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. 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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 ...

The protocol describes a method to generate an orthotopic mouse model of pancreatic cancer using a system that combines the injection of cells and the evaluation of engraftment and development of tumors. This technique has great reproducibility, is minimally invasive, allows to overcome many drawbacks caused by the surgical procedure, and complies with the 3Rs principles. The echo-guided injection could be used for other orthotopic models, such as injections in the biliary tree or breast cancer models.The use of such a technique could be coupled with the use of small molecules. Start preparing an 8-week-old 20 Athymic Nude-Foxn-1nu female mice for ultrasound-guided injection by administering tramadol intraperitoneally at a dose of 5 milligrams per kilogram using a tuberculin syringe with a 27-gauge needle. Next, turn on the imager and select Mouse, Small, Abdominal"on the application menu from the transducer panel.Ensure that the B-Mode, or Brightness Mode, imaging window appears and the system is ready to acquire B-Mode data. Go to the Study Browser to select New Study"and type the study name and information, such as the date of the study. Then, fill in all the necessary information in the Series Name, like animal strain, ID, date of birth.After the information is entered, tap Done"to prepare the program for B-Mode imaging. Place the anesthetized mouse on its right flank onto the handling table heated at 37 degrees Celsius with its snout in a nose cone and apply a drop of vet ointment on the mouse's eyes. Then, use an adhesive gauze to tape the right hand, right foot, and tail of the mouse onto the electrode pads on the animal platform.The mouse's respiration rate and electrocardiogram are recorded through the electrode pads. Before injecting the mouse with PANC1 cells, sanitize the mouse skin with 70%ethanol and keep the skin of the left flank stretched using adhesive gauze. Use a 20 milliliter syringe without the needle to apply ultrasound gel on the abdomen and left flank of the mouse.With the help of the height control knob of the ultrasound transducer, lower the transducer to touch the left flank of the mouse and place the transducer transversely to the body of the animal. Then, move the transducer to visualize the pancreas on the transducer display using B-Mode imaging. Prepare a 50 microliter Hamilton syringe, containing 10 million PANC1 cells suspended in 20 microliters of PBS with a sanitized 30-millimeter 28-gauge needle and place the syringe on the appropriate holder.Using the holder micromanipulator, lower the syringe to the mouse skin, with the needle bevel face up and in the same plane as the ultrasound transducer, forming an angle of 45 degrees with the transducer. Perforate the skin with the micromanipulator and insert the syringe needle into the pancreas while observing the ultrasound image on display to follow the trajectory. Once the needle is inserted into the pancreas, inject a 20 microliters bolus containing the cells by applying constant pressure on the syringe's plunger.The correct injection procedure can be checked by the presence of a small bubble in the pancreas and the flow of hypoechogenic fluid. After 5 to 10 seconds of the injection, retract the needle. Then, remove the ultrasound transducer and clean the gel from the flank.When done, place the mouse alone in a new cage under observation until it has regained sufficient consciousness to maintain sternal recumbency. The representative image shows the presence of a tumor mass in the tail of the pancreas after echo-guided cell injection, confirming the reproducibility of the protocol. The tumor volume of orthotopic pancreatic tumors derived from PANC1 cells was evaluated by ultrasound imaging once a week, starting from Day 8 after cell injection.At the end point of the experiment on Day 43, ultrasound images of the tumor masses were acquired. In the necropsy examination, tumor mass, a small portion of a healthy pancreas, spleen, stomach, and liver was observed. Additionally, the tumor masses were explanted for histologic analysis.It is important to position the mouse on its right side onto the table, and the skin must be properly stretched to avoid the risk of the cells spilling out of the pancreas. Future applications of the pancreatic cancer model obtained by the ultrasound-guided injection include the studies aimed at the development of appropriate therapeutic treatments or treatment combinations.

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