We would like to thank the TUM animal facility and the imaging core facility of the Department of Nuclear Medicine, Klinikum rechts der Isar, for excellent technical support. This study was supported by the German Cancer Consortium (DKTK), Deutsche Forschungsgemeinschaft (DFG SA 1374/4-2, DFG SA 1374/6-1, SFB 1321 Project-ID 329628492 P06, P11 and S01) to D.S., the Wilhelm Sander-Stiftung (2020.174.1 and 2017.091.2) to D.S., and the European Research Council (ERC CoG No. 648521, to D.S.).

The animal experiments were approved by the institutional animal care and use committees (IACUCs) of the local authorities of Technical University of Munich and Regierung von Oberbayern.
1. Information to consider prior to the procedure
<ol>
	<li>Ensure approval of the animal protocol and personnel by the local authorities before starting any animal experimentation.</li>
	<li>Select recipient mice.
	<ol>
		<li>Select mice of similar ages for implantation (C57Bl/6J recipient mice with an age range between 2 months and 4 months).</li>
		<li>Ensure that the sex and genetic background of the recipient animals match the sex and genetic background of the animals from which the cell line used for implantation originated (syngeneic allografts).</li>
	</ol>
	</li>
	<li>Prepare the mice for implantation. 
	NOTE: Handle the mice according to the hygienic standards of the local animal facility.
	<ol>
		<li>On the day of implantation, do not feed the mice 4 h before the procedure to avoid complications due to the administration of anesthetics.</li>
		<li>Weigh the mice prior to implantation to calculate the amount of the necessary anesthetics.</li>
		<li>Transfer the mice to the operation room.</li>
	</ol>
	</li>
	<li>Select the PDAC cell lines.
	<ol>
		<li>Make sure that the genetic background and sex of the mice from which the cell lines are derived match the background and sex of the recipient mice (e.g., the PDAC cell lines PDAC 1-3 previously generated from PDAC GEMMs on a C57Bl/6J background in the laboratory with the following genotypes, as described previously14: PDAC 1 and 2: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/+, PDAC 3: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/R172H).</li>
		<li>Select the cell lines that do not express proteins that are potentially immunogenic.</li>
	</ol>
	</li>
	<li>Culture the PDAC cell lines. 
	NOTE: Perform the cell culture work under a laminar flow hood. Disinfect the gloves, working space, and materials before working.
	<ol>
		<li>Thaw the cells 1 week before implantation by resuspending the pellet in 5 mL of cell culture medium. Spin down the cells, aspirate the supernatant, resuspend the pellet in 5 mL of phosphate-buffered saline (PBS), and spin down the cells again. Remove the supernatant and resuspend the cells in 5 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with 10% fetal bovine serum (FBS) and 0.1% penicillin-streptomycin (PS). Transfer the cell suspension to a 25 cm&#178;&#160;flask.</li>
		<li>Passage the cell lines at least once before implantation.
		<ol>
			<li>Remove the cell medium, wash 1x with PBS, and add 0.5 mL of trypsin to detach the cells from the flask. Incubate the cells at 37 &#176;C until they detach from the flask (depending on the cell line, this usually takes 5-10 min). When the cells are detached, resuspend the cells in 10 mL of DMEM with 10% FBS and 0.1% PS and transfer the cell suspension to a 75 cm&#178; flask. 
			NOTE: As FBS inactivates trypsin, it would hinder trypsinization.</li>
		</ol>
		</li>
		<li>Ensure confluency of ~70%-80% on the day of implantation.</li>
	</ol>
	</li>
	<li>Material needed for implantation
	<ol>
		<li>Autoclave all the surgical instruments.</li>
		<li>Keep anesthetics ready based on the animal experiment license.
		<ol>
			<li>Use meloxicam for analgesia at least 5 min in advance as a long-lasting analgetic with anti-inflammatory properties.</li>
			<li>For surgery, use a combination of midazolam, medetomidine, and fentanyl (MMF) for analgosedation.</li>
			<li>Use a combination of atipamezole, flumazenil, and naloxone (AFN) for antagonizing the MMF analgosedation after implantation.</li>
		</ol>
		</li>
		<li>Prepare a sterile drape, a heating mat, a shaver, gloves, eye cream, 0.9% sodium chloride, iodine or chlorhexidine-based surgical scrub, 80% ethanol, soluble suture, wound clips, a 50 &#181;L injection volume syringe, and tape.</li>
