Yazarlar Avner Pankreas Kanseri Vakfı'ndan destek aldılar.

Tüm prosedürler Yeni Güney Galler Üniversitesi Hayvan Bakım ve Etik Kurulu tarafından onaylandı (17/109A). Bu protokol için 8-10 haftalık 16-19 g ağırlığında dişi athymic Balb/c çıplak fareler kullanılmıştır. Fareler mikro izolatör kafeslerinde barındırıldı ve ticari olarak mevcut peletlenmiş yiyecek ve su ad libitumile beslendi.
1. Ortopedik pankreas kanseri implantasyonu
<ol><li>Hücreleri implantasyona hazırlayın. İlk olarak, prosedür için gerekli hücre sayısını hesaplayın (her hayvan için 1 x10 6 luciferaz etiketli AsPC-1 hücreleri ve 1 x10 6 kanser ilişkili insan pankreas yıldız hücreleri [CAhPSC'ler] gereklidir).<ol>
<li>Bu hücreleri nemlendirilmiş sıcaklık kontrollü bir CO2 inkübatöründe koruyun ve rutin mikoplazma testi gerçekleştirin. AsPC-1 ve CAhPSC'ler için kullanılan kültür ortamı RPMI 1640'tır (300 mg/ L L-glutamin ile, %20 v/v foetal sığır serumu, %1 v/v penisilin/streptomisinin) ve IMDM (4 mM L-glutamin, %10 v/v foetal sığır serumu, %1 v/v penisilin/streptomisan ile).</li>
		<li>Hücreleri hücre süspansiyonuna dönüştürmeyi denemek için standart hücre kültürü tekniklerini kullanın. Kullanılan tripsin çözeltisinin iki katı bir hacimde ilgili tam kültür ortamını kullanarak tripsin nötralize edin.</li>
		<li>Bu hücreleri fosfat tamponlu salin (PBS) ile iki kez yıkayın ve 50 μL hücre süspansiyonunda 1 x10 6 AsPC-1 hücresi ve 1 x10 6 CAhPSC içeren bir karışıma yeniden atın.</li>
		<li>Bu süspansiyonu kullanıma kadar buzda tutun.</li>
	</ol></li>
	<li>Prosedür için sınıf II biyogüvenlik kabini hazırlayın. Steril plastik bir örtü ile kaplanmış bir ısıtma paspası kullanın. İşlem sırasında büyütme için, 2,5x ila 3,5x büyütme cerrahi loupes bir çift kullanın.</li>
	<li>1 cm çapında bir deliği gazlı bez çubuğuna keserek çanta ipli sürüntüler hazırlayın. Bu deliği bir çanta ipli dikişle sabitleyin. Bunun için herhangi bir ince örgülü dikiş kullanılabilir (örneğin, 5/0 poliglik asit dikiş). Örgülü dikiş malzemesi, gevşek düğümün sıkıldıktan sonra yerinde kalmasını sağladığı için önerilir. Bu şekil 1a'dagösterilmiştir.</li>
	<li>Fareyi intraperitoneal enjeksiyonla 80 mg/kg ketamin ve 10 mg/kg ksilazin ile uyuşturun.</li>
	<li>5 mg/kg enrofloksasin antibiyotik profilaksisi, 2,5 mg/kg flunixin analjezi ve %0,9 salin deri altı 1 mL'si uygulanır.</li>
	<li>Uyuşturuldktan sonra, fareyi steril alana bir destek pozisyonunda yerleştirin ve povidone-iyot uygulayın ve ardından cilt hazırlığı için% 70 etanol uygulayın.</li>
	<li>Karnın sol kraniyal kadranının derisinde uzunlamasına bir kesi yapın ve ardından kas tabakasınıps arasında keserek karın içine girin.</li>
	<li>50 μL hücre süspansiyonu ile 29 G insülin şırıngası yükleyin - bu, enjekte başına 1 x10 6 CAhPSC ve 1 x10 6 luciferaz etiketli AsPC-1 hücrelerine eşittir. Enjeksiyon cihazına monte edin. Bu enjeksiyon cihazının tasarımı ve işlevi Şekil 1b ve göstergesinde ayrıntılı olarak açıklanmıştır.</li>
	<li>Çanta-string bezini laparotomi kesisinin üzerine yerleştirin ve ardından bu çubuğun açılmasıyla dalak ve pankreas kuyruğunu dış hale getirin. Pankreasın gövdesini hafifçe çevrelemek için çanta dizesini sıkın, pankreas kuyruğunu enjeksiyon için açığa koyun. Gazlı bez pankreası çevresine temas ederken aynı zamanda daraltmayacak kadar sıkı olmak önemlidir.</li>
	<li>Bir çift tokmaca kullanarak pankreasın kuyruğunu kavrayın ve üzerine hafifçe yanal gerginlik yerleştirin. Ventral periton yüzeyini iğne ile sığ bir açıyla delin ve ardından hücre süspansiyonu enjeksiyon cihazı ile yavaş ve kontrollü bir şekilde (10−15 sn'nin üzerinde) pankreasa enjekte edin.</li>
	<li>Enjeksiyon işlemi sırasında, hem enjeksiyon bölgesinin etrafında (reflüden) hem de pankreas lobülünün diğer tarafında (içinden ve içinden penetrasyon durumunda) sızıntıyı dikkatlice gözlemleyin. Görünür sızıntı meydana gelirse, şırınnada kalan enjekte hacmini kontrol ederek enjeksiyonu durdurun ve sızıntı hacmini not edin. Sızıntı küçük hacimdeyse (<10 μL) ve ardından herhangi bir sızıntıyı gazlı bezle emer ve enjeksiyonu tamamlamak için iğneyi farklı bir pankreas lobülüne yeniden konumlandırın.</li>
	<li>Dalak ve pankreası değiştirin ve karın duvarını sürekli bir şekilde 5/0 poliglkolik asit dikişi ile kapatın. Cildi klipslerle kapatın.</li>
	<li>Fareyi anesteziden kurtulana kadar ısıtılmış bir kafeste izleyin. Uyandıktan ve uyarıldıktan sonra fareyi kafesine geri taşıyın.</li>
