Authors have received support from the Avner Pancreatic Cancer Foundation.

All procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales (17/109A). Female athymic Balb/c nude mice, aged 8-10 weeks weighing 16-19 g, were used for this protocol. Mice were housed in micro-isolator cages and fed commercially available pelleted food and water ad libitum.
1. Orthotopic pancreatic cancer implantation
<ol>
	<li>Prepare the cells for implantation. First, calculate the number of cells required for the procedure (1 x 106 luciferase-tagged AsPC-1 cells and 1 x 106 cancer-associated human pancreatic stellate cells [CAhPSCs] are required for each animal).
	<ol>
		<li>Maintain these cells in a humidified temperature-controlled CO2 incubator and perform routine mycoplasma testing. Culture medium used for AsPC-1 and CAhPSCs are RPMI 1640 (with 300 mg/L L-glutamine, 20% v/v foetal bovine serum, 1% v/v penicillin/streptomycin) and IMDM (with 4 mM L-glutamine, 10% v/v foetal bovine serum, 1% v/v penicillin/streptomycin).</li>
		<li>Use standard cell culture techniques to trypsinize the cells into a cell suspension. Neutralize the trypsin using the respective complete culture medium at a volume twice that of the trypsin solution used.</li>
		<li>Wash these cells twice with phosphate buffered saline (PBS) and resuspend into a mixture containing 1 x 106 AsPC-1 cells and 1 x 106 CAhPSCs in a 50&#8239;&#956;L cell suspension.</li>
		<li>Keep this suspension on ice until use.</li>
	</ol>
	</li>
	<li>Prepare a class II biosafety cabinet for the procedure. Use a heating mat overlaid by a sterile plastic drape. For magnification during the procedure, use a pair of 2.5x to 3.5x magnification surgical loupes.</li>
	<li>Prepare purse-string swabs by cutting a hole, 1 cm in diameter, into a gauze swab. Secure this hole with a purse-string suture. Any fine braided suture can be used for this (e.g., 5/0 polyglycolic acid suture). Braided suture material is recommended as it allows the loose knot to stay in place after tightening. This is illustrated in Figure 1a.</li>
	<li>Anesthetize the mouse with 80 mg/kg of ketamine and 10 mg/kg of xylazine by intraperitoneal injection.</li>
	<li>Administer 5 mg/kg enrofloxacin antibiotic prophylaxis, 2.5 mg/kg flunixin analgesia and 1 mL of 0.9% saline subcutaneously.</li>
	<li>Once anesthetized, place the mouse on the sterile field in a supine position and apply povidone-iodine followed by 70% ethanol for skin preparation.</li>
	<li>Make a longitudinal incision in the skin of the left cranial quadrant of the abdomen, and then enter the abdomen by incising the muscular layer between forceps.</li>
	<li>Load a 29 G insulin syringe with 50 &#956;L of cell suspension&#8211;this equates to 1 x 106 CAhPSCs and 1 x 106 luciferase-tagged AsPC-1 cells per injectate. Mount it on the injection device. The design and function of this injection device is explained in detail in Figure 1b and its legend.</li>
	<li>Place the purse-string swab over the laparotomy incision and then exteriorize the spleen and pancreatic tail through the opening of this swab. Tighten the purse-string to gently encircle the body of the pancreas, exposing the pancreatic tail for injection. It is important to be tight enough that the gauze contacts the pancreas circumferentially while at the same time not constricting it.</li>
	<li>Using a pair of forceps, grasp the tail of the pancreas and gently place lateral tension on it. Puncture the ventral peritoneal surface with the needle at a shallow angle and then inject the cell suspension into the pancreas in a slow and controlled fashion (over 10&#8722;15 s) with the injection device.</li>
	<li>During the injection process, carefully observe for leakage&#8212;both around the injection site (from reflux) and on the other side of the pancreatic lobule (in case of through-and-through penetration). If visible leakage occurs, stop the injection and note the volume of leakage by checking the volume of remaining injectate in the syringe. If the leakage is of small volume (&#60;10 &#956;L), and then absorb any leakage with gauze and reposition the needle into a different pancreatic lobule to complete the injection.</li>
	<li>Replace the spleen and pancreas and close the abdominal wall with 5/0 polyglycolic acid suture in a continuous fashion. Close the skin with clips.</li>
	<li>Monitor the mouse in a warmed cage until recovered from the anaesthetic. Once awake and alert, move the mouse back to its cage.</li>
