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

RRW har modtaget støtte for dette arbejde fra Collaborative Translationel forskning tilskud finansieret af San Diego C3 Padres Pedal årsag (#PTC2017).

Alle dyreforsøg udført følgende denne protokol skal godkendes af de respektive institutionelle Animal Care og brug udvalg (IACUC). Alle procedurer beskrevet her er blevet godkendt af IACUC UCSD.
1. skaffe modtagerens dyr
Bemærk: KPC-Luc 4580 celle line blev etableret fra en svulst, der opstår i en LSL-KrasG12D / +; LSL-Trp53R172H / +; PDX1Cre / +; LSL-ROSA26 Luc / + mus (KPC), som er en genetisk manipuleret model af PC ...

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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. 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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. 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I denne undersøgelse, har vi vist en immunkompetente musemodel for PC, der kan bruges til at studere virkningerne af IRE på tumorvækst. I øjeblikket, bliver IRE brugt som en ikke-termiske ablation teknik kun i yderst valgte lokalt Avanceret PC patienter, der ikke har Fjern sygdomsprogression efter måneder af præoperativ behandling. Dets anvendelse har derfor været begrænset, fordi de fleste patienter med lokalt Avanceret PC udvikle Fjern metastatisk sygdom20. Denne model vil tjene som grun...

Pancreas duktalt adenokarcinom (PC) forventes for at blive den næststørste årsag til kræftdødsfald i USA omkring 20201. Langt størstedelen af patienter diagnosticeret med PC vil til sidst dø fra fjern metastatisk sygdom2. PC mikromiljø er notorisk immunosuppressive og chemoresistant. Dens desmoplastic stroma indeholder en knaphed på effektor (anti-tumor) T-celler og en fremhævelse af immunosuppressive leukocytter, herunder tumor-associerede makrofager (TAMs), myeloid afledte suppressor celler (MDSCs) og regulerende T-celler (Tregs)3 . Disse ligge til grund for behovet for at udvikle multimodale strategier, der modvirker disse virkninger af mikromiljø.
IRE er blevet udviklet som en ikke-termisk metode til tumor ablation. I modsætning til termisk ablation teknikker, IRE forårsager ikke hurtig coagulative nekrose, men i stedet resulterer i gradvis apoptotiske celle død4. Vigtigere for pancreas tumorer, IRE er ikke sårbar over for "heat sink" effekter og kan udføres lige ved siden af blodkar5. Denne teknologi har 510 clearance fra FDA6 og i øjeblikket bliver brugt klinisk, til udvalgte patienter med lokalt fremskreden eller borderline resektable kræft i bugspytkirtlen. I den største publicerede serie af IRE for PC7var den mediane overlevelse for patienter, der gennemgår IRE ca dobbelt overlevelse af patienter behandlet med moderne kemoterapi alene uden resektion8,9.
Flere undersøgelser har vist, der termisk ablation inducerer et systemisk immunrespons i andre tumortyper (gennemgik i Chu et al. 10). radiofrekvens ablation (RFA) i animalsk tumor modeller fører til øget T celle infiltreret11,12, herunder en stigning i aktiverede natural killer (NK) celler i hepatocellulært kræft patienter13, 14, og et fald i immunosuppressive Tregs i lunge cancer patienter15. Et langt mindre antal studier har undersøgt immun, microenvironmental, og skade svar til IRE16. IRE har vist sig at stimulere en systemisk immunrespons i immunkompetente musemodeller hvor væksten af sekundære (kontralaterale) renal celle allografts blev reduceret eller forhindret af IRE af en primær tumor to uger tidligere17. De bemærkede også at immunkompetente mus kræves mindre spænding for komplet regression end gjorde immunkompromitterede mus. Det har været en hypotese at VREDE kan resultere i forbedrede antigen præsentation i forhold til den coagulative nekrose af termisk ablation, men dette er ikke blevet specifikt undersøgt.
Vi har udviklet en syngeneic musen model af PC fra KPC-Luc 4580 cellelinje (gave fra J.J. Yeh ved University of North Carolina), som var afledt af en tumor, der er udviklet i en mandlig LSL-KrasG12D / +; LSL-Trp53R172H / +; PDX1Cre / +; LSL-ROSA26 Luc / + mus, at studere de lokale og systemiske virkninger af IRE18,19. Denne luciferase-udtrykker cellelinie er immunogen og også tumorfremkaldende hos immunkompetente C57BL/6 mus når injiceres SQ eller orthotopically og pålideligt producerer lever metastaser når injiceres i milten. Vi har udnyttet en programmerbar firkantbølge pulse generator til at levere 100 µs impulser af elektricitet på en spænding/distance forholdet mellem 1.500 V/cm ved hjælp af en to-nål array sonde (adskilt af 5 mm) eller platin pincet-trodes at SQ eller orthotopic tumorer, henholdsvis i mus til at modellere virkningerne af IRE i et lille dyr.