	</ol>
	</li>
</ol>
2. Preparing the cell lines before implantation
NOTE: Prepare the tumor cells only when the mice are ready for implantation. Ensure a short time frame between the harvesting or collection of cells and implantation.
<ol>
	<li>Warm all the liquids to 37 &#176;C before use.</li>
	<li>Prepare the cells (starting from 1x T75 (a 75 cm2 culture flask) as described in step 1.5.2.1 until they detach from the flask. Resuspend the cells in 10 mL of DMEM with 10% FBS and 0.1% PS and transfer the cell suspension to a 15 mL tube.</li>
	<li>Spin down the cells at 200 &#215; g for 5 min in a centrifuge. Aspirate the supernatant and resuspend the cell pellet in an adequate volume of DMEM without additives based on the required final concentration of the cells for injection. Use 10 &#181;L of the cell suspension to count the cells using a Neubauer chamber and calculate the number of available cells.</li>
	<li>Dilute the cells to the final required number of cells for injection (e.g., 2,500 cells in 20 &#181;L) in DMEM without additives. Transfer 1 mL of the diluted cell suspension to a 1.5 mL tube. Place the tube on a rotator until implantation to prevent the cells from aggregating.</li>
</ol>
3. Orthotopic implantation of PDAC cells
NOTE: In case of insufficient surgical experience, practice with cadavers first and get adequate training, for example, within an animal training protocol. The personnel performing the surgery and the animal experiments need to fulfill the criteria of the respective authorities and the institutional guidelines.
<ol>
	<li>Use meloxicam as a perioperative analgesic and apply 5 mg/kg body weight subcutaneously.</li>
	<li>Intraperitoneally inject MMF according to the body weight of each mouse (midazolam [5.0 mg/kg], medetomidin [0.5 mg/kg], and fentanyl [0.05 mg/kg]).</li>
	<li>Put the mouse back into the cage for 10-15 min.</li>
	<li>Apply eye cream when the mouse is asleep.
	<ol>
		<li>Check sufficient analgosedation after 10 min by testing the pedal withdrawal reflex (pinch on the foot pads of both hind feet). In case of a response, apply 1/3 of the original dose of analgosedation. After 10 min, repeat the procedure and proceed only in the absence of a pedal withdrawal reflex.</li>
	</ol>
	</li>
	<li>Shave the left lateral abdominal flank of the mouse. Place the mouse lying on the right side on a sterile heating mat and tape its legs to the surface. Disinfect the abdomen using an iodine or chlorhexidine-based surgical scrub followed by 80% ethanol in a circular motion and repeat the procedure three times. To maintain sterility of the surgical field, use a surgical drape.</li>
	<li>Wear new gloves and disinfect them. Use surgical scissors to cut the skin and subcutaneous fat tissue longitudinally on the left flank where the spleen is projected. Perform a cut of about 1 cm in length.</li>
	<li>Using scissors and forceps, separate the skin from the peritoneum to get an easier access to the peritoneum.</li>
	<li>Change the pair of scissors. Open the peritoneum at the location of the spleen with a 1 cm longitudinal cut.</li>
	<li>Mobilize the spleen and the attached pancreas using blunt, fine-tipped forceps. Make sure the organs are not attached to other tissue and check for supplying blood vessels to avoid injuring the vessels or the surrounding tissue when handling the pancreas. 
	NOTE: Do not grab the spleen directly to prevent bleeding.</li>
	<li>Carefully pull the pancreas out of the incision to enable easier access to the organ&#39;s tail and observe the spleen following the pancreas. Use the non-dominant hand to grab the pancreas with forceps and carefully cut the ligaments that prevent the pancreas from extracorporalization. Make sure the organ is not folded during injection and that the organ&#39;s capsule is held under tension at the site of needle penetration to avoid spillage.</li>
	<li>Use the dominant hand to carry out the injection. Carefully penetrate the organ&#39;s capsule with the syringe&#39;s needle following the longitudinal axis of the organ. Aim for a piece of pancreatic tissue between visible blood vessels to minimize the risk of puncturing or injecting into a vessel. Slowly inject an adequate number of cells according to the experimental plan (here, 2,500 cells in 20 &#181;L of DMEM) with a 27 G cannula and a 50 &#181;L syringe into the tail area of the pancreas. 
