Name: generation of an orthotopic xenograft of pancreatic cancer cells by ultrasound-guided injection
Uploaded: 2021-11-01T13:00:00
Description: Watch this Scientific Journal Video about Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection at JoVE.com

This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC, grant no. 15627, IG 21510, and IG 19766) to AA, PRIN Italian Ministry of University and Research (MIUR). Leveraging basic knowledge of ion channel network in cancer for innovative therapeutic strategies (LIONESS) 20174TB8KW to AA, pHioniC: European Union&#39;s Horizon 2020 grant No 813834 to AA. CD was supported by a AIRC fellowship for Italy ID 24020.

The present protocol received approval from the Italian Ministry of Health with the authorization number 843/2020-PR. In order to ensure aseptic conditions, the animals were maintained inside the barrier room of the research animal vivarium (Ce.S.A.L.) of the University of Florence. All procedures were performed in the same space where the mice were housed at the LIGeMA facility of the University of Florence (Italy).
1. Cell preparation
<ol>
	<li>Culture PCa cells from the P...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+orthotopic+pancreatic+tumor+mouse+models.">Development of orthotopic pancreatic tumor mouse models.</a> Methods in Molecular Biology. 980, 215-223 (2013).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Reviews. 17 (3), 279-284 (1998).</li><li>Qiu, W., Su, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+advances+in+mouse+modeling+for+human+pancreatic+tumorigenesis+and+metastasis.">Challenges and advances in mouse modeling for human pancreatic tumorigenesis and metastasis.</a> Cancer and Metastasis Reviews. 32 (1-2), 83-107 (2013).</li><li>Erstad, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+guidelines+for+using+isoflurane.">Advantages and guidelines for using isoflurane.</a> The Veterinary Clinics of North America Small Animal Practice. 22 (2), 328-331 (1992).</li><li>Huynh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+orthotopic+human+pancreatic+cancer+xenograft+model+using+ultrasound+guided+injection+of+cells.">Development of an orthotopic human pancreatic cancer xenograft model using ultrasound guided injection of cells.</a> PLoS One. 6 (5), 20330 (2011).</li><li>Duranti, C., 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=Harnessing+the+hERG1/&#946;1+Integrin+Complex+via+a+Novel+Bispecific+Single-chain+Antibody:+An+Effective+Strategy+against+Solid+Cancers.">Harnessing the hERG1/&#946;1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers.</a> Molecular Cancer Therapeutics. 20 (8), 1338-1349 (2021).</li></ol>

Although the use of US imaging is widespread in the clinic, tumor development in many preclinical mouse models is usually described using bioluminescent imaging11. The latter is an indirect way to evaluate tumor engraftment and expansion and it also does not provide a reliable tumor growth kinetics. In the present study, we have applied US imaging for performing cell injection as well as for monitoring tumor development. The protocol we have described and the results we have provided represent a r...

