasyon olarak aynıdır.</li>
	<li>Lizis donma çözülme tarafından hücre:-80 ° c için 1 h EP tüpler dilleri donma ve 3 kür için dilleri için 45 dakika oda sıcaklığında erimek.</li>
	<li>Cerrahi sütür cerrahi iğne kullanarak bir parça üzerine her dil yüklemek ve steril folyo küçük bir parça ile dikiş sonu sarın. % 75 etanol steril koşullarda içeren bir 3,5 cm veya 6 cm kültür tabağına işlemi. Not: Uygun da cerrahi sütür her parçası yaklaşık 20 cm uzunluğunda ve 0,3 cm2 hakkında (1 cm × 0,3 cm) folyo her parçasının uygun boyutudur. Dil folyo yüklü olmalıdır.</li>
	<li>Her dil 3 mL steril PBS ile 30 s. gerçekleştir steril koşullarda bu işlem için bir 3,5 cm veya 6 cm kültür tabak içinde yıkayın.</li>
	<li>Dilleri Ultrasaf Su ile yıkamak: ampicilin geniş ağız şişe içine steril Ultrasaf Su 250 mL ile 90 µg/mL nihai bir konsantrasyon için ekleyin. Dilleri içeren bir heyecan bar şişe içine koymak. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek. Not: en fazla 5 dilleri dikiş twining söz konusu aynı şişe içine koyabilirsiniz. Folyo şişe dil kapalı kaymasını önlemek için dikiş sonu altındadır. Dilleri şişe içinde kalan dikiş uzunluğunu ayarlayarak şişenin alttan 2 cm yüksekliğinde yerleştirilmelidir. Bu Not için adımları 1.8, 1.10, 1.12 ve 1,16 da.</li>
	<li>Manyetik karıştırıcı 12 h için şişe koymak.</li>
	<li>Dilleri NaCl ile yıkayın: ampicilin geniş ağız şişe içine steril 1 250 mL ile eklemek M NaCl 90 µg/mL nihai bir konsantrasyon için. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 24 h için şişe koymak.</li>
	<li>Hücre lizis Triton X-100 tarafından: 90 µg/mL 250 mL steril % 2 ile son bir konsantrasyon geniş ağız şişe içine ampicilin eklemek PBS Triton X-100. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 48 h için şişe koymak.</li>
	<li>Dilleri CaCl2/MgCl2yıkama: ampicilin geniş ağız şişe içine steril 5 mM CaCl2/MgCl2 250 mL ile 90 µg/mL nihai bir konsantrasyon için ekleyin. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 24 h için şişe koymak.</li>
	<li>DNaz tarafından sindirim: Hank'in 1 mL dengeli tuz solüsyonu (HBSS) her EP tüp için ekleyin. DNaz HBSS sırasıyla 300 son bir konsantrasyon için eklemek µM. taşımak her dil tüp dışında dikiş parçası ile her EP tüpün içine. Bu işlemi steril koşullarda gerçekleştirmek. Not: yukarıdaki adımı şişelerde içinde kalır dikiş parçası da bu adımda EP tüp içinde kalır emin olun ve önceki adımları şişelerde dışında kalır dikiş parçası da bu adımda EP tüp dışında kalır emin olun.</li>
	<li>EP tüpler için 24 h 37 ° C'de dilleri kuluçkaya.</li>
	<li>Dilleri PBS ile yıkayın: ampicilin geniş ağız şişe içine steril PBS 250 mL ile 90 µg/mL nihai bir konsantrasyon için ekleyin. Dilleri ve heyecan bar şişe içine taşıyın. Şişe kapağı şişe dışında kalan dikiş parçası ile sıkın. Bu işlemi steril koşullarda gerçekleştirmek.</li>
	<li>Manyetik karıştırıcı 24 h için şişe koymak.</li>
	<li>Hazırlanan TEM 4 ° C'de steril PBS kadar kullanmak saklayın.</li>
</ol>2. TSCC üç boyutlu (3D) sulandırma
<ol><li>
Statik TSCC modeli İnşaat
	<ol>
<li>Tohum 1.0 x 106 tek TSCC hücreler (Cal27) 3.5 cm kültür çanak içine. Dulbecco'nın 3 mL kartal orta/F12 (DF12) içeren % 10 fetal Sığır serum (FBS), 90 µg/mL ampicilin ve 90 µg/mL sefaloridin modifiye ekleyin.</li>
		<li>Cal27 hücreleri 37 ° C'de 2-3 gün için kültür. Hücreleri en az % 60 cover emin olun alan çanak alt.</li>
		<li>TEM Cal27 monolayer kültür tabağına üzerine yükleyin.</li>
		<li>Çanak 28 gün boyunca bir CO2 kuluçka 37 ° C'de koyun.</li>
		<li>Kültür Orta hücre kültür işlemi sırasında her gün yenileyin. Kuluçka CO2 konsantrasyonu % 5 var.</li>
	</ol>
</li>
	<li>
Karıştırılmış TSCC modeli İnşaat
	<ol>
<li>
Kendi yaptığınız karıştırılmış minibioreactor hazırlanması
		<ol>
<li>Dalgıç 10 mL şırınga dan çıkar.</li>
			<li>Çubuk daha düşük terminal bir delik (1 cm çapında) kazmak ve delik bir heyecan bar yük.</li>
			<li>Bir delik (çapı 0.5 cm) geniş ağız şişe Plastik şişe kapağı merkezinde kazmak ve piston kolu ile kap koymak.</li>
			<li>50 mL santrifüj tüpü yarısı kesilmiş ve şişe kapağı dış tarafında kaynak.</li>
			<li>Eşyaları için çubuk çubuk eşyaları için bağlı balıkçılık satırlar ile sarma tarafından iliştirin. Not: en fazla 4 eşyaları bir çubuk için eklenebilir.</li>
			<li>Otoklav kullanmadan önce kendi kendine yapılan karmaşık. Not: değil basınçlı kap plastik geniş ağız şişe var. Yeni bir steril plastik geniş ağız şişe hücre kültürü çalışmalarının ise kullanın.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Dinamik hücre kültürü
	<ol>
<li>Tohum 1.0 x 106 tek Cal27 hücrelerde kendi kendine yapılan minibioreactor. %10 FBS, 90 µg/mL ampicilin ve 90 µg/mL sefaloridin içeren DF12 orta 150 mL ekleyin.</li>
		<li>TEM çubuk bağlı eşyaları kullanarak minibioreactor üzerine yükleyin.</li>
		<li>Şişe kapağı sıkın ve minibioreactor bir manyetik karıştırıcı tarih koymak. CO2 kuluçka 37 ° C'de 200 devirde minibioreactor 7-14 gün için etkinleştirin. Not: CO2 kuluçka CO2 konsantrasyonu % 5 olduğunu.</li>
	</ol>
</li>
</ol>