	NOTE: A transparent bubble forms upon successful injection into the tissue.</li>
	<li>Keep the pancreas and the inserted syringe still for approximately 1 min to avoid spilling of the tumor cells. Remove the needle carefully from the injection site. 
	NOTE: Change needles between mice and use a new sterile syringe for the injection of a new cell line.</li>
	<li>Rearrange the organs inside the abdomen carefully. Take care not to injure the tissue to avoid spilling of the cells. Pour 1 mL of 0.9% sodium chloride onto the organs to avoid organ adhesion.</li>
	<li>Carefully suture the peritoneum using the simple interrupted suture technique and close the skin using two to three 9 mm stainless steel wound clips. 
	NOTE: Remove the wound clips 7 days after implantation if the surgical incision has healed.</li>
	<li>If necessary, prevent dehydration of the animals due to the procedure and the anesthesia by injecting 0.5 mL of sodium chloride subcutaneously.</li>
	<li>After implantation, antagonize the analgosedation by MMF with a subcutaneous injection of AFN (atipamezol [2.5 mg/kg], flumazenil [0.5 mg/kg], naloxon [1.2 mg/kg]), according to the body weight of the mouse.</li>
	<li>Place the mouse in a 37 &#176;C heated chamber until it is awake and completely active. Then, place the mouse back into its original cage. Monitor the health status of the mouse by checking the fur, skin color, and activity and repeat this 3-4 h after surgery.</li>
</ol>
4. Aftercare for the mice
<ol>
	<li>Give the mice access to water and food when back in the animal facility.</li>
	<li>Continue subcutaneous or oral application of meloxicam (5 mg/kg) every 6-12 h for 2-3 days after surgery.</li>
	<li>Monitor the health status of the mice regularly according to the animal protocol and the institutional guidelines.</li>
	<li>Depending on the goal of the experiment, monitor tumor growth every week or more frequently by palpating, ultrasound imaging, bioluminescence (BLI), or magnetic resonance imaging (MRI). 
	&#8203;NOTE: Based on the method of choice, the growth rate, and the number of cells implanted, a tumor can be detected as early as 1 week after the injection.</li>
</ol>
5. Harvesting tumor grafts
<ol>
	<li>Harvest the tumors at the end of the experiment or when the mice need to be euthanized following the criteria of the institutional animal care and use committees or animal experiment license.</li>
	<li>Euthanize the mouse using a method permitted by the animal experiment license. 
	NOTE: Carry out steps with a minimal time loss to minimize the ischemia time. Therefore, cervical dislocation is recommended as it is a quick method and the time of death can be determined exactly, in contrast to terminal bleeding or chemical methods.</li>
	<li>Place the mouse supine on a sterile mat and tape its legs to the surface. Use 80% ethanol to disinfect the abdomen.</li>
	<li>Wear new gloves and disinfect them. Use surgical scissors to cut the skin longitudinally on the central abdomen.</li>
	<li>Using scissors and forceps, separate the skin from the peritoneum to get an easier access to the peritoneum.</li>
	<li>Change the pair of scissors. Open the peritoneum at the central abdomen with a 5 cm longitudinal cut.</li>
	<li>Mobilize the spleen and the attached pancreas using blunt, fine-tipped forceps. Carefully detach the tumor from the surrounding tissue using forceps and surgical scissors.</li>
	<li>Store the tumor tissue in a medium suitable for downstream experiments. Process the tissues immediately or, if necessary, keep the sample at 4 &#176;C for a maximum of 6 h until further processing.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Syngeneic mouse orthotopic allografts represent a robust model for preclinical studies due to their cost-effectiveness, reproducibility, and relatively simple experimental procedures13,15. These models not only allow the study of tumor-host interactions but also guarantee the preservation of the genetic heterogeneity of the tumors they originate from when primary mouse cell cultures are used for the experiment.
This protocol presents a...

