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>

<ol>
	<li>Rahib, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projecting+cancer+incidence+and+deaths+to+2030:+the+unexpected+burden+of+thyroid,+liver,+and+pancreas+cancers+in+the+United+States.">Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States.</a> Cancer Res. 74 (11), 2913-2921 (2014).</li><li>Howlader, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SEER Cancer Statistic Review, 1975-2013. , (1975).</li><li>Clark, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+immune+reaction+to+pancreatic+cancer+from+inception+to+invasion.">Dynamics of the immune reaction to pancreatic cancer from inception to invasion.</a> Cancer Res. 67 (19), 9518-9527 (2007).</li><li>Lee, E. W., Loh, C. T., Kee, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+guided+percutaneous+irreversible+electroporation:+ultrasound+and+immunohistological+correlation.">Imaging guided percutaneous irreversible electroporation: ultrasound and immunohistological correlation.</a> Technol Cancer Res Treat. 6 (4), 287-294 (2007).</li><li>Charpentier, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible+electroporation+for+the+ablation+of+liver+tumors:+are+we+there+yet?.">Irreversible electroporation for the ablation of liver tumors: are we there yet?.</a> Arch Surg. 147 (11), 1053-1061 (2012).</li><li>Narayanan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible+Electroporation.">Irreversible Electroporation.</a> Seminars in Interventional Radiology. 32 (4), 349-355 (2015).</li><li>Martin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+200+locally+advanced+(stage+III)+pancreatic+adenocarcinoma+patients+with+irreversible+electroporation:+safety+and+efficacy.">Treatment of 200 locally advanced (stage III) pancreatic adenocarcinoma patients with irreversible electroporation: safety and efficacy.</a> Ann Surg. 262 (3), 486-494 (2015).</li><li>Belfiore, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+CT-guided+irreversible+electroporation+followed+by+chemotherapy+as+a+novel+neoadjuvant+protocol+in+locally+advanced+pancreatic+cancer:+Our+preliminary+experience.">Percutaneous CT-guided irreversible electroporation followed by chemotherapy as a novel neoadjuvant protocol in locally advanced pancreatic cancer: Our preliminary experience.</a> Int J Surg. 21 Suppl 1, S34-S39 (2015).</li><li>Belfiore, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+chemotherapy+alone+versus+irreversible+electroporation+followed+by+chemotherapy+on+survival+in+patients+with+locally+advanced+pancreatic+cancer.">Concurrent chemotherapy alone versus irreversible electroporation followed by chemotherapy on survival in patients with locally advanced pancreatic cancer.</a> Med Oncol. 34 (3), 38 (2017).</li><li>Chu, K. F., Dupuy, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+ablation+of+tumours:+biological+mechanisms+and+advances+in+therapy.">Thermal ablation of tumours: biological mechanisms and advances in therapy.</a> Nat Rev Cancer. 14 (3), 199-208 (2014).</li><li>Wissniowski, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+tumor-specific+T+lymphocytes+by+radio-frequency+ablation+of+the+VX2+hepatoma+in+rabbits.">Activation of tumor-specific T lymphocytes by radio-frequency ablation of the VX2 hepatoma in rabbits.</a> Cancer Res. 63 (19), 6496-6500 (2003).</li><li>Eros de Bethlenfalva-Hora, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiofrequency+ablation+suppresses+distant+tumour+growth+in+a+novel+rat+model+of+multifocal+hepatocellular+carcinoma.">Radiofrequency ablation suppresses distant tumour growth in a novel rat model of multifocal hepatocellular carcinoma.</a> Clin Sci (Lond). 126 (3), 243-252 (2014).</li><li>Zerbini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiofrequency+thermal+ablation+for+hepatocellular+carcinoma+stimulates+autologous+NK-cell+response.">Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response.</a> Gastroenterology. 138 (5), 1931-1942 (2010).</li><li>Ali, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+dendritic+cells+by+local+ablation+of+hepatocellular+carcinoma.">Activation of dendritic cells by local ablation of hepatocellular carcinoma.</a> J Hepatol. 43 (5), 817-822 (2005).</li><li>Fietta, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+inflammatory+response+and+downmodulation+of+peripheral+CD25+Foxp3++T-regulatory+cells+in+patients+undergoing+radiofrequency+thermal+ablation+for+lung+cancer.">Systemic inflammatory response and downmodulation of peripheral CD25+Foxp3+ T-regulatory cells in patients undergoing radiofrequency thermal ablation for lung cancer.</a> Hum Immunol. 70 (7), 477-486 (2009).</li><li>Jiang, C., Davalos, R. V., Bischof, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+basic+to+clinical+studies+of+irreversible+electroporation+therapy.">A review of basic to clinical studies of irreversible electroporation therapy.</a> IEEE Trans Biomed Eng. 62 (1), 4-20 (2015).</li><li>Neal, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+local+and+systemic+anti-tumor+efficacy+for+irreversible+electroporation+in+immunocompetent+versus+immunodeficient+mice.">Improved local and systemic anti-tumor efficacy for irreversible electroporation in immunocompetent versus immunodeficient mice.</a> PLoS One. 8 (5), e64559 (2013).</li><li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer Cell. 7 (5), 469-483 (2005).</li><li>Safran, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+reporter+strain+for+noninvasive+bioluminescent+imaging+of+cells+that+have+undergone+Cre-mediated+recombination.">Mouse reporter strain for noninvasive bioluminescent imaging of cells that have undergone Cre-mediated recombination.</a> Mol Imaging. 2 (4), 297-302 (2003).</li><li>Martin, R. C., McFarland, K., Ellis, S., Velanovich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irreversible+electroporation+in+locally+advanced+pancreatic+cancer:+potential+improved+overall+survival.">Irreversible electroporation in locally advanced pancreatic cancer: potential improved overall survival.</a> Ann Surg Oncol. 20 Suppl 3, S443-S449 (2013).</li><li>Neal, R. E., Garcia, P. A., Robertson, J. L., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Characterization+and+Numerical+Modeling+of+Tissue+Electrical+Conductivity+during+Pulsed+Electric+Fields+for+Irreversible+Electroporation+Treatment+Planning.">Experimental Characterization and Numerical Modeling of Tissue Electrical Conductivity during Pulsed Electric Fields for Irreversible Electroporation Treatment Planning.</a> IEEE Transactions on Biomedical Engineering. 59 (4), 1076-1085 (2012).</li></ol>