</ol>2. Kanser rezeksiyon cerrahisi: Distal pankreastomi ve splenektomi
<ol><li>İmplantasyon ile ilgili rezeksiyon zamanlaması deneysel protokole bağlı olarak değişebilir. Genel olarak, tümörlerin rezeksiyondan önce en az 3 hafta büyümesine izin verin, ancak bunu belirli implante edilmiş kanser hücresi hattı için ampirik olarak optimize edin.</li>
	<li>Rezeksiyon ameliyatından bir gün önce, lokalize primer tümörün varlığını doğrulamak için hayvanlar üzerinde biyolüminesans görüntülemesi gerçekleştirin. Bu görüntüleme çalışmasının, bariz ekstra pankreas hastalığı olan fareleri rezeksiyondan dışlamak için kullanıldığını unutmayın. Rezeksiyon için uygunluğu belirlemek için eşik olarak ne boyut ne de parlak akı kullanılmamalıdır.<ol>
<li>Fareleri tartın ve D-luciferin intraperitoneally (150 mg / kg) ile enjekte edin.</li>
		<li>Luciferin kinetik eğrisinin performansına göre her deney için luciferin enjeksiyonu ile ilgili görüntüleme adımının zamanlamasını belirleyin. Işıltılı akısının maksimumun% 90'ının üzerinde olduğu süre, biyolüminesans görüntüleme için en uygun zamanı temsil eder (bu deneyde, enjeksiyon sonrası 18 ila 26 dakika)</li>
		<li>Anesteziye neden olur ve izofluran (sırasıyla% 4 ve% 3 oksijen ile) kullanmaya devam eder ve biyolüminesan görüntüleme cihazı (örneğin, IVIS Lumina II) kullanarak görüntüleme gerçekleştirir. Otomatik pozlama ve binning ayarlarını kullanın (ancak bu, beklenen parlak akı için optimize edilebilir).</li>
	</ol>
</li>
	<li>Sınıf II biyogüvenlik kabinini prosedür için hazırlayın. Steril plastik bir örtü ile kaplanmış bir ısıtma paspası kullanın. Diseksiyon sırasında büyütme için, 2,5x ila 3,5x büyütme cerrahi loupes bir çift kullanın.</li>
	<li>Fareyi intraperitoneal enjeksiyonla 80 mg/kg ketamin ve 10 mg/kg ksilazin ile uyuşturun.</li>
	<li>5 mg/kg enrofloksasin antibiyotik profilaksisi, 2,5 mg/kg flunixin analjezi ve %0,9 salin deri altı 1 mL'si uygulanır.</li>
	<li>Fareyi steril alana bir destek pozisyonunda yerleştirin ve povidone-iyot ve ardından cilt hazırlığı için% 70 etanol uygulayın.</li>
	<li>Karnın sol kraniyal kadranının derisinde, tercihen önceki kesi bölgesinden uzunlamasına bir kesi yapın.</li>
	<li>Alttaki kas karın duvarındaki deriyi künt bir şekilde parçalara ayrıştırın ve ardından cilt yarasını açık tutmak için bir Alm kendi kendini koruyan retraktör yerleştirin.</li>
	<li>Perküsler tabakayı, önceki operasyonun dikiş hattının hemen bir tarafına kadar perküler tabakayı inkütleyin ve daha sonra kesiği genişleterek önceki dikiş hattının tamamını kesin.</li>
	<li>Dalak ve distal pankreası dışlayın ve kraniyal olarak geri çekin. Pankreasın kaudal yönünde, kolon film yapışmaları ile tutturulmuş olarak bulunabilir. Bu bulunursa, kolonu açıkça parçalara ayırın.</li>
	<li>Pankreas ve dalak damarlarının vücuduna dikkatlice bir çift tokmak dorsal geçirin ve bu alanı açın. Bu, sonraki ligasyon için bir pankreas segmentini serbest bıraktı.</li>
	<li>Pankreasın proksimal gövdesini bir titanyum ligasyon klipsi ile tümöre yapıştırın ve sonra pankreas distalini koter ile buna çevirin. Pankreas kütüğünü kontrol etmenin alternatif bir yolu, transeksiyondan önce 5/0 poliglkolik asit dikişi ile süreklilik içinde lige etmektir.</li>
	<li>Pankreası kaudal olarak geri çekin ve dalak ve midenin kraniyal kutbu arasındaki gastrosplenik damarları dağlayın.</li>
	<li>Örneği çıkarın ve hemostazı onaylayın.</li>
	<li>Karın duvarını 5/0 poliglkolik asit dikişi ile sürekli bir şekilde kapatın. Cildi klipslerle kapatın.</li>
</ol>3. Ameliyat sonrası yönetim
<ol><li>Anestezi sonrası hemen dönemde (yukarıdaki prosedürlerin her ikisi için), fareyi anesteziden kurtulana kadar ısıtılmış bir kafeste izleyin. Uyandıktan ve uyarıldıktan sonra fareyi kafesine geri taşıyın. Ameliyat sonrası dönemde, hayvanları ağrı ve sıkıntı belirtileri için izleyin. Deri altı enjeksiyonu ile 0,05 mg/ kg buprenorfin uygulayın ve hayvanları 12 saat boyunca yakından gözlemleyin.</li>
	<li>Daha sonra, fareleri günlük olarak kilo, yiyecek alımı ve aktivite için izleyin. Kesi bölgelerini ve palpate tümör büyüklüğü için inceleyin. Ameliyat sonrası yedinci günde cilt klipslerini çıkarın.</li>
	<li>İnsancıl uç noktalara ulaşılırsa fareyi ötenazi edin. Bu insancıl uç noktalar şunlardır: vücut ağırlığının kaybı >20%, tedavi edilemeyen sıkıntı özellikleri (kambur duruş, hareket eksikliği veya tımar dahil) ve dış palpasyon tarafından tahmin edildiği gibi 1 cm3'ten büyük tümör boyutu.</li>
</ol>

