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.
Significance with respect to existing methods 
Despite the importance of adjuvant and neoadjuvant therapies in pancreatic cancer, there are few well-described orthotopic resectional mouse models in the literature. These described resectional models varied in their fidelity of replication of the clinical situation in humans. These previous models can be broadly classified into: (i) tumor excision only, with fluorescence guidance; (ii) subtotal pancreatic resection with no splenectomy; (iii) distal pancreatectomy/splenectomy.
Tumor excision with fluorescence guidance has been described in the greatest number of reports15,17,18,19,20,21. Many of these papers originated from the same research group. Unfortunately, in humans, local excision of the tumor alone (enucleation) is not performed for pancreatic adenocarcinoma (PC) due to the high likelihood of local recurrence, as well as the inability to assess lymph node status22,23. Therefore, the use of a non-clinically relevant comparator group (non-fluorescence guided enucleation) clouds the reporting of the oncological outcomes in papers describing this technique. Not surprisingly, the non-fluorescence enucleation groups invariably had excessive rates of local recurrence15,20,21. In contrast, Torgenson et al.14 described a similar fluorescence-guided resection technique, and reported a reasonably low recurrence rate of 58% (at eight weeks post-resection). Overall, these studies appear to demonstrate the utility of fluorescence guidance for visualization of residual disease during surgery. However, this is not yet the standard of care in humans, which is a limitation in terms of its use in a mouse model aiming to replicate the clinical scenario. Of course, this may change if fluorescence-guided surgery were to be widely adopted in clinical practice.
Another resection model was based on subtotal pancreatectomy without splenectomy for a tumor implanted into the body of the pancreas13,24. The clinical relevance of this is also called into question as the operation described was neither a pancreaticoduodenectomy nor distal pancreatectomy as performed in humans. Not surprisingly, these mice also suffered from high rates of tumor recurrence, both distant and local. Of particular note is that splenic recurrence was common, suggesting either inadequate resection or possible peritoneal tumor seeding at implantation24.
Ni et al.16 described a distal pancreatectomy/splenectomy model performed with fluorescence imaging guidance. Disappointingly, despite the use of a clinically relevant operation (with fluorescence guidance), the survival was very short (mean survival of 18 days), even in the distal pancreatectomy group. This degree of progressive disease appears to be even worse than palliative treatment models25,26,27, suggesting the possible presence of gross residual disease after resection. Most recently, Giri et al.28 reported a distal pancreatectomy and partial splenectomy mouse model. This study is notable in that it represents an immunocompetent mouse model of cancer. However, this study reported almost universal local and other intraperitoneal tumor recurrence, possibly indicating occult iatrogenic metastasis at implantation.
The use of mouse models where there is gross residual disease post resection for testing adjuvant treatments may be inappropriate. The issue is that treatment for gross residual disease cannot truly be classified as adjuvant treatment but rather should be considered to be treatment with palliative intent. In that case, such mouse models offer no advantage compared to non-resectional models with low volume disease.
Tips and pitfalls of critical steps 
Tumor implantation procedure 
In order to replicate the clinical scenario, there are distinct challenges in this model which relate to the implantation and resection procedures. For the implantation procedure, the major challenges which need to be overcome are successful implantation and prevention of leakage. These two issues are interrelated as failure of injection would result in gross leakage of the tumor cell suspension into the abdominal cavity. This would produce a mouse model with peritoneal metastasis, which will progress regardless of pancreatic resection. This reflects the well-known clinical scenario in humans where pancreatic resection in metastatic PC does not affect the patient outcome. This is the basis of the staging laparoscopy in humans29.
The success of implantation of the tumor can be seen intraoperatively as the successful generation of a &#8220;bubble&#8221; of cell suspension without obvious leakage. Of most importance in achieving a good result is the accurate placement of the needle within the pancreatic parenchyma. This could only be achieved by &#8220;stretching out&#8221; the pancreas so that the peritoneal surface is taut. Puncture should occur with the needle bevel facing upwards (ventrally). Once the needle punctures the peritoneal surface, it should be advanced while the needle tip is slightly lifted-up so that the beveled surface glides just beneath the peritoneum. This will prevent inadvertent through-and-through puncture of the pancreas, a common pitfall due to the small dimensions of mouse pancreatic lobules. Once the entire bevel is within the substance of the pancreas, the cell suspension is injected. Magnification of vision with surgical loupes is highly desirable to visualize accurately the depth of the needle penetration.
A number of techniques can be used to further minimize the risk of inadvertent leakage. 
Selection of a large lobule for injection. Small lobules require higher pressures to inflate (following Laplace&#8217;s law), thereby increasing the risk of leakage around the needle at the puncture site. 
Optimization of the speed of injection. The use of an injection device (Figure 1b) which allows the cell suspension to be injected over 10-15 seconds serves three purposes. First, it decreases the rate of change of pressure in the pancreas, giving the tissues time to deform and reduces the risk of reflux of the suspension. Second, it allows the injection process to be monitored and, if necessary, stopped and needle repositioned. Any leakage can be mopped up by a povidone-iodine-soaked gauze. Third, it frees the operator from needing to depress the plunger, allowing the operator to focus on keeping the needle tip within the pancreas while the assistant injects the cell suspension. 