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			<td>Fisher Scientific&#160;</td>
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			<td>Fisher Scientific&#160;</td>
			<td>NC1292810</td>
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			<td height="40" style="height:40px;">D-luciferrin</td>
			<td>Perkin Elmer</td>
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			<td height="40" style="height:40px;">IVIS&#160; Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
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			<td height="40" style="height:40px;">Mouse strain C57BL/6J</td>
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			<td>Sigma-Aldrich, Inc.</td>
			<td>Z192406</td>
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			<td>BD</td>
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			<td height="40" style="height:40px;">Digital calipers</td>
			<td>ThermoFisher Scientific</td>
			<td>14-648-17</td>
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			<td>VWR</td>
			<td>21909</td>
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			<td>VWR</td>
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			<td>Harvard Apparatus</td>
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			<td>Harvard Apparatus</td>
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			<td>BD</td>
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			<td>KENT SCIENTIFIC CORP.</td>
			<td>TP-3E</td>
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			<td>KENT SCIENTIFIC CORP.</td>
			<td>CL8787</td>
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			<td>Harvard Apparatus</td>
			<td>59-7421</td>
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			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
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a syngeneic pancreatic cancer mouse model to study the effects of irreversible electroporation

Kræft i bugspytkirtlen (PC), en sygdom, der dræber ca 40.000 patienter hvert år i USA, har med held unddragne flere terapeutiske metoder, herunder de lovende immunterapeutisk strategier. Irreversibel elektroporation (IRE) er en ikke-termiske ablation teknik, der inducerer tumor celledød uden ødelæggelse af tilstødende kollagen strukturer, således at proceduren, der udføres i tumorer meget tæt på blodkarrene. I modsætning til termisk ablation teknikker, IRE resulterer i gradvis apoptotisk celledød, sammen med øjeblikkelig ablation induceret nekrose, og er i øjeblikket i klinisk brug for udvalgte patienter med lokalt Avanceret PC. En ablativ, ikke-målarter specifikke procedure som IRE kan fremkalde et utal af svar i tumor mikromiljø. Et par undersøgelser har behandlet IRE indvirkning på tumorvækst i andre tumortyper, men ingen har fokuseret på PC. Vi har udviklet en syngeneic musen model af PC hvor subkutan (SQ) og orthotopic tumorer held kan behandles med IRE i et stærkt kontrolleret indstilling, at lette forskellige longitudinelle studier post procedure. Dette dyr model fungerer som et robust system til at studere virkningerne af VREDE og metoder til at forbedre den kliniske effekt af IRE.

Vi fulgt proceduren beskrevet ovenfor og genereret SQ tumorer på 5-6 ugers gammel vildtype C57BL/6 mus podet med 1 x 106 celler med 50% BMM. Når tumorstørrelse nåede 5-6 mm i diameter, et par af mus blev aflivet, deres tumorer blev skåret ud og implanteret orthotopically i en modtagende C57BL/6 mus. IRE var udført 10 dage post implantation som vist i tidslinjen på figur 1. IRE blev udført på de resterende mus forsynet med SQ tumorer.
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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

En Syngeneic bugspytkirtelkræft musen Model til at studere virkningerne af irreversibel elektroporation

Irreversibel elektroporation (IRE) er en ikke-termiske ablation teknik, der anvendes til behandling af lokalt fremskreden kræft i bugspytkirtlen. At være en forholdsvis ny teknik, er virkningerne af IRE på tumorvækst dårligt forstået. Vi har udviklet en syngeneic musemodel, der gør det lettere at studere virkningerne af IRE på kræft i bugspytkirtlen.

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.

Research

JoVE Journal

Cancer Research

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