<ol>
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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=Hepatocyte+growth+factor+inhibition:+a+novel+therapeutic+approach+in+pancreatic+cancer.">Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer.</a> British Journal of Cancer. 114 (3), 269-280 (2016).</li><li>Giri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Immunocompetent+Model+of+Pancreatic+Cancer+Resection+and+Recurrence.">An Immunocompetent Model of Pancreatic Cancer Resection and Recurrence.</a> Journal of Gastrointestinal Surgery. , (2020).</li><li>Allen, V. B., Gurusamy, K. S., Takwoingi, Y., Kalia, A., Davidson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+accuracy+of+laparoscopy+following+computed+tomography+(CT)+scanning+for+assessing+the+resectability+with+curative+intent+in+pancreatic+and+periampullary+cancer.">Diagnostic accuracy of laparoscopy following computed tomography (CT) scanning for assessing the resectability with curative intent in pancreatic and periampullary cancer.</a> Cochrane Database of Systematic Reviews. (11), 009323 (2013).</li><li>Vaillant, F., Lindeman, G. J., Visvader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jekyll+or+Hyde:+does+Matrigel+provide+a+more+or+less+physiological+environment+in+mammary+repopulating+assays.">Jekyll or Hyde: does Matrigel provide a more or less physiological environment in mammary repopulating assays.</a> Breast Cancer Research. 13 (3), 108 (2011).</li><li>Jesnowski, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+pancreatic+stellate+cells+as+an+in+vitro+model+of+pancreatic+fibrosis:+deactivation+is+induced+by+matrigel+and+N-acetylcysteine.">Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine.</a> Laboratory Investigation. 85 (10), 1276-1291 (2005).</li><li>Phillips, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+novel+aspect+of+pancreatic+stellate+cell+biology.">Cell migration: a novel aspect of pancreatic stellate cell biology.</a> Gut. 52 (5), 677-682 (2003).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Egberts, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superiority+of+extended+neoadjuvant+chemotherapy+with+gemcitabine+in+pancreatic+cancer:+a+comparative+analysis+in+a+clinically+adapted+orthotopic+xenotransplantation+model+in+SCID+beige+mice.">Superiority of extended neoadjuvant chemotherapy with gemcitabine in pancreatic cancer: a comparative analysis in a clinically adapted orthotopic xenotransplantation model in SCID beige mice.</a> Cancer Biology &amp; Therapy. 6 (8), 1227-1232 (2007).</li></ol>

