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 PCa cell line in a 100 mm Petri dish containing Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 2% L-glutamine and 10% Fetal Bovine Serum (FBS).</li>
	<li>Incubate the cells in normoxia at 37 &#176;C with 5% CO2.</li>
	<li>Detach the cells with trypsin. Count, and resuspend 1 x 106 cells in 20 &#181;L of PBS, 1 h before inoculation.</li>
</ol>
2. Mouse preparation for ultrasound-guided injection (US-GI)
NOTE: Following steps were performed under sterile conditions. The entire procedure of US-guided injection, from the beginning of the anesthesia until the mouse is removed from the animal platform, takes around 10-12 min plus 5 min for complete mouse recovery.
<ol>
	<li>Just before the intervention, administer carprofen (NSAID) subcutaneously (s.c.) at a final dose of 5 mg/kg or tramadol at a final dose of 5 mg/kg using a tuberculin syringe with a 27 G needle. 
	NOTE: For the present protocol, 20 Athymic Nude-Foxn1nu female mice were used. The mice were 8 weeks old and weighed 20-22 g.</li>
	<li>Turn on the imager and select Mouse (Small) Abdominal on the application menu from the transducer panel. Ensure that the B-Mode (Brightness Mode) imaging window appears, and the system is ready to acquire B-Mode data. 
	NOTE: B-Mode is the system&#39;s default imaging mode. The system displays echoes in a two-dimensional (2D) view by assigning a brightness level based on the echo signal amplitude. B-Mode is the most effective mode for locating anatomical structures.
	<ol>
		<li>Go to the Study Browser.</li>
		<li>Select New Study and type the study name and information, i.e., date of the study, etc.</li>
		<li>Fill in all the necessary information in the Series Name, i.e., animal strain, ID, date of birth, etc.</li>
		<li>Tap Done; the program is ready for B-mode imaging.</li>
	</ol>
	</li>
	<li>Anesthetize the mouse in the anesthesia induction chamber&#160;using 4% isoflurane with a gas flow of 2 L/min of O2. 
	NOTE: Approximately 4 min are sufficient for proper anesthesia (breathing around 50-60 breaths per min).</li>
	<li>Once the mouse is anesthetized, change the connection of the anesthetic machine to direct the isoflurane toward the mouse handling table.</li>
	<li>Place the anesthetized mouse on its right flank onto the handling table (heated at 37 &#176;C) with its snout in a nose cone to ensure that the mouse is anesthetized using 2% isoflurane with a gas flow of 0.8 L/min of O2 (Figure 1A).</li>
	<li>Apply a drop of artificial tears on the mouse&#39;s eyes to prevent dryness while under anesthesia.</li>
	<li>Tape the right hand, right foot, and tail firmly onto the electrode pads on the animal platform with adhesive gauze. 
	NOTE: The mouse&#39;s respiration rate and electrocardiogram (ECG) are recorded through the electrode pads.</li>
</ol>
3. Injection of PANC1 cells in the pancreas by US-GI method
<ol>
	<li>Sanitize the mouse skin with 70% ethanol and keep the skin of the left flank stretched using adhesive gauze. 
	NOTE: Keeping the skin stretched is important to reduce resistance to the insertion of the needle and to prevent needle deformations.</li>
	<li>Apply ultrasound gel on the abdomen and left flank of the mouse using a 20 mL syringe (without the needle).</li>
	<li>Using the height control knob of the US transducer, lower the transducer to touch the left flank of the mouse and place it transversely to the body of the animal.</li>
	<li>Move the transducer to visualize the pancreas on the transducer display using B-Mode imaging (Figure 1B).</li>
	<li>Prepare a 50 &#181;L Hamilton syringe, containing 1 x 106 PANC1 cells suspended in 20 &#181;L of PBS, with a 30 mm 28 G needle and place the syringe on the appropriate holder (Figure 1C). 
	NOTE: Before using, sanitize the syringe needle with 70% ethanol for a period of 5-10 minutes.</li>
	<li>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&#176; with the transducer (Figure 1D). 
	NOTE: From this step onward, proceed by monitoring the US image on the display.</li>
	<li>Using the micromanipulator, perforate the skin and insert the syringe needle into the pancreas and observe the US image on the display, to follow its trajectory (Figure 1E). 
	NOTE: Before injection, take the tail of the pancreas as a reference which is located behind the spleen and close to the left kidney.</li>
	<li>Once the needle is inserted into the pancreas, inject a 20 &#181;L bolus containing the cells directly into the pancreas by applying constant pressure on the syringe plunger (Figure 1F). 
	NOTE: The correct injection procedure is checked by the presence of a small bubble in the pancreas and the flow of hypoechogenic fluid, which is barely visible from the needle tip.</li>
	<li>Leave the needle in place for 5-10 s after the whole bolus is injected, and then slowly retract it.</li>
	<li>Remove the US transducer, clean the gel from the flank, and place the mouse alone in a new cage. Observe the animal until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
4. 3D US imaging for monitoring pancreatic tumors in mice
NOTE: The evaluation of tumor development was performed starting 8 days after the cell injection, using the same instrument used for the US-guided injection (listed in Table of Materials). Hence, some procedures, such as the system ignition (step 2.2.), anesthesia (steps 2.3. - 2.6.), and mouse placement on the animal platform (step 2.7.), fully match what was described above in the protocol.
<ol>
	<li>Before starting the US imaging, set up the workstation as shown in Figure 2A.</li>
	<li>Fix the transducer on the 3D motor system (Figure 2A).</li>
	<li>Turn on the imager and select New Study on the Study Browser.</li>
	<li>Place the mouse in the anesthesia induction chamber (4% isoflurane).</li>
	<li>Place the anesthetized mouse on its right flank onto the handling table heated at 37 &#176;C with its snout in the anesthetic tube and reduce isoflurane to 2% (Figure 2B). 
	NOTE: It is important that the mouse is placed in the same position as the US-guided injection, to maintain the same anatomical references.</li>
	<li>Apply a drop of vet ointment on the mouse&#39;s eyes.</li>
	<li>Apply a layer of ultrasound gel on the mouse&#39;s abdomen and left flank.</li>
	<li>Position the 55 MHz transducer in the transverse orientation to touch the left flank skin such that the pancreas is approximately centered (Figure 2C).</li>
	<li>Use the 3D motor to acquire images of the whole pancreas in the transverse orientation, ideally gathering 90-100 frames per acquisition (number of frames may vary depending on personal choice).
	<ol>
		<li>Select the 3D Motor Position on the imager touchpad.</li>
		<li>Indicate the scanning distance by moving the cursor to acquire images of the whole pancreas from both extremities.</li>
		<li>Select Scan Frames and begin 3D acquisition.</li>
	</ol>
	</li>
	<li>Once 3D imaging is done, remove the transducer, clean the gel from the skin, and place the mouse alone in a new cage for recovery. Observe the animal until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>