Use of a double-layered purse-string gauze. This gauze forms a collar around the pancreatic tail which will absorb any leakage of the cell suspension and therefore minimize contamination in the abdominal cavity.
Some studies in the literature have used an extracellular matrix mixture (Matrigel) which solidifies with time after injection13,15,24. This may reduce the risk of leakage post-injection. However, a potential disadvantage of this strategy is that Matrigel or other similar extracellular matrix solutions may exert non-physiological effects on PSCs30. For instance, Matrigel has been shown to render PSCs quiescent thereby potentially negating the effects of PSCs in the model31,32. An alternative to injection of cancer cells is the orthotopic implantation of tumor tissue (either directly from patients or from subcutaneous mouse models). However, these approaches have their own disadvantages. First, heterogeneity may arise from sampling error or from variations in the volume of tissue implanted. Such heterogeneity may reduce the power of subsequent treatment comparisons. Second, passaging of tumor tissue with a subcutaneous mouse model may lead to selection of sub-clones which have different biological behaviours to the original patient tumor.
Tumor resection procedure 
In this model, we have utilized a distal pancreatectomy/splenectomy procedure akin to that performed in humans. The challenges relating to the resectional surgery depend on pathological and anatomical factors.
The key pathological factor is tumor dissemination. Low volume local spread can be resected at the time of pancreatic resection, although it may indicate the possibility of more distant peritoneal and other metastasis. We routinely excise the suture line from the first operation as it is a possible area of local recurrence. If the tumor is attached to surrounding structures, such as the abdominal wall or the left lobe of the liver, these can be resected en bloc. Anatomically, the key step is dissecting the plane dorsal to the body of the pancreas. The splenic vein can often be visualized behind the pancreas once the pancreas is exteriorized. This is a key landmark, as the embryological bloodless plane is immediately dorsal to this.
There are two other potential anatomical pitfalls in the model described here. The colon may be adherent to the caudal aspect of the pancreatic body. Failure to mobilize this structure away could lead to inadvertent colonic injury at the time of pancreatic division or ligation. The gastrosplenic vessels are small and may easily bleed if avulsed or inadequately cauterized. Furthermore, once avulsed, the bleeding point often retracts deep into the abdomen behind the greater curve of the stomach, making subsequent control of bleeding more challenging. Therefore, careful retraction of the spleen and cautery of the gastrosplenic vessels are required. One approach for successful hemostasis is to cauterize these vessels on the hilar aspect of the spleen which minimizes the risk of inadvertent thermal injury to surrounding hollow viscera.
We have found that using a titanium ligation clip, widely used in human surgery for ligation of vessels, is a rapid and effective way of controlling the pancreatic stump, with consequent reduction in total operative time compared to the use of ligatures. This was also used by Giri et al.28.
Limitations of the technique 
There are limitations to this resectional model of the pancreas. One limitation relates to the time allowed to produce recurrence/metastasis. On the one hand, one needs to maximize the development of metastatic disease, but on the other hand, one needs to resect the tumor before it became locally advanced. The period between implantation and resection may therefore need to be adjusted for the particular clinical scenario one wishes to replicate. Another limitation relates to the inadvertent spillage and subsequent peritoneal metastasis of cancer cells which is discussed above.
A major challenge of adjuvant treatment models is dissecting the adjuvant treatment effect from the surgical treatment effect. Clearly, a well-designed study which is randomized, with a control group undergoing resection surgery is required. To further improve the assessment of the relative treatment effects, we suggest assessing tumor burden in vivo (for example, by using luciferase-tagged cancer cells and performing in vivo bioluminescence imaging). Despite the semi-quantitative nature of this assessment in orthotopic models (as the bioluminescence signal is attenuated by passage through the overlying tissues), this approach allows longitudinal assessment of tumor burden, including the assessment of the post-surgical residual disease.
Modifications and future applications 
The implanted cell line and/or cell numbers with or without pancreatic stellate cells could be modified to reflect the target clinical scenario12. The duration between implantation and resection could also be modified to change the risk of metastasis formation. Other variations could include implantation of patient- or mice-derived xenografts or organoids33.
Neoadjuvant therapy can also be tested within the basic features of the model described here. It would simply require commencement of drug treatment prior to surgical resection34. Similarly, both neoadjuvant and adjuvant therapy could be studied in the same mice.
Finally, while we have described the use of athymic Balb/c nude mice which represents an immunodeficient model, an alternative immunocompetent model may involve KPC tumor cells implanted into C57B6 mice28. This may be a useful alternative for the testing of adjuvant/neoadjuvant immune therapies.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario and does not require specialized equipment. This model may be useful for the testing of both adjuvant and neoadjuvant treatments.

