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Abstract
Introduction
Protocol
Results
Discussion
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Materials
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Cancer Research

En Ortotopisk endometriecancer model med retroperitoneal lymfadenopati fremstillet af in vivo opformeret og kulturperler VX2-celler

Published: September 12, 2019
doi:
10.3791/59340
, , , , , , , , ,
1Institute of Medical Science,University of Toronto, 2Department of Obstetrics and Gynecology,University of Toronto, 3Guided Therapeutics Laboratory, TECHNA Institute,University Health Network, 4Department of Pathology, Princess Margaret Cancer Center,University Health Network, 5Department of Medical Biophysics,University of Toronto, 6Princess Margaret Cancer Center,University Health Network, 7Techna Institute,University Health Network, 8Department of Surgery,University of Toronto, 9Department of Otolaryngology, Head and Neck Surgery,University of Toronto, 10Division of Gynecologic Oncology, University of Toronto /Princess Margaret Cancer Center,University Health Network, 11Princess Margaret Cancer Center,University Health Network

Summary

Denne protokol præsenterer en standardiseret metode til at dyrke VX2-celler i kulturen og til at skabe en ortotopisk VX2-model af endometriecancer med retroperitoneal lymfeknude metastaser hos kaniner. Ortotopiske endometriale Cancer modeller er vigtige for den prækliniske undersøgelse af nye billedbehandlings metoder til diagnosticering af lymfeknudemetastaser.

Abstract

Endometriecancer er den mest almindelige gynækolog malignitet i Nordamerika og forekomsten er stigende på verdensplan. Behandling består af kirurgi med eller uden adjuverende behandling afhængigt af lymfeknude involvering som bestemt af lymfadadenektomi. Lymfadadenektomi er en morbid procedure, som ikke har vist sig at have en terapeutisk fordel hos mange patienter, og dermed en ny metode til at diagnosticere lymfeknude metastaser er påkrævet. For at teste nye billedbehandlings midler er der behov for en pålidelig model af endometriecancer med retroperitoneal lymfeknude metastaser. Den VX2-endometriale Cancer model er blevet beskrevet hyppigt i litteraturen; der er dog betydelige forskelle med hensyn til metoden for etablering af modeller. Endvidere har ingen undersøgelser rapporteret om brugen af dyrkede VX2-celler til at skabe denne model, da kun celler, der er formeret in vivo, tidligere er blevet anvendt. Heri, præsenterer vi en standardiseret kirurgisk metode og postoperativ overvågning metode til etablering af VX2-endometriecancer model og rapport om den første anvendelse af dyrkede VX2-celler til at skabe denne model.

Introduction

Endometriecancer, eller kræft i slimhinden i livmoderen, er den næstmest almindelige gynækolog malignitet på verdensplan og den mest almindelige malignitet i udviklede nationer1. Forekomsten af endometriecancer er steget støt og stiger med 2,3% pr. år mellem 2005-2013 med en tilsvarende stigning i dødelighed på 2,2%1,2,3. Diagnosen lymfeknude metastaser er altafgørende, da tilstedeværelsen af positive lymfeknuder er en stærk negativ indikator for overlevelse4,5,6,7 og kan vejlede administrationen af adjuverende behandling8,9,10,11,12,13. Lymfeknude metastaser diagnosticeres i øjeblikket ved kirurgisk fjernelse af lymfevævet, der ligger over de store blodkar i bækkenet og maven. Denne procedure, kendt som en lymfadektomi, er kontroversiel på grund af modstridende overlevelse data fra to store forsøg14,15,16,17,18 og den kendte risiko for intra-operative15,19,20 og post-operative sygelighed21,22,23. Da nuværende ikke-invasive billedbehandlings metoder ikke har den nødvendige følsomhed og specificitet til at erstatte lymfeknude dissektion24, har der været et skub til at udvikle nye diagnostiske Imaging teknikker. For at afprøve disse nye teknikker i en præklinisk indstilling er en pålidelig model af endometriecancer med retroperitoneal lymfeknude metastaser påkrævet.

Kanin VX2-tumor model er en veletableret model, som har været anvendt i udstrakt grad at studere flere menneskelige solide organ tumorer25 herunder lunge26, hoved og nakke27,28, leveren29, nyre30, knogle31,32, hjernen33, bugspytkirtlen34 oguterus 35,36,37. Den VX2-model blev oprindeligt udviklet i 1940 af Kidd og Rous38 ved succesfuld Trans plantering af en cottontail kanin papilloma virus opdaget af shope i 193339. Siden den tid, VX2-model er blevet opretholdt in vivo, kræver seriel passage i quadriceps muskel af hvide New Zealand kaniner40. Mere for nylig dog, flere grupper har med succes vokset VX2-celler in vitro40,41,42 og påvist den bevarede tumorigenecity af den dyrkede cellelinje31, 42af43. VX2-tumorer er histologisk defineret som Anaplastisk planocellulært karcinomer44 og indeholder glandulære funktioner, der ligner adenokarcinom26. Tumorer er karakteriseret ved nem implantation, hurtig vækst og hyper-vaskularisering44,45 og pålideligt metastasize, mest almindeligt til regionale og fjerne lymfeknuder45. Ligheder i uterin vaskulær og lymfe anatomi46 samt den ortotopiske vækst site sikre, at det metastatiske mønster af kanin VX2-karcinom efterligner den humane endometriecancer, hvilket gør VX2-model en pålidelig model for at studere Human metastatisk sygdom. Desuden tyder histologiske egenskaber såsom unormal mikrovaskulær spredning47 , samt immunologisk48 og genetiske ligheder49,50 mellem mennesker og kaniner på, at tumoren mikromiljø kan afspejle den humane endometriecancer.

Flere grupper har rapporteret om brugen af VX2-til at skabe en model af endometriecancer med retroperitoneale metastaser med en høj rapporterede hastighed af succes36,51,52; der findes dog betydelige variationer i den nuværende litteratur med hensyn til model skabelens metode. Cellesuspension doser så lavt som 4 x 105 celler/uterine horn51 og så højt som 5 x 109 celler/uterine horn37,53 er blevet rapporteret uden standard konsensus om den krævede VX2-cellulære dosis. Samt, en række inokulering metoder er blevet rapporteret herunder mikro-kirurgisk implantation af tumor i uterin myometrium36, injektion af VX2-cellesuspension37,44,52 , 53 og i nogle tilfælde tilsætning af uterin horn suturering før innoculation52. Endelig har ingen grupper rapporteret brugen af dyrkede VX2-celler til at skabe denne model. Således formålet med denne undersøgelse er at demonstrere en vellykket standardiseret metode til VX2-model skabelse og til at rapportere den første anvendelse af dyrkede VX2-celler til at skabe en model af endometriecancer med retroperitoneale metastaser i en kanin.

Protocol

Dyb abdominal væg lukning: identificere spidsen af peritonealdialyse indsnit og forstå peritoneum, rectus femoris muskler og fascia med væv pincet. Alle dyreforsøg blev udført i dyre ressource Center (ARC) godkendte faciliteter på universitetets sundhedsnetværk og i overensstemmelse med godkendte dyre brugs-og pleje protokoller (AUP #3994/#4299). VX2-Cell line blev hentet fra Dr. Aken Lab på University Health Network. 1. oprettelse af in vitro VX2-cellelinje Høst VX2-tumor f…

Representative Results

Tyve-otte kaniner blev anvendt til oprettelse af endometriecancer model. Kaniner havde en gennemsnitsvægt på 2,83 kg (2.71-3.58 kg) på tidspunktet for forsøget. Uterine tumorer med succes voksede i 21 kaniner for en samlet model succesrate på 75%. Forud for optagelsen af uterin suturering i protokollen, succesraten var 57% sammenlignet med 81% efter uterin suturering blev tilføjet. Uterine suturering blev føjet til protokollen efter den 7th kanin som reaktion på den opr…