PCa, and its most common form, the Pancreatic Ductal Adeno Carcinoma (PDAC), is one of the most common causes of cancer-related death with a 1-year survival rate lower than 20% and a 5-year survival rate of 8%, regardless of the stage1,2. The disease is almost always fatal, and its incidence is forecasted to continuously grow in the next years, unlike other cancer types, whose incidence is declining3. Factors such as late cancer detection, the tendency of rapid progression, and lack of specific therapies lead to a poor prognosis of PCa4. Great advances in cancer research have been obtained, thanks to the development of more accurate preclinical mouse models. The models have provided appropriate insights to the understanding of the molecular mechanism underlying cancer and to the development of new treatments5. These advances poorly apply to PCa, which, despite great recent efforts, remains resistant to current chemotherapeutic therapies1. For these reasons, the development of novel approaches to improve patients&#39; prospects is mandatory.
Over the years, many PCa mouse models have been developed, including xenografts, which are the most widely used models nowadays5. Xenograft models are classified as subcutaneous heterotopic and orthotopic, depending on the location of the implanted tumor cells. Subcutaneous heterotopic xenografts are easier and cheaper to accomplish but miss certain characteristic features of PCa (i.e., the peculiar tumor microenvironment, characterized by the accumulation of fibrotic tissue, hypoxia, acidity, and angiogenesis)6,7. This explains why subcutaneous xenografts often fail to provide robust data for therapeutic treatments leading to failures when translated to the clinical setting8. On the other hand, orthotopic xenografts resemble the tumor microenvironment more closely, leading to better mimicking of the natural development of the disease. In addition, orthotopic xenografts are more suitable for studying the metastatic process and the invasive features of PCa, which almost do not occur in subcutaneous models9. Overall, orthotopic xenograft mouse models are nowadays preferred to perform preclinical drug testing9,10. Orthotopic xenografts usually rely on surgical procedures to implant either cells or very small tumor tissue pieces into the pancreas. Indeed, several papers based on surgical models of PCa have been published in the last few decades11. However, the quality and the outcome of the surgical procedure for the establishment of an orthotopic tumor model strongly depend on the technical skill of the operator. Another key point for a successful orthotopic PCa xenograft for a translational clinical approach is the possibility to establish localized disease with predictable growth kinetics.
To address these issues, here we describe an innovative procedure to produce an orthotopic PCa xenograft, exploiting ultrasound (US)-guided injection of human PCa cells into the tail of the pancreas in immunodeficient mice. This procedure generates a reliable PCa mouse model. The tumor growth is followed in vivo by US imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Sarstedt, Germany</td>
			<td>P5856</td>
			<td></td>
		</tr>
		<tr>
			<td>3D-Mode package</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td></td>
			<td>Includes the 3D Motor; necessary for volumetric imaging</td>
		</tr>
		<tr>
			<td>Aquasonic 100, Sonypack 5 lt Ultrasound Transmission Gel</td>
			<td>PARKER LABORATORIES, INC.</td>
			<td>150</td>
			<td>Gel for ultrasound</td>
		</tr>
		<tr>
			<td>Athymic Mice (Nude-Foxn1nu)</td>