Yazarların bu projeyle ilgili açıklayacak hiçbir şeyi yoktur.

Pankreas kanserinin rezeksiyonlu ortotopik fare modeli önemlidir, çünkü adjuvan ve neoadjuvan tedavilerin testine izin verir. Bu özellikle cerrahinin en etkili tedavi olmaya devam ettiği ancak yüksek nüks riski ile ilişkili olduğu pankreas kanserinde önemlidir. Bu makalede, neoadjuvan / adjuvan tedavinin gerekli olduğu klinik senaryoyu çoğaltarak, rezeksiyonla tedavi edilebilen pankreas kanserini güvenilir bir şekilde üretecek bir yöntem açıklanmaktadır.
Mevcut yö...

Pankreas düktal adenokarsinom (pankreas kanseri [PC]) zayıf bir prognoz ile ilişkilidir1. Cerrahi rezeksiyon PC için potansiyel olarak iyileştirici tek tedavi olmaya devam etmektedir ve erken evre hastalığı olan hastalar için düşünülmelidir. Ne yazık ki, R0 rezeksiyonu (yani tümör içermeyen rezeksiyon marjları) ile bile, tekrarlama oranı (lokal veya tespit edilmemiş metastatik hastalıktan) yüksektir2,3. Bu nedenle, rezeksiyon uygulanan hemen hemen tüm hastalarda sistemik adjuvan tedavi endikedir4. Ayrıca, neoadjuvan tedavi artık sadece borderline-rezeke edilebilir kanserler için tavsiye edilirken, endikasyonları rutin kullanımı çok klinik araştırmaların odak noktası olacak şekilde genişliyor5,6,7,8. PC için rezeksiyon içeren yeni terapötik yaklaşımlar geliştirmek için, bu yaklaşımların öncelikle klinik ayarları doğru bir şekilde yeniden deneyen klinik öncesi modellerde değerlendirilmesi gerekir.
PC'nin ortotopik fare modelleri geçmişte ilaç tedavilerini test etmek için sıklıkla kullanılmıştır9,10. Bunların çoğu, kanser hücrelerinin tek başına fare pankreasına enjekte ederek üretildi ve PC'nin karakteristik özelliği olan belirgin stromadan yoksun tümörlerle sonuçlandı. Daha yakın zamanda, ilk olarak insan PC ve insan pankreas yıldız hücrelerinin bir karışımını enjekte ederek geliştirdiğimiz gibi ortak enjeksiyon ortotopik modeller (PC'deki kollajenöz stromanın birincil üreticileri olan PSC'ler) düzenli olarak kullanıma girdi11,12. Kanser ve stromal hücrelerin bu tür ko-enjeksiyonu ile üretilen tümörler (i) hem kanser elementlerini hem de PC'nin karakteristik stromal (desmoplastik) bileşenini ve (ii) gelişmiş kanser hücresi çoğalması ve metastazını sergiler11. Bu nedenle, bu model insan PC'ye çok benziyor. Ortotopik PC'nin bir dizi rezeksiyon modeli tanımlanmış olsa da13,14,15,16, hiçbiri insanlarda pankreas rezeksiyonunun klinik gerçeklerini bu model kadar doğru yansıtmamıştır ve bu nedenle adjuvan veya neoadjuvan tedavileri test etmek için suboptimal olmuştur.
Sunulan fare modelinin amacı şu şekildeydi: (i) yanlışlıkla periton yayılımını en aza indirirken ortotopik pankreas kanserinin başarılı bir şekilde implante edildiğini ve (ii) daha sonra kanserin tamamen resect olduğunu göstermekti. Makale, bu tekniğin ipuçlarını ve potansiyel tuzaklarını vurgulamaktadır.