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 studies to evaluate the effects of IRE on PC using various extended treatment parameters and combinations.
The most important thing to consider during the protocol is the size of the tumor at which IRE is performed. The commercially available probes for mice models are limited by their electrode distance (5 -&#160;10 mm). Hence, in tumors significantly larger than the electrode distance, incomplete ablation takes place. For SQ tumors, the use of 2-needle array electrodes over tweezer-trodes is recommended as the skin surrounding the tumor increases resistance to the flow of current in tweezer-trodes. The size limitation of the 2-needle array electrodes can be overcome by performing the IRE at various depths and angles in the same tumor; however, this approach makes it difficult to treat tumors uniformly. Alternatively, for larger tumors (up to 10 mm), an incision can be made on the skin below the tumor to a length slightly longer than the tumor diameter. The tumor can then be externalized through the incision by inverting the overlying skin using forceps. The tumor can then be treated with tweezer-trodes, similar to the orthotopic tumors described above. The electroporated tumor can then be reinserted under the skin, and the skin sutured with a 4-0 non-absorbable suture as an interrupted stitch. However, the effects of the incision may confound the effects of IRE, and we have found that performing IRE when the tumors are within the working distance of the two-needle array electrodes (~ 5 - 6 mm) provides the best standardization.
Regardless of the approach, the number of pulses needs to be determined empirically depending on the tumor type. Published studies have utilized between 150&#160;- 300 pulses at this voltage17. However, we determined the optimal number of pulses in a preliminary dose-response experiment. There are models to predict the zone of complete ablation based on voltage and electrode distance, but tumor types can vary greatly in vascularity and fibrosis, which may affect the response to electroporation21.
Each set of 10 pulses was separated by 10 s in order to avoid the heating effects that occur if too many pulses are delivered too quickly. Over-treatment can result in thermal ablation, which may prevent accurate characterization of the effects of non-thermal IRE. The 10 s interval also allows us to frequently confirm electrode positioning accurately since muscle contractions can cause electrode displacement. Our IACUC has not yet allowed the use of paralytic agents in small animals, which can greatly reduce muscle contractions during IRE. In case of orthotopic tumors, the tweezer-trodes allows us to hold the tumors tightly and have a larger electrode surface area compared to the needle electrode.
Although IRE is currently only used as an ablative procedure in the clinical setting, electroporation in general has a wide spectrum of applications ranging from nerve and muscle activations to delivery of various drugs and oligonucleotides. By carefully analyzing the pathophysiological changes occurring during and after IRE using this model, it is possible to develop countless therapeutic strategies for PC.