			<td>ENVIGO, Italy</td>
			<td>69</td>
			<td>20 females, 8 weeks old, Athymic Nude-Foxn1nu, 20-22 g body weight</td>
		</tr>
		<tr>
			<td>CO2 Incubator Function Line</td>
			<td>Heraeus Instruments, Germany</td>
			<td>BB16-ICN2</td>
			<td></td>
		</tr>
		<tr>
			<td>Display of ECG, Respiration Waveform and body temperature</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>11426</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#8217;s Modified Eagle Medium)</td>
			<td>Euroclone Spa, Italy</td>
			<td>ECM0101L</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#8217;s Phosphate Buffered Saline)</td>
			<td>Euroclone Spa, Italy</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf (1.5mL)</td>
			<td>Sarstedt, Germany</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Euroclone Spa, Italy</td>
			<td>ECS0170L</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Needle Pointstyle 4, lenght 30 mm, 28 Gauge</td>
			<td>Permax S.r.l., Italy</td>
			<td>7803-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe 705RM 50 &#181;L</td>
			<td>Permax S.r.l., Italy</td>
			<td>7637-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflo (250 mL)</td>
			<td>Ecuphar</td>
			<td>7081219</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 100X</td>
			<td>Euroclone Spa, Italy</td>
			<td>ECB3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Handling table II</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>50249</td>
			<td></td>
		</tr>
		<tr>
			<td>MX550D: 55 MHz MX Series Transducer</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>51069</td>
			<td>Ultrasound Transducers</td>
		</tr>
		<tr>
			<td>Oxygen/isofluorane mixer</td>
			<td>Angelo Franceschini S.r.l.</td>
			<td>LFY-I-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>PANC1 cell line</td>
			<td>American Type Culture Collection (ATCC), USA</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl (carprofen)</td>
			<td>Pfizer</td>
			<td>11319</td>
			<td>20 mL, injection solution</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1X in PBS</td>
			<td>Euroclone Spa, Italy</td>
			<td>ECB3052D</td>
			<td></td>
		</tr>
		<tr>
			<td>Vet ointment for eyes, Systane nighttime</td>
			<td>Alcon</td>
			<td>509/28555-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Compact Dual Anesthesia System (Tabletop Version)</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>VS-12055</td>
			<td>complete with gas chamber</td>
		</tr>
		<tr>
			<td>Vevo Imaging Station 2</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>VS-11983</td>
			<td>Imaging WorkStation 1 plus Imaging Station Extension with injection mount</td>
		</tr>
		<tr>
			<td>Vevo Lab</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>VS-20034</td>
			<td>Data Analysis Software</td>
		</tr>
		<tr>
			<td>Vevo LAZR-X Photoacoustic Imaging System</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>VS-20054</td>
			<td>Includes analytic software package for B-mode</td>
		</tr>
		<tr>
			<td>Vevo Photoacoustic Enclosure</td>
			<td>Visualsonics Fujifilm, Italy</td>
			<td>53157</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the protocol described above, mice were first anesthetized in an isoflurane chamber, and placed on the animal platform (Figure 1A). The pancreas was visualized with ultrasound imaging (Figure 1B). A 50 &#181;L Hamilton syringe was loaded with 1 x 106 PANC1 cells suspended in 20 &#181;L of PBS and placed on the needle holder (Figure 1C). The optimal angle between the syringe and the US transducer was 45&#176; (<s...