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Yazarlar ifşa gerek yok.

Decellularized ECM imalat için köklü bir protokol dokularda hücresel bileşenleri kaldırma sırasında yerel ECM kompozisyon tutmak gerekir neredeyse tamamen21. Perfüzyon konvektif taşıma ile hücresel malzemeleri kaldırmak için damarlara aracılığıyla gerektiren şu anda bildirilen decellularization protokolleri rağmen mekanik ajitasyon burada, geleneksel basit ve ucuz Yöntem22 bilinen kabul edilmiştir , 23 , <s...

Dil deglutition, eklem ve tatma gibi çeşitli önemli işlevleri vardır. Böylece, dil işlev bozukluğu hastaların yaşam kalitesi1üzerinde büyük etkisi vardır. Oral kavite içinde en yaygın malignite dil Skuamöz Hücre alkol veya sigara tütün2insan genellikle oluşan Karsinomu (TSCC), olduğunu.
Son yıllarda, TSCC üzerinde temel araştırma az bir gelişme sağlanmıştır. Verimli vitro araştırma eksikliği en büyük sorunlardan biri kalır modelleri. Böylece, hücre dışı matriks (ECM) olası bir çözüm olarak ortaya çıktı. ECM son derece organize matris bileşenlerden oluşan bir karmaşık ağ çerçeve olduğu bir ECM benzeri yapıda ve kompozisyon sahip İskele malzemeleri kanser araştırmaları için yetkili gerekir. Decellularized ECM mükemmel niş için hangi ECM en önemli avantajı için çıkıyor aynı kökenli vitro, hücrelerden sağlar.
ECM doku deterjanlar ve enzimler kullanarak decellularization aracılığıyla gelen kaldırılmakta olan hücresel bileşenleri ile muhafaza edilebilir. Kolajen, fibronektin ve laminin decellularized matris içinde de dahil olmak üzere çeşitli ECM bileşenleri yerel doku gibi microenvironment kültürlü hücreler için hayatta kalma, yayılması ve hücreleri3farklılaşma teşvik sağlar. Ayrıca, ECM hücresel bileşenleri yokluğu ile en az düzeye immünojenisite nakli için azaltılabilir.
Şimdiye kadar decellularized ECM imalat yöntemleri farklı doku ve organlarında, kalp4,5,6,7, karaciğer8,gibi9,10 denendi ,11, akciğer12,13,14,15,16,17ve böbrek18,19 , 20. ancak, ilgili hiçbir araştırma olmak buldu en iyi bildiğimiz kadarıyla dilinde benzer iş.
Bu çalışmada, decellularized dil hücre dışı matriks (TEM) verimli ve uygun fiyatla rezervasyon yaptırın fiziksel, kimyasal ve enzimatik tedavi bir dizi tarafından fabrikasyon. Sonra TEM TSCC vitro, özetlemek için TSCC davranış ve Kalkınma için uygun bir simülasyon gösterilen kullanıldı. TEM iyi biyouyumluluk hücreleri TEM TSCC araştırma3' te büyük potansiyeli olabilir gösterir doku özel niş Kılavuzu yeteneği vardır. Burada gösterilen iletişim kuralı Patogenez ya da TSCC klinik tedaviler üzerinde eğitim araştırmacılar için bir seçim sağlar.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
			<td style="width:292px;">Sun Yat-sen University Laboratory Animal Center</td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">HB15-GR-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A601988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">PHR1487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CO2 incubator</td>
			<td style="width:292px;">SHEL LAB</td>
			<td style="width:119px;">SCO5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">227270</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cal27 cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Tongue squamous cell carcinoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">U2OS cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
			<td style="width:292px;">BBI</td>
			<td style="width:119px;">A600264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:292px;">Hyclone</td>
			<td style="width:119px;">SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 &#176;C fridge</td>
			<td style="width:292px;">Haier</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water purifier</td>
			<td style="width:292px;">ELGA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:292px;">BLAU</td>
			<td style="width:119px;">717805</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