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>&lt;strong&gt;Animals, Materials and Equipment for Implantation Procedure&lt;/strong&gt;</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&#039;s modified Dulbecco&#039;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 &amp;mu;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) &amp;times; 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>&lt;strong&gt;Equipment for Resection Procedure&lt;/strong&gt;</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 &amp;ldquo;Weck style&amp;rdquo; 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>&lt;strong&gt;Drugs for Procedures&lt;/strong&gt;</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 &amp;mu;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/splenectomy with no issues of haemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice.
At the time of resection, local metastasis was found in 9/45 (20%) mice &#8211; mostly in the suture line (discontinuous with the primary tumor) with three of the nine showing additional isolated nodules on the greater curve of the stomach and one showing a subcapsular nodule on the liver. The primary pancreatic tumor was adherent to the suture line in five (11%) mice and to the liver in one (2%) mouse. These adherent structures were excised en bloc.
The mean (SEM) surgery time (induction to closure) was 22 (0.9) minutes. None of the animals died within 1 week after resection.
One-week post resection, mice underwent bioluminescence imaging to detect residual disease. The ratio of the maximum radiance over the ventral surface of the mouse was compared to that of the background. Thirty-two (71%) mice had a maximum radiance ratio (mouse:background) of &#60;10, indicating minimal or no residual disease.
<img alt="Figure 1B" class="xfigimg" src="/files/ftp_upload/61726/61726fig1v2.jpg" /> 
Figure 1: Custom-made devices to facilitate tumor implantation.&#160;(a) Purse-string gauze swab: (i) Central hole, approximately 1 cm in diameter, through which the pancreatic tail will be placed at the time of injection; (ii) Purse-string suture around the hole; (iii) Double-layered gauze; (iv) Single throw knot; (v) One limb of the suture material is secured to the gauze with sterilising indicator tape; (vi) A handle, made from indicator tape, is fashioned on the other end of the suture material. (b) Injection device: (I) Actuating syringe. Slots cut through the body of this syringe allows the injection syringe (with the cell suspension injectate; not shown) to be mounted on this syringe body; (II) Controller syringe. This is filled with water. Depression of the plunger on the smaller controller syringe by the surgical assistant causes displacement of the larger actuating syringe plunger. The displacement of the actuating plunger is smaller, but with a mechanical advantage which allows the injection to overcome the resistance associated with the injection syringe mechanism as well as the tissue&#8217;s resistance to expansion by the injectate. This allows for precise and smooth injection of 50 &#956;L over 10&#8211;15 seconds; (III) Polytetrafluoroethylene (PTFE) connection tubing with internal diameter of 0.5 mm. <a href="https://www.jove.com/files/ftp_upload/61726/61726fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.2K 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 ...

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 ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

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 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Medicine

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 ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice

Source: Chai, M. G., et al. <a href="https://www.jove.com/v/50395/" target="_blank">Bioluminescent Orthotopic Model of Pancreatic Cancer Progression</a>.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and&#160;in vivo&#160;bioluminescence imaging for non-invasive monitoring of tumor progression. The orthotopic model is suited to both syngeneic and xenograft models and could be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Source: Chai, M. G., et al. Bioluminescent Orthotopic Model of Pancreatic Cancer Progression.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Summary

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Chapters in this video

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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

Orthotopic Mouse Model of Colorectal Cancer

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

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

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice