Name: a syngeneic pancreatic cancer mouse model to study the effects of irreversible electroporation
Uploaded: 2018-06-08T12:30:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation at JoVE.com

RRW has received support for this work from a Collaborative Translational Research Grant funded by the San Diego C3 Padres Pedal the Cause (#PTC2017).

All animal experiments performed following this protocol must be approved by the respective Institutional Animal Care and Use Committee (IACUC). All procedures described here have been approved by IACUC UCSD.
1. Procure Recipient Animals
NOTE: The KPC-Luc 4580 cell line was established from a tumor arising in a LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse (KPC), which is a genetically engineered model of PC on a C57BL/6 background. The advantage of this cell line is that it constitutively expresses luciferase-enabling tumor monitoring in orthotopic models. However, other cell lines may also be used as long as they are compatible with the genetic background of the recipient mice.
<ol>
	<li>Determine the experimental groups and the number of animals per group in advance.</li>
	<li>Choose the age (6 - 10-week old mice recommended) and sex of the animals being used according to the hypothesis and the cell line used.</li>
</ol>
2. Culture Cells
<ol>
	<li>Use Dulbecco&#39;s modified eagle medium (DMEM): nutrient mixture F12 (50:50) media containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic (Complete growth media) to grow KPC-Luc 4580 cells.</li>
	<li>If frozen, thaw the cells 4 days prior to tumor implantation.</li>
	<li>Grow the cells at 37 &#176;C with 5% CO2 and 95% humidity under aseptic conditions.</li>
	<li>Trypsinize the cells with 0.25% Trypsin for 5 min at 37 &#176;C before they reach &#62;80% confluency and neutralize the trypsin by adding twice the volume of complete growth media. 
	NOTE: These cells do not establish tumors efficiently in mice if they are used at 100% confluency or if they are passaged for more than 12 times. Hence, it is advisable to freeze the cells at earlier passages under optimal confluency.</li>
	<li>Count the cells using a hemocytometer and proceed to tumor induction if adequate numbers are present (0.5 - 1 x 106 cells/mouse). If not, expand the cells for one more passage until adequate cell numbers are obtained.</li>
</ol>
3. Induction of SQ Tumors
<ol>
	<li>Prepare the materials required: P1000 and P100 pipettes and tips, 1-mL syringe, 25G needles, basement membrane matrix (BMM), DMEM basal media, 1.5-mL centrifuge tubes. Keep all materials sterile and cold (4 &#176;C) until the end of the procedure. Also keep hair clippers and alcohol swabs ready for animal use.</li>
	<li>Trypsinize (as per step 2.4) and count the cells, and resuspend them at a concentration of 2 - 4 x 107 cells/mL of cold DMEM.</li>
	<li>Add an equal volume of 100% BMM to make a final cell suspension in 50% BMM. The final volume should amount to 50 &#181;L of cell suspension for each mouse required. Prepare 10% in excess to accommodate the dead space in the syringe and for any pipetting errors.</li>
	<li>Aliquot the suspension in 550 &#181;L volumes in 1.5 mL centrifuge tubes placed in ice.</li>
	<li>Restrain the animals (6-week old male C57BL/6 mice) using a restrainer or manually. 
	NOTE: Alternatively, mice can be anaesthetized with 2.5% isoflurane in oxygen gas at a flow rate of 2 L/min using a precision vaporizer.</li>
	<li>Shave the site of injection, which is preferably on a flank above a leg, using a clipper. Using an alcohol swab, clean the site of injection twice.</li>
	<li>Draw 250 &#181;L of cell suspension at a final concentration of 1 - 2 x 107 cells/mL in a 1-mL cold syringe and remove any air bubbles by moving the piston up and down. Attach a 25G needle to the syringe and make sure to fill the space within the needle.</li>
	<li>Carefully insert the needle under the skin at a 30&#176; angle to the body near the flanks, making sure that the needle does not pierce the peritoneal cavity or the musculature of the limbs.</li>
	<li>Flatten the needle parallel to the body, and flatten wrinkles on the skin. Slowly inject 50 &#181;L of cell suspension. Hold the needle at the site of injection for 10 s to allow the BMM to solidify to prevent any leakage.</li>
	<li>Slowly withdraw the needle keeping it parallel without any sideways movement. Gently clean the site of injection with an alcohol swab and release the mouse to its housing. 
	NOTE: If the mouse was anesthetized during the procedure, monitor the animal until it regains its righting reflexes.</li>
	<li>Monitor the growth of the tumor regularly using calipers until it reaches 5 mm in diameter.</li>
</ol>
4. Induction of Orthotopic Tumors
<ol>
	<li>Prepare the materials required. Autoclave the surgical tools such as scissors, scalpels, needle drivers, and pointed and flat-tipped forceps. Keep handy sterile sutures (absorbable 6-0 and non-absorbable 4-0), alcohol swabs, hair clippers, depilatory cream, eye lubricant, cotton gauze, 10% povidone iodine solution, surgical drapes, heating pads, buprenorphine, and anesthetic agents.</li>
	<li>Euthanize a donor mouse (preferably of the same background required for the orthotopic model) carrying a SQ KPC tumor using a slow fill CO2 chamber. Excise the SQ tumor carefully under sterile conditions in a BSL-2 hood using sterile forceps and microscissors shortly before orthotopic implantation. During tumor excision take care not to puncture the peritoneal cavity and wash the tumor immediately with 2 exchanges of sterile PBS.</li>
	<li>Using sterile scalpels, mince the excised tumor into 1 mm3 pieces, place them on a Petri dish containing cold sterile DMEM basal media, and store on ice until implantation.</li>
	<li>Administer the recipient mouse 0.05 - 1 mg/kg buprenorphine analgesic SQ, 30 min prior to surgery.</li>
	<li>Anesthetize the recipient mice with 2 - 3% isoflurane in oxygen (2 L/min) using a precision vaporizer (or other anesthetic agents). Keep mice on a 37 &#176;C heating pad for the entirety of the procedure. Apply lubricant to the eyes to prevent desiccation. Test the depth of anesthesia by lack of startling reflex initially, and confirm surgical plane anesthesia by the lack of pedal reflex to a gentle toe pinch. Maintain the anesthesia during the entire surgical procedure.</li>
	<li>Place the mouse on its back and gently turn it to its right side so that the left side of the abdomen is exposed. Remove the abdominal hair of the mouse using depilatory cream and clean with gauze to ensure no free hair enters the abdomen post incision. Prepare the left abdomen for surgery using 3 cycles of 10% povidone iodine followed by alcohol wipes to disinfect the skin.</li>
	<li>Using a sterile scalpel, make a 1.5-cm transverse or oblique incision in the skin, 1-cm to the left of the midline, below the ribcage, slightly medial to the spleen. Then, extend the incision through the abdominal musculature, mirroring the overlying superficial incision.</li>
	<li>Locate the spleen using flat tipped forceps and gently externalize it from the abdominal cavity. Retract the spleen using a sterile cotton applicator, and find the tail of the pancreas attached to the bottom of the spleen.</li>
	<li>Using flat-tipped forceps, retract the tail of the pancreas laterally.</li>
	<li>Pick up one finely minced tumor piece from the donor mouse with the tip of a fine (6-0) sterile suturing needle and position it using sterile forceps.</li>
	<li>While gently retracting the tail/body of the pancreas laterally, insert the needle of the suture containing tumor fragment into the tail of the pancreas. Pass the suture slowly through the tissue, holding the tumor in contact with the pancreatic tail. Apply one or two additional sutures depending on the fragment&#39;s orientation in relation to the tissue.</li>
	<li>Remove the needle completely from the tissue and keep the pancreas/spleen externalized for an additional 60 s while inspecting for any signs of hemorrhage or leak. Then, internalize them gently into the abdominal cavity using blunt forceps.</li>
	<li>Close the abdominal musculature using a 6-0 absorbable suture with either a continuous or interrupted stitch, and close the overlying skin using a 3-0 to 6-0 non-absorbable interrupted suture. Discontinue the anesthesia at this point.</li>
	<li>Allow the mouse to recover in its home cage with free access to food and water. Place the cage on a heating pad to facilitate recovery. Monitor vital signs such as breathing, perfusion etc., during the recovery process.</li>
	<li>Confirm the signs of righting reflex once the mouse recovers, and then return the cage can to regular housing.</li>
	<li>Administer buprenorphine 0.05 - 0.1 mg/kg 8 - 12 h after the surgery, and every 8 - 12 h after the procedure as needed for signs of pain.</li>
	<li>Monitor tumor growth using an in vivo bioluminescence imaging system (see the Table of Materials).
	<ol>
		<li>Follow tumor growth by imaging starting on day 4 after orthotopic implantation, and perform imaging twice a week.</li>
		<li>On the day of imaging, administer (intraperitoneal injection) 30 mg/kg D-luciferin to an anesthetized (2 - 3% isoflurane in oxygen (2 L/min)) recipient mouse 10 min prior to imaging.</li>
		<li>Image for luciferase activity using the luminescence setting without any emission filters for a minimum of 5 s exposure in a luminescence imager with auto-fluorescence free heated stage and while still maintaining the anesthesia for the mice.</li>
	</ol>
	</li>
</ol>
5. IRE of SQ Tumors
<ol>
	<li>Prepare the materials required: square wave electroporator, safety foot switch, electrodes (needle array versus tweezer-trode), and adaptors. Keep sterile sutures (non-absorbable 4-0), alcohol swabs, hair clippers, depilatory cream, eye lubricant, cotton gauze, buprenorphine, and anesthetics also nearby.</li>
	<li>Sterilize the tweezer or needle array electrodes using gas sterilization. Autoclaving is not recommended. Use a glass bead sterilizer to sterilize the electrodes between animals.</li>
	<li>Administer buprenorphine 0.05 - 0.1 mg/Kg to the tumor-bearing mice 30 min prior to the procedure.</li>
	<li>Follow steps 4.4 - 4.5 to successfully induce anesthesia in mice once the SQ tumor implant reaches 5 mm diameter.</li>
	<li>Place the anesthetized mouse on its side to access the SQ tumor on the flank. Remove the hair using clippers and clean skin using an alcohol swab.</li>
	<li>For SQ tumors, use 2-needle array electrodes. Elevate the skin directly under the tumor site using forceps and insert the electrodes through the skin parallel to the body making sure they do not penetrate the peritoneal cavity. Once through the skin, position the electrodes in such a way that they bracket the tumor.</li>
	<li>Program the electroporator to deliver 100 &#181;s pulses at a frequency of 1 Hz at 1,500 V/cm for a total of 150 pulses. Deliver the pulses using the foot pedal. Separate each set of 10 pulses by 10 s, in order to allow for heat dissipation and confirm the correct position of the electrodes. 
	NOTE: Without the use of paralytic agents, mice will experience muscle contractions with IRE that can cause displacement of the electrodes unless manually secured.</li>
	<li>Remove the electrodes after complete dosing, which should not exceed 200 s. Record the actual voltage delivered, which is displayed on the electroporator. Allow the mice to recover following IRE as per steps 4.14 - 4.16.</li>
</ol>
6. IRE of Orthotopic Tumors
NOTE: IRE of orthotopic tumors involves a second survival surgery on the same mouse thus requiring special approval from local IACUC before beginning.
<ol>
	<li>Prepare required materials: autoclave the surgical tools such as scissors, scalpels, needle drivers, pointed forceps, and flat tipped forceps. Keep the following nearby as well: sterile sutures (absorbable 6-0 and non-absorbable 4-0), alcohol swabs, hair clippers, depilatory cream, eye lubricant, cotton gauze, 10% povidone iodine solution, surgical drapes, heating pads, square wave electroporator, safety foot switch, electrodes and their adaptors, buprenorphine, and anesthetics.</li>
	<li>Assess the mice for orthotopic tumor growth by in vivo imaging (see step 4.17) by injecting 30 mg/kg of D-Luciferin intraperitoneally. 
	NOTE: The orthotopic tumors are ideal for IRE treatment 8 to 10 days post implantation when the tumors are clearly visible and still confined to the pancreas as can be seen on the Luciferase luminescence images (see the Representative Results).</li>
	<li>Follow steps 4.4 - 4.9, to locate the implanted tumor. If the tumor is not easy to locate, use blunt nosed forceps to move the stomach and spleen gently to identify the tumor.</li>
	<li>Externalize the tumor with blunt nosed forceps and capture the tumor tightly with the platinum electrodes of the tweezer-trode. Deliver the electroporation pulses as programmed in step 5.7 using the square wave electroporator in sets of 10 pulses controlled by the foot switch.</li>
	<li>Keep the tumor externalized for at least 60 s post IRE to monitor for any signs of hemorrhage. Insert the tumor back into the abdominal cavity and close the incision as described earlier (steps 4.13 - 4.16) and monitor the mouse until it recovers.</li>
	<li>Monitor the effects of IRE on tumor growth using in vivo luciferase imaging as per step 4.17.</li>
</ol>

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The authors have no relevant financial disclosures.

In this study, we have demonstrated an immunocompetent mouse model for PC that can be used to study the effects of IRE on the tumor growth. Currently, IRE is being used as a non-thermal ablation technique only in highly selected locally advanced PC patients who do not have distant disease progression after months of preoperative therapy. Its use has therefore been limited because most patients with locally advanced PC develop distant metastatic disease20. This model will serve as a basis for studi...

Pancreatic ductal adenocarcinoma (PC) is projected to become the second leading cause of cancer deaths in the US around 20201. The vast majority of patients diagnosed with PC will eventually die from distant metastatic disease2. The PC microenvironment is notoriously immunosuppressive and chemoresistant. Its desmoplastic stroma contains a scarcity of effector (anti-tumor) T cells and a prominence of immunosuppressive leukocytes, including tumor-associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs), and regulatory T cells (Tregs)3. These underlie the need to develop multimodal strategies that counteract these effects of the microenvironment.
IRE has been developed as a non-thermal method of tumor ablation. Unlike thermal ablation techniques, IRE does not cause rapid coagulative necrosis but instead results in gradual apoptotic cell death4. Importantly for pancreatic tumors, IRE is not vulnerable to "heat sink" effects and can be performed right next to blood vessels5. This technology has 510(k) clearance from the FDA6 and is currently being used clinically, for selected patients with locally advanced or borderline resectable pancreatic cancer. In the largest published series of IRE for PC7, the median survival of patients undergoing IRE was approximately double the survival of patients treated with modern chemotherapy alone without resection8,9. 
Several studies have demonstrated that thermal ablation induces a systemic immune response in other tumor types (reviewed in Chu et al.10). Radiofrequency ablation (RFA) in animal tumor models leads to increased T cell infiltrates11,12, including an increase in activated natural killer (NK) cells in hepatocellular cancer patients13,14, and a decrease in immunosuppressive Tregs in lung cancer patients15. A much smaller number of studies have examined immune, microenvironmental, and injury responses to IRE16. IRE has been shown to stimulate a systemic immune response in immunocompetent mouse models in which the growth of secondary (contralateral) renal cell allografts was reduced or prevented by IRE of a primary tumor two weeks earlier17. They also observed that immunocompetent mice required less voltage for complete regression than did immunocompromised mice. It has been hypothesized that IRE may result in improved antigen presentation compared to the coagulative necrosis of thermal ablation, but this has not been specifically studied.
We have developed a syngeneic mouse model of PC from the KPC-Luc 4580 cell line (gift from J.J. Yeh at University of North Carolina), which was derived from a tumor that developed in a male LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse, to study the local and systemic effects of IRE18,19. This luciferase-expressing cell line is immunogenic and also tumorigenic in immunocompetent C57BL/6 mice when injected SQ or orthotopically and reliably produces liver metastases when injected into the spleen. We have utilized a programmable square wave pulse generator to deliver 100-&#181;s pulses of electricity at a voltage/distance ratio of 1,500 V/cm using a two-needle array probe (separated by 5 mm) or platinum tweezer-trodes to SQ or orthotopic tumors, respectively, in mice to model the effects of IRE in a small animal.

<table><tbody><tr><td>ECM 830 square wave electroporator</td><td>Harvard Apparatus</td><td>BTX # 45-0002 ( 58018-004 )</td><td></td></tr><tr><td>2 needle array electrode</td><td>Harvard Apparatus</td><td>45-0167</td><td></td></tr><tr><td>Safety foot switch</td><td>Harvard Apparatus</td><td>45-0211</td><td></td></tr><tr><td>Platinum Tweezer-trode</td><td>Harvard Apparatus</td><td>&amp;nbsp;45-0486</td><td></td></tr><tr><td>DMEM-F12 media</td><td>ThermoFisher Scientific</td><td>11320-033</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher Scientific</td><td>10437028</td><td></td></tr><tr><td>Trypsin</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Matrigel</td><td>Corning&amp;nbsp;</td><td>354230</td><td></td></tr><tr><td>Isoflurane</td><td>Sigma-Aldrich, Inc.</td><td>792632</td><td></td></tr><tr><td>Lacrilube</td><td>Fisher Scientific&amp;nbsp;</td><td>19090646</td><td></td></tr><tr><td>Buprenorphine</td><td>Fisher Scientific&amp;nbsp;</td><td>NC1292810</td><td></td></tr><tr><td>D-luciferrin</td><td>Perkin Elmer</td><td>122799</td><td></td></tr><tr><td>IVIS&amp;nbsp; Spectrum In Vivo Imaging System</td><td>Perkin Elmer</td><td>124262</td><td></td></tr><tr><td>Mouse strain C57BL/6J</td><td>The Jackson Laboratory&amp;nbsp;</td><td>000664/Black 6</td><td></td></tr><tr><td>Cell line (KPC-Luc 4580)</td><td>J.J. Yeh Lab at University of North Carolina</td><td></td><td></td></tr><tr><td>BD Precisionglide syringe needles</td><td>Sigma-Aldrich, Inc.</td><td>Z192406</td><td></td></tr><tr><td>Alcohol Swab(70% isopropyl alcohol )</td><td>BD</td><td>326895</td><td></td></tr><tr><td>Digital calipers</td><td>ThermoFisher Scientific</td><td>14-648-17</td><td></td></tr><tr><td>Disposable Scalpels, Sterile</td><td>VWR</td><td>21909</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>VWR</td><td>89198</td><td></td></tr><tr><td>Suture Needle, 45 cm, Size 6-0</td><td>Harvard Apparatus</td><td>72-3308</td><td></td></tr><tr><td>Suture Needle, 45 cm, Size 4-0</td><td>Harvard Apparatus</td><td>72-3314</td><td></td></tr><tr><td>Povidone-iodine 10%</td><td>BD</td><td>29900-404</td><td></td></tr><tr><td>Disposable Warming Pad&amp;nbsp;</td><td>KENT SCIENTIFIC CORP.</td><td>TP-3E</td><td></td></tr><tr><td>Mouse Hair Clipper</td><td>KENT SCIENTIFIC CORP.</td><td>CL8787</td><td></td></tr><tr><td>Surgical Drape</td><td>Harvard Apparatus</td><td>59-7421</td><td></td></tr><tr><td>Phosphate-buffered Saline</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr></tbody></table>

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.

We followed the procedure described above and generated SQ tumors on 5 - 6 week old wild type C57BL/6 mice inoculated with 1 x 106 cells with 50% BMM. When the tumor size reached 5 - 6 mm in diameter, a few of the mice were euthanized, their tumors were excised, and implanted orthotopically in a recipient C57BL/6 mice. IRE was performed 10 days post implantation as shown in the timeline on Figure 1. IRE was performed on the remaining mice bearing S...

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

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 ...

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Kansai Medical University

Research

JoVE Journal

Cancer Research

11.4K Views.  University California San Diego. 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.

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

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 ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

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

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

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

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

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

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

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

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, ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Medicine

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 ...

JoVE Encyclopedia of Experiments

Pancreatic Cancer

Assays & techniques

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www.jove.com/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...

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

Summary

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Chapters in this video

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

A Preclinical Murine Model of Hepatic Metastases

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

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models