<table>
	<tbody>
		<tr>
			<td>Animals, Materials and Equipment for Implantation Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid)</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td></td>
			<td>supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan</td>
		</tr>
		<tr>
			<td>Autoclip wound clips, 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW, Australia</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cancer associated human pancreatic stellate cells</td>
			<td>Pancreatic Research Group cell bank</td>
			<td></td>
			<td>In house cell bank</td>
		</tr>
		<tr>
			<td>Cryogenic tubes, 1.0 mL</td>
			<td>Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia</td>
			<td>366656</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female</td>
			<td>Australian Bioresources, Moss Vale, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) with 4mM L-glutamine and no phenol red</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Penicillin 10,000 U/mL, streptomycin 10,000 &#956;g/mL</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>11875085</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25300054</td>
			<td>For pancreatic stellate cells</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25200056</td>
			<td>For ASPC-1 cells</td>
		</tr>
		<tr>
			<td>U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) &#215; 13 mm needle</td>
			<td>Terumo Medical Corporation, Elkton, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for Resection Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alm self-retaining retractor</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Autoclip wound clips 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td>08-559NP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Hand-held high temperature fine tip cautery</td>
			<td>Bovie Medical Corporation, Melville, NY, USA</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II Bioluminescent Imaging Device</td>
			<td>Caliper Life Sciences, Hopkinton, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Titanium &#8220;Weck style&#8221; Ligaclip, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Ligaclip applier for open surgery, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volatile anaesthetic machine, including vapouriser and induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Generic vapouriser and induction chamber</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for Procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% w/w ethanol solution</td>
			<td>Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia</td>
			<td></td>
			<td>Applied topically as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 0.05 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>D-Luciferin (1 U/g)</td>
			<td>PerkinElmer, Inc., Waltham, MA, USA</td>
			<td>122799</td>
			<td>diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p</td>
		</tr>
		<tr>
			<td>Enrofloxacin 50 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Flunixin 50 mg/mL</td>
			<td>Norbrook Laboratories Australia, Tullamarine, VIC, Australia</td>
			<td></td>
			<td>Dose: 2.5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis Australia Pty Ltd., Rhodes, NSW, Australia</td>
			<td></td>
			<td>Dose (vapourised with oxygen): 4% induction, 3% maintenance</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Maylab, Slacks Creek, QLD, Australia</td>
			<td></td>
			<td>Dose: 80 mg/kg i.p.</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% w/v solution</td>
			<td>Perrigo Australia, Balcatta, WA, Australia</td>
			<td>RIO00802F</td>
			<td>Applied topically to the anterior abdomen as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w)</td>
			<td>Allergan Australia Pty Ltd, Gordon, NSW, Australia</td>
			<td></td>
			<td>Applied to both eyes</td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% w/v</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>9481P</td>
			<td>Dose: 900 &#956;L s.c.</td>
		</tr>
		<tr>
			<td>Water for injections BP</td>
			<td>Pfizer Australia, Sydney, NSW, Australia</td>
			<td></td>
			<td>For dilution of drugs</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 10 mg/kg i.p.</td>
		</tr>
	</tbody>
</table>