Discussion

Heri, vi har rapporteret en standardiseret kirurgisk metode til etablering af en VX2-endometriecancer model og rapporterede om den første brug af dyrkede VX2-celler til at skabe denne model. Tumoren tager sats på 75% er lavere end 100% procentsats tidligere rapporteret i litteraturen35,37,53,56; men THW 90% af patologisk bekræftede lymfeknuder metastaser er i overensstemmelse med tidligere u…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Denne undersøgelse blev finansieret af Terry Fox Research Institute (PPG # 1075), det canadiske Institut for sundhedsforskning (Foundation Grant #154326), den canadiske Cancer Society Research Institute (704718), Natural Sciences og Engineering Research Council of Canada, Сanada Foundation for innovation og prinsesse Margaret Cancer Foundation.

Jeg vil gerne takke Dr. Marguerite Akens for at give de indledende VX2-celler til etablering af den oprindelige VX2-model og de frosne VX2-tumorblokke. Jeg vil gerne takke Marco DiGrappa for at hjælpe med at udføre indledende VX2-cellekultur eksperimenter og Lili Ding for at hjælpe med VX2-cellekultur.

Materials

11-blade scalpel, Sterile, Disposible Aspen Surgical (VWR) 80094-086
22-gague ear vein catheter CDMV 14332
3-0 absorbable poly-filament suture (Polysorb) Covidien 356718
3-0 braided absorbable suture (Polysorb) Covidien 356718
70uM cell strainer, Individually wrapped, Nylon Falcon 352350
Acepromazine (Atravet) CDMV 1047
Betadine soap (Poviodone iodine 7.5%) CDMV 4363
Betadine solution (Poviodone iodine 10%) UHN Stores 457955
Buprenorphine McGill University
Cefazolin UHN in-patient pharmacy No Cat # Needed
Chlorhexidine solution CDMV 119872
Corning BioCoatCellware, Collagen Type I, 100mm dishes Corning 354450 brand not important
Corning BioCoatCellware, Collagen Type I, 24-well plates Corning 354408 brand not important
Corning BioCoatCellware, Collagen Type I, 6-well plates Corning 354400 brand not important
Corning Matrigel Basement Membrane Matrix, *LDEV-free, 10 mL Corning 354234
DMEM/HAM F12 1:1 Life Technologies 11320 brand not important
DMSO Caledon Lab Chem 1/10/4100
Enrofloxacin (Baytril injectable) CDMV 11242
Falcon Tube Corning Centri-Star 430828
Fetal Bovine Serum, Qualified, Canadian Origin, 500ml Life Technologies 12483020 brand/source not important
Isoflurane UHN in-patient pharmacy No Cat # Needed
Isohexol contrast GE Healthcare 407141210
Meloxicam (Metacam 0.5%) CDMV 104674
Normal Saline House Brand (UofT Medstore) 1011
PBS Multicell or Sigma 331-010-CL or D8537-500mL
Penicillin/Streptomycin (100mL; 10000U Penicillin, 10000ug Streptomycin) Corning-Cellgro CA45000-652
Sterile Hanks Balanced Salt Solution (-Ca++, -Mg++, -Phenol Red) T.C.M.F (Dr Bristow) 28-Jan-11
Surgical Glue (Tissue Adhesive) 3M Vetbond 14695B
Trypsin (0.25%), Proteomics Grade Sigma T-6567-5X20UG
Trypsin-EDTA, 0.05%, 100ml Wisent Inc 325-542-EL brand not important
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Tags

Keywords: Orthotopic Endometrial Cancer ModelVX2 CellsRetroperitoneal Lymph Node MetastasisImage-guided SurgeryIn Vitro PropagationIn Vivo PropagationCell CultureCell SuspensionCell CountingCell Injection
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Philp, L., Chan, H., Rouzbahman, M., Rostami, A., Ding, L., Bratman, S. V., Akens, M. K., Irish, J. C., Bernardini, M. Q., Zheng, G. An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells. J. Vis. Exp. (151), e59340, doi:10.3791/59340 (2019).
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