<ol>
	<li>Zeng, 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=Chemoresistance+in+pancreatic+cancer.">Chemoresistance in pancreatic cancer.</a> International Journal of Molecular Sciences. 20 (18), 4504 (2019).</li><li>Bray, 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=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><li>Rahib, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projecting+cancer+incidence+and+deaths+to+2030:+The+unexpected+burden+of+thyroid,+liver,+and+pancreas+cancers+in+the+united+states.">Projecting cancer incidence and deaths to 2030: The unexpected burden of thyroid, liver, and pancreas cancers in the united states.</a> Cancer Research. 74 (11), 2913-2921 (2014).</li><li>Rawla, P., Sunkara, T., Gaduputi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+pancreatic+cancer:+Global+trends,+etiology+and+risk+factors.">Epidemiology of pancreatic cancer: Global trends, etiology and risk factors.</a> World Journal of Oncology. 10 (1), 10 (2019).</li><li>Herreros-Villanueva, M., Hijona, E., Cosme, A., Bujanda, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+pancreatic+cancer.">Mouse models of pancreatic cancer.</a> World Journal of Gastroenterology. 18 (12), 1286-1294 (2012).</li><li>Hofschroer, V., 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=Ion+channels+orchestrate+pancreatic+ductal+adenocarcinoma+progression+and+therapy.">Ion channels orchestrate pancreatic ductal adenocarcinoma progression and therapy.</a> Frontiers in Pharmacology. 11, 586599 (2021).</li><li>Adamska, A., Domenichini, A., Falasca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+ductal+adenocarcinoma:+Current+and+evolving+therapies.">Pancreatic ductal adenocarcinoma: Current and evolving therapies.</a> International Journal of Molecular Sciences. 18 (7), 1338 (2017).</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=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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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+and+heterotopic+murine+models+of+pancreatic+cancer+and+their+different+responses+to+FOLFIRINOX+chemotherapy.">Orthotopic and heterotopic murine models of pancreatic cancer and their different responses to FOLFIRINOX chemotherapy.</a> DMM Disease Models and Mechanisms. 11 (7), (2018).</li><li>Lottini, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-ultrasound,+non-linear+contrast+mode+with+microbubbles+and+Optical+Flow+software+tool:+together+for+a+new+translational+method+in+the+study+of+the+tumoral+rheology+microenvironment.">Micro-ultrasound, non-linear contrast mode with microbubbles and Optical Flow software tool: together for a new translational method in the study of the tumoral rheology microenvironment.</a> WMIC 2017: Imaging the Future from Molecules to Medicine. , (2017).</li><li>Ludders, J. 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>

The authors have nothing to disclose.

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 ...

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2.8K Views.  University of Florence. 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.

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

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.

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 ...

Endobronchial Ultrasound-guided Intratumoral Injection of Cisplatin for the Treatment of Isolated Mediastinal Recurrence of Lung Cancer

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients do not have many treatment options and are usually offered palliative chemotherapy or best supportive care. Endobronchial ultrasound (EBUS)-guided intratumoral injection of cisplatin (ITC) is a novel approach for these patients. The procedure is performed under conscious sedation. The lesion is located with a bronchoscopy using EBUS, and a 22-gauge EBUS needle is advanced through the working channel of the scope and locked in position. Under ultrasound guidance, the wall of the tracheobronchial tree is punctured and the needle is moved into the target lesion. The needle stylet is then removed and cisplatin (40 mg/40 mL) is injected into the lesion. One to two sites are treated per session. Details of the procedure are described in the protocol section of paper. At our center, 50 sites were treated in 36 patients (19 males, 17 females). The mean age of our cohort was 61.9 &#177;8.5 years. We performed final analyses on 35 patients and 41 sites. 24/35 (69%) had complete or partial response (responders), whereas 11/35 (31%) had stable or progressive disease (non-responders). Overall, survival in our group was 8 months (95% CI of 6-11 months), with patients who responded having significantly better survival than the ones who did not.

The management of isolated recurrent lung cancer in a previously-irradiated field is challenging. Here, we describe an endobronchial ultrasound (EBUS)-guided cisplatin injection for the management of patients with localized lung cancer recurrence in a previously-radiated field.

Isolated hilar and mediastinal recurrence (IMHR) following external beam radiation therapy (EBRT) in patients with lung cancer is common. These patients ...

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.

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

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.

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

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

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

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

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

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

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

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

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.

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.

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

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

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