</ol>
2. Cancer resection surgery: Distal pancreatectomy and splenectomy
<ol>
	<li>The timing of resection in relation to implantation can vary depending on the experimental protocol. In general, allow the tumors to grow at least for 3 weeks prior to resection, but optimize this empirically for the particular implanted cancer cell line.</li>
	<li>On the day prior to the resection surgery, perform bioluminescence imaging on the animals to confirm the presence of a localized primary tumor. Note that this imaging study is simply used to exclude mice with obvious extra-pancreatic disease from resection. Neither size nor radiant flux should be used as thresholds for determining eligibility for resection.
	<ol>
		<li>Weigh mice and inject with D-luciferin intraperitoneally (150 mg/kg).</li>
		<li>Determine the timing of the imaging step in relation to luciferin injection for each experiment by the performance of a luciferin kinetic curve. The period of time where the radiant flux is above 90% of its maximum represents the optimal time for bioluminescence imaging (in this experiment, 18 to 26 minutes post-injection)</li>
		<li>Induce anaesthesia and maintain using isoflurane (4% and 3% with oxygen, respectively) and perform imaging using a bioluminescent imaging device (e.g., IVIS Lumina II). Use automatic exposure and binning settings (this can, however, be optimized for the expected radiant flux).</li>
	</ol>
	</li>
	<li>Prepare the class II biosafety cabinet for procedure. Use a heating mat overlaid by a sterile plastic drape. For magnification during dissection, use a pair of 2.5x to 3.5x magnification surgical loupes.</li>
	<li>Anesthetize the mouse with 80 mg/kg of ketamine and 10 mg/kg of xylazine by intraperitoneal injection.</li>
	<li>Administer 5 mg/kg enrofloxacin antibiotic prophylaxis, 2.5 mg/kg flunixin analgesia and 1 mL of 0.9% saline subcutaneously.</li>
	<li>Place the mouse on the sterile field in a supine position and apply povidone-iodine followed by 70% ethanol for skin preparation.</li>
	<li>Make a longitudinal incision in the skin of the left cranial quadrant of the abdomen, preferably through the previous incision site.</li>
	<li>Bluntly dissect the skin off the underlying muscular abdominal wall, and then place an Alm self-retaining retractor to hold the skin wound open.</li>
	<li>Incise the muscular layer between forceps just to one side of the suture line of the previous operation, and then extend the incision to excise the entire previous suture line.</li>
	<li>Exteriorize the spleen and distal pancreas and retract it cranially. At the caudal aspect of the pancreas, the colon may be found attached by filmy adhesions. If this is found, bluntly dissect the colon off.</li>
	<li>Carefully pass a pair of forceps dorsal to the body of the pancreas and splenic vessels and open this space. This frees up a segment of pancreas for subsequent ligation.</li>
	<li>Ligate the body of the pancreas proximal to the tumor with a titanium ligation clip, and then transect the pancreas distal to this with cautery. An alternative way to control the pancreatic stump is to ligate it in continuity with 5/0 polyglycolic acid suture before transection.</li>
	<li>Retract the pancreas caudally and cauterize the gastrosplenic vessels between the cranial pole of the spleen and the stomach.</li>
	<li>Remove the specimen and confirm haemostasis.</li>
	<li>Close the abdominal wall with 5/0 polyglycolic acid suture in a continuous fashion. Close the skin with clips.</li>
</ol>
3. Postoperative management
<ol>
	<li>In the immediate post anaesthetic period (for both of the above procedures), monitor the mouse in a warmed cage until recovered from the anaesthetic. Once awake and alert, move the mouse back to its cage. In the postoperative period, monitor the animals for pain and signs of distress. Administer 0.05 mg/kg&#160;buprenorphine by subcutaneous injection and closely observe the animals for 12 hours.&#160;</li>
	<li>Subsequently, monitor mice daily for weight, food intake and activity. Examine incision sites and palpate for tumor size. Remove skin clips on the seventh postoperative day.</li>
	<li>Euthanize the mouse if humane endpoints are reached. These humane endpoints include: loss of body weight &#62;20%, features of untreatable distress (including hunched posture, lack of movement or grooming) and tumor size greater than 1 cm3 as estimated by external palpation.</li>
</ol>

<ol>
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The authors have nothing to disclose with respect to this project.

A resectional orthotopic mouse model of pancreatic cancer is important because it allows for the testing of adjuvant and neoadjuvant treatments. This is particularly important in pancreatic cancer where surgery remains the most effective treatment but is associated with high risk of recurrence. This paper describes a method which will reliably produce a pancreatic cancer which is potentially curable with resection, replicating the clinical scenario where neoadjuvant/ adjuvant therapy is required.
<p class="jove_conte...

Pancreatic ductal adenocarcinoma (pancreatic cancer [PC]) is associated with a poor prognosis1. Surgical resection remains the only potentially curative treatment for PC and should be considered for patients presenting with early stage disease. Unfortunately, even with R0 resection (i.e., resection margins free of tumor), the recurrence rate (local or from undetected metastatic disease) is high2,3. Therefore, systemic adjuvant therapy is indicated in almost all patients who undergo resection4. Furthermore, while neoadjuvant therapy is now recommended only for borderline-resectable cancers, its indications are expanding such that its routine use is the focus of much clinical research5,6,7,8. In order to develop novel therapeutic approaches for PC involving resection, these approaches need to be first assessed in pre-clinical models that accurately recapitulate clinical settings.
Orthotopic mouse models of PC have been frequently used in the past to test drug treatments9,10. Many of these were produced by injection of cancer cells alone into mouse pancreas, resulting in tumors that lacked the prominent stroma that is characteristic of PC. More recently, co-injection orthotopic models, such as the one we first developed by injecting a mixture of human PC and human pancreatic stellate cells (PSCs, the primary producers of the collagenous stroma in PC), have come into regular use11,12. The tumors produced by such co-injection of cancer and stromal cells exhibit (i) both the cancer elements and the characteristic stromal (desmoplastic) component of PC, and (ii) enhanced cancer cell proliferation and metastasis11. Thus, this model closely resembles human PC. While a number of resectional models of orthotopic PC have been described13,14,15,16, none have reflected the clinical realities of pancreatic resection in humans as accurate as this model, and therefore have been suboptimal for testing adjuvant or neoadjuvant treatments.
The aims of the mouse model presented were to demonstrate how to: (i) successfully implant orthotopic pancreatic cancer while minimizing inadvertent peritoneal dissemination and (ii) subsequently completely resect the cancer. The paper highlight tips and potential pitfalls of this technique.

<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

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.

Fifty-nine consecutive mice underwent implantation surgery. Gross leakage occurred in eight (14%) mice. The degree of leakage at the time of injection is estimated as described above in the protocol section. After three weeks to allow these implanted tumors to grow, pre-resection bioluminescence imaging was performed to exclude mice with gross metastatic disease prior to resection. Forty-five (76%) mice underwent surgical resection.
All 45 (100%) mice underwent successful distal pancreatectomy...

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

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 ...

an-orthotopic-resectional-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

9.9K Views.  University of New South Wales. 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.

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.

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 ...

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 ...

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.

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.

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.

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).

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.

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.

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 ...

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.

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