Dil Skuamöz Hücre Karsinomu (TSCC) vitroiçin etkili ve gerçekçi bir model oluşturmak için yöntemleri TSCC İnşaat için fonksiyonel iskele sağlayan decellularized üretmek dil hücre dışı matriks (TEM) oluşturulmuştur. TEM vitro niş hücre büyümesi, farklılaşma ve hücre geçiş sağlar. Yerel hücre dışı matriks (ECM) ve biyokimyasal besteleri decellularized matris içinde muhafaza microstructures doku özel niş hücreleri demirleme için sağlar. TEM imalatı deoksiribonükleaz (DNaz) sindirim bir ciddi organik veya inorganik Önarıtma ile eşlik tarafından gerçekleştirilebilir. Bu iletişim kuralı kullanımı kolay ve decellularization için yüksek verimlilik sağlar. TEM inşaat TSCC modeli sağlayan olumlu cytocompatibility TSCC hücrelerin çalkalanmış mı statik kültür koşullarda gösterdi. Kendini yetiştirmiş bir biyoreaktör Ayrıca hücre kültürü için kalıcı karıştırılmış koşul için kullanıldı. Yeniden oluşturulan TSCC TEM kullanarak özellikleri ve klinik TSCC histopatoloji, benzer özellikleri TSCC araştırma potansiyeli düşündüren gösterdi.

Bu iletişim kuralı TEM hazırlanması için verimli ve uygun olduğu ortaya çıkıyor. TEM anadili kurcalayarak kıyasla mükemmel decellularization gösterdi. Decellularization etkinliğini hematoksilen-(Şekil 1A-B) boyama eozin tarafından (o) doğrulandı. Sonuçları boyama o nükleer TEM (Şekil 1B) boyama tam ortadan kalkması saptandı. Ayrıca, önceki işten DNA içerik miktar DNA TE...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Dil hücre dışı matriks ve dil Skuamöz Hücre Karsinomu Vitro sulandırma imalatı

Bir yöntem etkili decellularization ile dil hücre dışı matriks (TEM) hazırlanması için burada gösterilir. TEM fonksiyonel iskele dil Skuamöz Hücre Karsinomu (TSCC) manken çalkalanmış mı statik kültür koşullar altında yeniden inşası için kullanılabilir.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

fabrication-tongue-extracellular-matrix-reconstitution-tongue

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

6.2K Görüntüleme.  Sun Yat-sen University. Bir yöntem etkili decellularization ile dil hücre dışı matriks (TEM) hazırlanması için burada gösterilir. TEM fonksiyonel iskele dil Skuamöz Hücre Karsinomu (TSCC) manken çalkalanmış mı statik kültür koşullar altında yeniden inşası için kullanılabilir.

Video: Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Baş ve Boyun Skuamöz Hücre Karsinomu içinde perinöral invazyon İçin Bir Model

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Kanser Araştırmaları

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Preklinik Tedaviler Nicel ve Boyuna Değerlendirme A sinjeneik Fare Modeli Yapmış Bir Renal Hücreli Karsinom

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

Arşiv Formalin Sabitlenmiş, Parafin Gömülü İnsan Orofaringeal Skuamöz Hücreli Karsinoma Örneklerinde T-hücresi Subpopülasyonlarının Dört Renkli Floresan İmmunohistokimyası

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Baş ve boyun Skuamöz hücreli karsinom için bir küresel tabanlı 3D hücre kültür modelinde test terapi

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Skuamöz Hücre Karsinomu Hücre Tümörü bağışıklık profil oluşturma için fareler ve tedaviye yanıt değerlendirme intramucosal aşı

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Üç boyutlu kemik hücre dışı matriks modeli osteosarkom için

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Ekstrasellüler matris Hidrogeller kullanarak normal doku radyasyon etkileri eğitim

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Eş-Kültürlerde M1 Makrofaj-4T1 Fare Mammary Karsinom Hücrelerinde Fagositik Aktivitenin Zaman Atlamalı 2D Görüntülemesi

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Fare Böbreği ve Tavuk Koryoallantoik Membranda Kurulan Metastatik Berrak Hücreli Renal Hücreli Karsinom Modelinin Karşılaştırılması

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Akciğer Skuamöz Karsinom Progresyonunu Modellemek Için Çok Boyutlu Coculture Sistemi

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...