Recently, PDAC became the third leading cause of cancer-related deaths in the western world1. It causes the highest death rate among all cancers and a 10 year overall survival rate of ~1%, which has not changed for decades2. Due to the lack of progress in PDAC treatment, this disease is expected to become the second leading cause of cancer-related deaths by the next decade3.
PDAC tumors are complex entities characterized by a diverse tumor microenvironment (TME) composed of a heterogeneous assembly of stroma, vascular, immune, and extracellular matrix components4. Differences in the composition of the TME influence disease prognosis and response to therapy4,5,6. Indeed, many studies have shown that the basal-like, mesenchymal subtype of PDAC is associated with a highly immunosuppressive TME and shows decreased survival and lack of response to therapies7,8,9,10,11,12. Therefore, a deeper understanding of the differences in TME composition and how these features influence tumor biology remains an important factor for the development of molecularly precise therapies. To better understand the biology behind this complex phenotype and identify therapeutic strategies able to overcome the barrier that the TME of PDAC constitutes, in vivo models are indispensable.
A key aspect for any cancer preclinical model system is that it should mimic human phenotypes, recapitulating both the genetic heterogeneity and the milieu incorporating the multitude of stromal and immune populations that make up the TME. Therefore, when choosing mouse models for preclinical research, several aspects must be taken into consideration. To investigate the tumor-immune interaction, histocompatible cancer cell lines can be injected into syngeneic immunocompetent mice. In most cases, these are subcutaneously injected into the flank of the mouse, allowing easy tumor monitoring by palpation or visual inspection. However, the resulting models do not mimic the growth of tumor cells in their organ of origin. Therefore, orthotopic transplantations became the gold standard for allograft models.
Mouse orthotopic allografts have several advantages: they are cost-effective, can be generated with a relatively simple procedure, and result in models with known molecular makeup, as well as a reproducible and predictable tumor progression and phenotype. Indeed, while patient-derived xenograft models represent the behavior of human PDAC cells accurately, the need for implantation into immunodeficient mice to avoid graft rejection limits the analysis of the tumor-immune and tumor-stroma interactions, allowing researchers to capture only a partial image of the complexity of these tumors. Syngeneic orthotopic allografts of PDAC hold an advantage in this regard also in comparison to genetically engineered mouse models (GEMMs). GEMMs accurately recapitulate human PDAC tumorigenesis and the heterogeneity observed in PDAC patients. However, because of these features, GEMM tumors can show high variance in their genetic makeup, tumor progression, aggressiveness, histological differentiation, and TME composition. While this can be an advantage in certain studies, it limits genotype-to-phenotype studies and the focused investigation of PDAC phenotypes13. Therefore, mouse orthotopic allografts constitute a good tradeoff and model to perform tumor-host and treatment studies in vivo. This paper outlines a protocol for orthotopic transplantation experiments of murine PDAC cells into the mouse pancreas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>27 G cannula</td>
			<td>B.Braun</td>
			<td>08915992</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezole (Antisedan 5 mg/mL)</td>
			<td>Orion Corporation</td>
			<td>23554.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip Stainless Steel Wound Clips, 9 mm</td>
			<td>Braintree Scientific</td>
			<td>NC9334081</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco`s Modified Eagle Medium&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D5796-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye cream (Bepanthen)</td>
			<td>Bayer Vital GmbH</td>
			<td>1578675</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl (50 &#181;g/mL)</td>
			<td>Eurovet Animal Health BV</td>
			<td>9113473</td>
			<td></td>
		</tr>
		<tr>
			<td>Flumazenile (Flumazenil-hameln 0.1 mg/mL)</td>
			<td>Hameln pharma</td>
			<td>09611975</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine (Sedator 1 mg/mL)</td>
			<td>Eurovet Animal Health BV</td>
			<td>400926.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam 5 mg/mL)</td>
			<td>Boehringer Ingelheim Vetmedica GmbH</td>
			<td>3937902</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringe</td>
			<td>Hamilton</td>
			<td>HT80908</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam (5 mg/mL)</td>
			<td>Hexal</td>
			<td>00886423</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>B. Braun</td>
			<td>2737756</td>
			<td></td>
		</tr>
		<tr>
			<td>Naloxone (Naloxon-hameln 0.4 mg/mL)</td>
			<td>hameln pharma</td>
			<td>04464535</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P7059-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (Ethilon)</td>
			<td>Ethicon</td>
			<td>9999034</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypZean Solution 1x</td>
			<td>Sigma-Aldrich</td>
			<td>T3449</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In the context of a large-scale drug-response study, we successfully implanted more than 170 mice (C75Bl6/J recipient mice, male and female mice sex matched to the PDAC cell lines injected) using the above-described protocol, exemplified in its main steps in Figure 112. In this protocol, we orthotopically implanted three KrasG12D-driven PDAC cell lines (PDAC 1 and 2: Ptf1aCre/+;LSL-KrasG12D/+;LSL-Trp53R172H/+</e...

Watch this Scientific Journal Video about Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer at JoVE.com

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

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, ...

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Research

JoVE Journal

Cancer Research

2.4K Views.  German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK). 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. 

Video: Syngeneic Mouse Orthotopic Allografts to Model 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 Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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 ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

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

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

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 ...