an orthotopic resectional mouse model of pancreatic cancer

Pankreas kanseri (PC) cerrahisi için düşünülen hastalarda adjuvan ve/veya neoadjuvan tedaviyi incelemek için tatmin edici hayvan modellerinin eksikliği vardır. Bu eksikliği gidermek için, PC'nin ortotopik implantasyonunu ve ardından distal pankreastomi ve splenektomi içeren bir fare modelini tarif ediyoruz. Modelin, adjuvan ve neo adjuvan ortamlarda çeşitli terapötik yaklaşımların incelenmesi için güvenli ve uygun şekilde esnek olduğu gösterilmiştir.
Bu modelde, pankreas tümörü ilk olarak balb/c athymic çıplak farelerin distal pankreasına insan pankreas kanseri hücrelerinin (luciferaz etiketli AsPC-1) ve insan kanseri ilişkili pankreas yıldız hücrelerinin bir karışımının yerleştirilmesiyle üretilir. Üç hafta sonra, kanser yeniden laparotomi, distal pankreastomi ve splenektomi ile resected edilir. Bu modelde biyolüminesans görüntüleme, kanser gelişiminin ilerlemesini ve rezeksiyon/tedavilerin etkilerini takip etmek için kullanılabilir. Rezeksiyondan sonra adjuvan tedavi verilebilir. Alternatif olarak, rezeksiyondan önce neoadjuvan tedavi verilebilir.
45 fareden temsili veriler sunulmaktadır. Tüm farelere hemostaz sorunu olmayan başarılı distal pankreastomi / splenektomi uygulandı. 43'te 5 mm'den büyük makroskopik proksimal pankreas marjı elde edildi (%96) Fare. Pankreas rezeksiyonunun teknik başarı oranı %100, erken mortalite ve morbiditesi %0 idi. Rezeksiyondan sonraki hafta boyunca hayvanların hiçbiri ölmedi.
Özetle, klinik senaryoyu taklit eden farelerde pankreas kanserinin cerrahi rezeksiyon modeli için sağlam ve tekrarlanabilir bir teknik tanımlıyoruz. Model hem adjuvan hem de neoadjuvan tedavilerin test edilmesi için yararlı olabilir.

Ardışık 59 fare implantasyon ameliyatı geçirdi. Brüt sızıntı sekizde meydana geldi (%14) Fare. Enjeksiyon anında sızıntı derecesi, protokol bölümünde yukarıda açıklandığı gibi tahmin edilmektedir. Bu implante tümörlerin büyümesine izin vermek için üç hafta sonra, rezeksiyon öncesi biyolüminesans görüntülemesi, rezeksiyondan önce brüt metastatik hastalığı olan fareleri dışlamak için yapıldı. Kırk beş (%76) farelere cerrahi rezeksiyon uygulandı.
Hep...

Watch this Scientific Journal Video about An Orthotopic Resectional Mouse Model of Pancreatic Cancer at JoVE.com

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Klinik bağlamda lokalize pankreas kanseri olan hastalar pankreastektomi ve ardından adjuvan tedavi görecektir. Burada bildirilen bu protokol, pankreas kanserinin ortotopik implantasyonu ve ardından distal pankreasektomi ve splenektomi yoluyla çıplak farelerde bu klinik senaryoyu modellemek için güvenli ve etkili bir yöntem oluşturmayı amaçlamaktadır.

Pankreas Kanserinin Ortotopik Rezeksiyonlu Fare Modeli

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

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Research

JoVE Journal

Cancer Research

10.0K Görüntüleme.  University of New South Wales. Klinik bağlamda lokalize pankreas kanseri olan hastalar pankreastektomi ve ardından adjuvan tedavi görecektir. Burada bildirilen bu protokol, pankreas kanserinin ortotopik implantasyonu ve ardından distal pankreasektomi ve splenektomi yoluyla çıplak farelerde bu klinik senaryoyu modellemek için güvenli ve etkili bir yöntem oluşturmayı amaçlamaktadır.

Video: An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pankreas Kanseri Floresan Ortotopik Fare Modeli

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Kanser Araştırmaları

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Kolorektal kanserli bir Ortotopik Fare Modelinde Dolaşan Tümör Hücrelerinin İzolasyonu

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Geri dönüşü olmayan Elektroporasyon etkilerini incelemek için bir Syngeneic pankreas kanseri fare modeli

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Orthotopic Homogref (Syngeneic) fare birincil KPC pankreas duktal adenokarsinom, akış sitometresi tarafından Immunophenotyping

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Retroperitoneal Lenfadenopati li Ortotopik Endometriyal Kanser Modeli In Vivo Propagated ve Kültürlü VX2 Hücrelerinden Yapılmıştır

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Hepatik İnflamasyon ve Fibrozis Ortamında Ortaya Çıkan Hepatosellüler Kanserin Onkojenik Hepatositkaynaklı Ortotopik Fare Modeli

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Ortotopik Meme Kanseri Modeli Kullanarak Üçlü Negatif Meme Kanseri Eğitimi

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Murine Pankreatik Duktal Adenokarsinomun Ultrason Eşliğinde Ortotopik İmplantasyonu

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Ortotopik Pankreas Tümörlerinin Üretimi ve Tümör Efiltrasyonu T Hücre Sitotoksisitesinin Ex vivo Karakterizasyonu

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Ultrason Eşliğinde Enjeksiyon ile Pankreas Kanseri Hücrelerinin Ortotopik Ksenograftının Oluşturulması

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...