Watch this Scientific Journal Video about Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection at JoVE.com

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

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.

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

Protokol, hücrelerin enjeksiyonunu ve tümörlerin engraftmanının ve gelişiminin değerlendirilmesini birleştiren bir sistem kullanarak pankreas kanserinin ortopik bir fare modelini oluşturmak için bir yöntemi açıklar. Bu teknik büyük tekrarlanabilirliğe sahiptir, minimal invazivdir, cerrahi prosedürün neden olduğu birçok dezavantajın üstesinden gelmeye izin verir ve 3R ilkelerine uygundur. Yankı rehberliğinde enjeksiyon, safra ağacındaki enjeksiyonlar veya meme kanseri modelleri gibi diğer ortotopik modeller için kullanılabilir.Böyle bir tekniğin kullanımı, küçük moleküllerin kullanımı ile birleştirilebilir. 27 gauge iğneli bir tüberkülin şırıngası kullanarak tramadol intraperitoneal olarak kilogram başına 5 miligramlık bir dozda uygulayarak ultrason rehberliğinde enjeksiyon için 8 haftalık 20 Atimik çıplak-Foxn-1nu dişi fare hazırlamaya başlayın. Ardından, görüntüleyiciyi açın ve dönüştürücü panelinden uygulama menüsünde Fare, Küçük, Abdominal'i seçin.B Modu veya Parlaklık Modu görüntüleme penceresinin göründüğünden ve sistemin B-Modu verilerini almaya hazır olduğundan emin olun. Yeni Etüt'ü seçmek için Etüt Tarayıcısına gidin ve çalışma adını ve çalışmanın tarihi gibi bilgileri yazın. Ardından, Seri Adı'nda hayvan suşu, kimlik, doğum tarihi gibi gerekli tüm bilgileri doldurun.Bilgiler girildikten sonra, programı B-Mode görüntülemeye hazırlamak için "Bitti" ye dokunun. Anestezi uygulanan fareyi sağ kanadında, burnu burun konisi içinde olacak şekilde 37 santigrat derecede ısıtılmış tutma masasına yerleştirin ve farenin gözlerine bir damla veteriner merhem uygulayın. Ardından, farenin sağ elini, sağ ayağını ve kuyruğunu hayvan platformundaki elektrot pedlerine bantlamak için yapışkan bir gazlı bez kullanın.Farenin solunum hızı ve elektrokardiyogramı elektrot pedleri aracılığıyla kaydedilir. Fareye PANC1 hücreleri enjekte etmeden önce, fare cildini% 70 etanol ile sterilize edin ve yapışkan gazlı bez kullanarak sol kanadın cildini gerilmiş halde tutun. Karın ve farenin sol kanadına ultrason jeli uygulamak için iğnesiz 20 mililitrelik bir şırınga kullanın.Ultrason dönüştürücüsünün yükseklik kontrol düğmesinin yardımıyla, farenin sol kanadına dokunmak için dönüştürücüyü indirin ve dönüştürücüyü hayvanın vücuduna enine olarak yerleştirin. Ardından, B-Mode görüntülemeyi kullanarak dönüştürücü ekranındaki pankreası görselleştirmek için dönüştürücüyü hareket ettirin. Dezenfekte edilmiş 30 milimetre 28 gauge iğne ile 20 mikrolitre PBS'de asılı 10 milyon PANC1 hücresi içeren 50 mikrolitrelik bir Hamilton şırıngası hazırlayın ve şırıngayı uygun tutucuya yerleştirin.Tutucu mikromanipülatörü kullanarak, şırıngayı fare derisine, iğne eğimi yukarı bakacak şekilde ve ultrason dönüştürücü ile aynı düzlemde, dönüştürücü ile 45 derecelik bir açı oluşturarak indirin. Mikromanipülatör ile cildi delin ve yörüngeyi takip etmek için ekrandaki ultrason görüntüsünü gözlemlerken şırınga iğnesini pankreasın içine yerleştirin. İğne pankreas içine yerleştirildikten sonra, şırınganın pistonuna sabit basınç uygulayarak hücreleri içeren 20 mikrolitrelik bir bolus enjekte edin.Doğru enjeksiyon prosedürü, pankreasta küçük bir kabarcığın varlığı ve hipoekojenik sıvının akışı ile kontrol edilebilir. Enjeksiyondan 5 ila 10 saniye sonra, iğneyi geri çekin. Ardından, ultrason dönüştürücüsünü çıkarın ve jeli yandan temizleyin.İşiniz bittiğinde, fareyi tek başına gözlem altındaki yeni bir kafese, sternal yassılığı korumak için yeterli bilinci yeniden kazanana kadar yerleştirin. Temsili görüntü, eko rehberliğinde hücre enjeksiyonundan sonra pankreasın kuyruğunda bir tümör kütlesinin varlığını gösterir ve protokolün tekrarlanabilirliğini doğrular. PANC1 hücrelerinden türeyen ortotopik pankreas tümörlerinin tümör hacmi, hücre enjeksiyonundan sonraki 8. günden başlayarak haftada bir kez ultrason görüntüleme ile değerlendirildi.43. günde deneyin son noktasında, tümör kitlelerinin ultrason görüntüleri elde edildi. Nekropsi muayenesinde tümör kitlesi, sağlıklı pankreas, dalak, mide ve karaciğerin küçük bir kısmı gözlendi. Ek olarak, tümör kitleleri histolojik analiz için eksize edildi.Fareyi sağ tarafında masanın üzerine yerleştirmek önemlidir ve hücrelerin pankreastan dökülme riskini önlemek için cilt uygun şekilde gerilmelidir. Ultrason eşliğinde enjeksiyon ile elde edilen pankreas kanseri modelinin gelecekteki uygulamaları, uygun terapötik tedavilerin veya tedavi kombinasyonlarının geliştirilmesine yönelik çalışmaları içermektedir.

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Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Fareler meme yağ yastığı içine meme kanser hücrelerinin Orthotopic enjeksiyon

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Karşılaştırmalı Ilaç değerlendirmesi için ev sahipleri olarak zebrafish larvaları kullanarak pankreas kanserinin hasta türevi heterojen xenograft modeli

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

İmmünficient Farelerde Karaciğer Ortotopik İnsan Uveal Melanom Xenograft Platformu Üretimi

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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

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

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 ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

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 ...