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 border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">ECM 830 square wave electroporator</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:344px;">BTX # 45-0002 ( 58018-004 )</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2 needle array electrode</td>
			<td>Harvard Apparatus</td>
			<td>45-0167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Safety foot switch</td>
			<td>Harvard Apparatus</td>
			<td>45-0211</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Platinum Tweezer-trode</td>
			<td>Harvard Apparatus</td>
			<td>&#160;45-0486</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM-F12 media</td>
			<td>ThermoFisher Scientific</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matrigel</td>
			<td>Corning&#160;</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>792632</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lacrilube</td>
			<td>Fisher Scientific&#160;</td>
			<td>19090646</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Buprenorphine</td>
			<td>Fisher Scientific&#160;</td>
			<td>NC1292810</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-luciferrin</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IVIS&#160; Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mouse strain C57BL/6J</td>
			<td>The Jackson Laboratory&#160;</td>
			<td>000664/Black 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell line (KPC-Luc 4580)</td>
			<td colspan="2">J.J. Yeh Lab at University of North Carolina</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BD Precisionglide syringe needles</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>Z192406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alcohol Swab(70% isopropyl alcohol )</td>
			<td>BD</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital calipers</td>
			<td>ThermoFisher Scientific</td>
			<td>14-648-17</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable Scalpels, Sterile</td>
			<td>VWR</td>
			<td>21909</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cotton Tipped Applicators</td>
			<td>VWR</td>
			<td>89198</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Suture Needle, 45 cm, Size 6-0</td>
			<td>Harvard Apparatus</td>
			<td>72-3308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture Needle, 45 cm, Size 4-0</td>
			<td>Harvard Apparatus</td>
			<td>72-3314</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-iodine 10%</td>
			<td>BD</td>
			<td>29900-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Warming Pad&#160;</td>
			<td>KENT SCIENTIFIC CORP.</td>
			<td>TP-3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Hair Clipper</td>
			<td>KENT SCIENTIFIC CORP.</td>
			<td>CL8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Drape</td>
			<td>Harvard Apparatus</td>
			<td>59-7421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 SQ tumors.
In SQ tumors, the IRE voltage and pulse duration were kept constant at 1,500 V/cm and 100 &#181;s, respectively, but the number of pulses varied. Figure 2 shows that the SQ tumors in a few mice regressed completely after 150 pulses of IRE but not as well with 75 pulses. In total, 4 out of 9 mice showed complete tumor regression after 150 pulses of IRE. Histological analysis of tumor tissue 1 week post IRE showed large regions of central tumor necrosis that were not seen in control untreated tumors. This necrotic core was flanked by live tumor tissue in cases of incomplete tumor regression (Figure 3). Successful implantation of orthotopic tumor was also achieved, and the growth rate was monitored using in vivo bioluminescence imaging showing tumor at day 10 and day 15 (Figure 4) after implantation. IRE at 150 pulses also proved to be effective in orthotopic tumors (Figure 5) showing reduced tumor volumes. Overall, these results demonstrate the ability of this model to simulate effects of IRE in an immunocompetent mouse model, thereby providing a platform to test various IRE conditions and combinations to treat PC.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57265/57265fig1.jpg" /> 
Figure 1: Schematic representation shows the time course of tumor implantation and IRE. For SQ tumors, IRE is performed as soon as the tumor reaches 5 - 6 mm in diameter. <a href="/files/ftp_upload/57265/57265fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57265/57265fig2.jpg" /> 
Figure 2: Luciferase-bioluminescence imaging shows a reduction in tumor growth post IRE in SQ model. KPC-Luc cell line constitutively expresses luciferase making it feasible to monitor tumor growth in response to IRE in real-time. Day 14 bioluminescence imaging post IRE shows the complete regression of tumor in one of the mice treated with 150 pulses of IRE, whereas incomplete regression was seen with 75 pulses of IRE when compared to the untreated control. <a href="/files/ftp_upload/57265/57265fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57265/57265fig3.jpg" /> 
Figure 3: Hematoxylin and eosin staining of tumor tissue shows the changes in the tissue architecture post IRE. SQ tumors exposed to IRE showed large regions of necrosis centrally, surrounded by regions of live tissue indicating inadequate IRE coverage in some cases. <a href="/files/ftp_upload/57265/57265fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57265/57265fig4.jpg" /> 
Figure 4: Bioluminescence imaging shows the successful implantation of orthotopic tumor and its growth over time. Images were taken using a commercial bioluminescence imaging instrument with the luminescence captured at 1 min exposure. The mice were injected with 30 mg/kg of D-luciferin solution intraperitoneally 10 min before imaging. Mice were kept anaesthetized using 2% isoflurane during the procedure. <a href="/files/ftp_upload/57265/57265fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57265/57265fig5.jpg" /> 
Figure 5: IRE induces tumor growth reduction in orthotopic PC tumors. (A) Bioluminescecnce images of mice harboring orthotopic PC showed reduced luciferase signal 7 days post-IRE compared to sham surgery suggesting reduced live tumor burden as a result of IRE. (B) Mean volume of excised orthotopic tumors (+ standard error) in control mice vs. mice that underwent IRE. <a href="/files/ftp_upload/57265/57265fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation at JoVE.com

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 ...

a-syngeneic-pancreatic-cancer-mouse-model-to-study-effects

Nanyang Technological 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 ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

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 ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

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 ...

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 ...

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 ...