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 ...

<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 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.

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		<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>
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			<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>
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		<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 S...

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 ...

The overall goal of this model is to study the effects of irreversible electroporation or IRE, on pancreatic cancer in immunocompetent mice. This model can help answer many key questions regarding the role of IRE in pancreatic cancer. Such as, how does IRE affect the local tumor microenvironment?The main advantage of this model is that it facilitate accurate tumor growth assessment in an immunocompetent system, without requiring knockout or mutant mouse models. This method can provide insight into the effects of IRE on the immune system, in an immunocompetent mouse model of pancreatic cancer. For subcutaneous tumor induction, first, shave the injection site on an anesthetized six-week old, male, C57 black six mouse, and use an alcohol swab to clean the site two times.Next, load a cold one-milliliter syringe with one to two times ten to the seven cells per milliliter and 250 microliters of basement membrane matrix. Attach a 25-gauge needle to the syringe, and load the needle with the tumor cell suspension. Press and retract the plunger a few times to remove any air bubbles.Carefully insert the needle under the skin of the injection site, at a 30 degree angle. With the needle parallel to the body, slowly inject 50 microliters of the tumor cells. After 10 seconds, slowly withdraw the needle, keeping it parallel without any sideways movement, and gently clean the injection site with an alcohol swab, before returning the mouse to its cage.When the tumor reaches five to seven millimeters, use sterile forceps and micro scissors to excise the subcutaneous tumor, under sterile conditions in a biosafety level two hood, taking care not to puncture the peritoneal cavity. Immediately wash the excised tumor, two times, in sterile PBS. And use sterile scalpels to mince the tumor into one to two cubed millimeter pieces.Then, transfer the tumor pieces into a Petri dish, containing cold, sterile DMEM basal medium on ice. To implant the tumor fragments, after confirming a lack of response to toe pinch, apply ointment to the anesthetized recipient animal's eyes, and place the mouse on its right side, so that the left side of the abdomen is exposed. Remove the abdominal hair with depilatory cream, and remove the cream with gauze.After disinfecting the exposed skin with three sequential 10%povidone plus iodine and alcohol wipes, use a sterile scalpel to make a 1.5 centimeter incision in the skin, one centimeter to the left of the midline, below the rib cage, and slightly medial to the spleen. Gently externalize the spleen with flat-tipped forceps and a sterile cotton applicator, and use the forceps to laterally retract the tail of the pancreas, at the bottom of the spleen. Using sterile forceps, grasp one finely-minced tumor piece, and pass a fine 6-O sterile suturing needle through the piece.Insert the needle containing tumor fragment into the tail of the pancreas, passing the suture slowly through the tissue. Take care to avoid contact between the tumor and the abdominal wall musculature and peritoneum, to prevent tumor seeding. When the tumor fragment has been secured, completely remove the needle from the tissue, and keep the organs completely externalized, for an additional 60 seconds, while inspecting for any signs of hemorrhage.If no bleeding is observed, use blunt forceps, or a cotton tip applicator, to gently return the spleen, pancreas, and tumor to the abdominal cavity. Before the tumor reaches six millimeters in diameter, place the anesthetized animal on its right side to allow access to the tumor, and use clippers and an alcohol swab to clean the tumor site. Using forceps, elevate the skin directly under the tumor, and insert two needle array electrodes through the skin, parallel to the body, to bracket the tumor, taking care that the electrodes do not penetrate the peritoneal cavity.Set the electroporator to deliver 100 microsecond pulses at a one hertz frequency, at 1500 volts per centimeter, for a total of 150 pulses, and use the foot pedal to deliver the pulses, separating each set of 10 pulses by 10 seconds, to allow heat dissipation between each set of pulses. As muscle twitching occurs during the IRE delivery, and is more pronounced during the initial set of pulses, ensure that the electrodes are still in the desired location, and that the animal is still sedated after each set of pulses. After no more than 200 seconds of dosing, remove the electrodes and record the actual voltage delivered, as displayed on the electroporator.Then, return the mouse to its cage. For IRE of an orthotopic tumor, eight to 10 days post tumor implantation, use blunt-nosed forceps to externalize the tumor with the forceps, and tightly capture the tumor with the platinum electrodes of the Tweezertrode, deliver the electroporation pulses, as just demonstrated, keeping the tumor externalized for the last set of pulses, for at least 60 seconds, to monitor for any signs of hemorrhage. Then, return the tumor to the abdominal cavity and close the incision, as demonstrated, monitoring the animal until it has fully recovered.In this representative experiment, subcutaneous tumors, in nearly 50%of the mice, regressed completely after 150 IRE pulses, with only minor regression after 75 pulses. Histological analysis of the tumor tissue, one week post IRE, revealed large regions of central tumor necrosis, that were not observed in control, untreated tumors, and that were flanked by live tumor tissue, in cases of incomplete tumor regression. Monitoring implanted orthotopic tumors growth rates revealed a consistent tumor growth over time in recipient animals, that could also be disrupted by IRE, demonstrating the ability of this model to simulate the effects of IRE, in an immunocompetent mouse model.Once mastered, this technique can be completed in 15 minutes per mouse, if it is performed properly. While attempting this procedure, it is important to remember to keep the tumor size within the working distance of the IRE electrodes available, as larger tumors will produce inconsistent responses due to incomplete ablation. Other treatment approaches, such as radiation or chemotherapies, can also be used to answer additional questions about whether combining treatments can improve tumor responses to IRE.After watching this video, you should have a good understanding of how to establish subcutaneous and orthotopic pancreatic cancers in mice, and how to perform IRE on these tumors.

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Research

JoVE Journal

Cancer Research

11.3K visualizações.  University California San Diego. The overall goal of this model is to study the effects of irreversible electroporation or IRE, on pancreatic cancer in immunocompetent mice. This model can help answer many key questions regarding the role of IRE in pancreatic cancer. Such as, how does IRE affect the local tumor microenvironment?The main advantage of this model is that it facilitate accurate tumor growth assessment in an immunocompetent system, without requiring knockout or mutant mouse models. This method can provide ...