Mærkning af Breast Cancer Patient-afledte Xenografter med Sporbare Journalister for tumorvækst og Metastase Studies
Summary
Vi beskriver en fremgangsmåde til stabil mærkning af patient-afledte xenotransplantater (PDXs) med lentivirale partikler, der udtrykker grønt-fluorescerende protein og luciferase reportere. Denne fremgangsmåde muliggør sporing af væksten af PDXs ved det primære sted, samt detektering spontane og eksperimentelle metastaser anvendelse af in vivo-billeddannelse.
Abstract
The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients’ tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.
Introduction
Udviklingen af patient-afledt tumorxenoplantater (PDXs), hvor kirurgisk resektion tumor prøver indpodede direkte i immunsvækkede mus, tilbyder flere fordele i forhold til standard celle-line xenograftmodeller og repræsenterer et stort fremskridt i kræftforskning 1,2. PDXs kan opretholdes og ekspanderes ved successive passager med minimal ændring af de genetiske og biologiske karakteristika af tumoren dyrket ved den første passage; og mere præcist afspejler tumor heterogenitet end xenografter fra humant cancer cellelinjer 3-8. Disse modeller er nu flittigt brugt som platform for personalisering kræftmedicin 9,10, som en præklinisk platform i lægemiddeludvikling 6,11 og som en eksperimentel værktøj til at studere kræft biologi 4,12.
De fleste PDXs implanteres og opformeret subkutant, der indpasses tillader måling af tumorvækst over tid ved hjælp af calipre. Imidlertid, Metastatisk sygdom har været vanskeligere at modellere bruge PDXs. Specifikt for brystkræft, har xenografter med metastatisk kapacitet til forskellige organer blevet beskrevet 3,5,13, men hyppigheden af spontan formidling til metastatiske steder er meget lav. Hvor rapporteret, identifikation og kvantificering af metastatisk byrde er afhængig i møjsommelige histologisk undersøgelse af målorganer efter slagtning. Cancercellelinier udtrykker bioluminiscerende (luciferase, Luc) eller fluorescerende (grønt fluorescerende protein GFP) -gen reportere er almindeligt anvendt i eksperimentelle modeller af brystkræft metastaser til hjerne-, lunge-, knogle- og lever efter intrakardial, hale-vene, intrafemoral og milt injektion 14-16. Mens disse modeller omgå formidling fra de primære tumorer, de er værdifulde til at undersøge mekanismerne for orgel tropisme og metastatisk kolonisering. celler afledt af primære patient tumorer og PDXs, kan dog have lav transfektion eller transduktion satser using standardprocedurer. Et alternativ er at etablere PDX-afledte cellelinier in vitro 17, som kan derefter mærket under anvendelse af konventionelle vævskulturplader protokoller. Denne fremgangsmåde er imidlertid ikke egnet til mærkning fleste PDXs, for hvilke cellelinje afledning er vanskelig og kan ændre fænotypen af cellerne. Her præsenterer vi en protokol for transduktion af PDX-dissocierede tumorceller med lentivirusvektorer egnede til in vivo billeddannelse. Derudover beskriver vi eksperimentel metastase hjælp intrakardial injektion af dissocierede luc-GFP mærkede PDX celler hos immunkompromitterede mus.
En grundlæggende protokol for transduktion af PDX-dissocierede organoids med gen-reporter udtrykker lentivirus er tidligere blevet beskrevet 18. I den nuværende protokol beskriver vi yderligere fremgangsmåder til berigelse for humane tumorceller og opnå nær 100% transduktionseffektivitet, samt anvendelsen af mærkede PDXs til detektering eksperimentelle brystcancermetastaser. Denne protokol kan tilpasses til mærkning flere cancertyper af PDXs med forskellige selvlysende og fluorescerende markører samt modulation af genekspression (dvs. shRNA knockdown af gener af interesse).
Protocol
Representative Results
Discussion
Kritiske trin i protokollen:
Brugen af høj titer lentivirale partikler (> 10 8 TU / ml) er et afgørende skridt for succesen af denne protokol, som giver mulighed for omhyggelig kontrol med medierne sammensætning under in vitro transduktion. Mens flere fremgangsmåder til fremstilling af høj-titer viruspartikler er blevet godt beskrevet 18,19; denne protokol bruger lentivirale partikler produceret som beskrevet i detaljer på <a href="htt…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Dr. Darrel Kotton ved Boston University for at levere fag-EF1aL-dsRed-UBC-GFP-W vektor og protokoller for høj titer lentiviral produktion anvendt i disse undersøgelser. Dette arbejde blev finansieret af DOD BCRP W81XWH-11-1-0101 (DMC), ACS IRG # 57-001-53 (DMC), NCI K22CA181250 (DMC) og R01 CA140985 (CAS) .NCI P30CA046934 center tilskud understøttet in vivo imaging og vævskultur cores ved University of Colorado AMC.
Materials
| DMEM/F12 (1:1) | Hyclone | SH30023.01 | |
| bFGF | BD Biosciences | 354060 | |
| EGF | BD Biosciences | 354001 | |
| Heparin | Sigma | H4784 | |
| B27 | Gibco/Thermo Fisher | 17504-44 | |
| Anti-fungi-antibiotics | Hyclone | SV30010 | |
| Accumax | Innovative Cell Technologies | AM-105-500 | Digestion Buffer |
| FBS | Atlanta Biologicals | S11550 | |
| HBSS Red Ca++/Mg++ free | Hyclone | SH30031.02 | |
| Hepes | |||
| 10X PBS | Hyclone | SH30258.01 | |
| Cultrex | Cultrex | 3433-005-01 | Basement Matrix Extract (BME) |
| 30C shaker | NewBrunswick Scientific CO. INC | Series 25 Incubator Shaker | |
| 70um filters | Falcon | 7352350 | |
| scalpels | Fisher | 22079690 | |
| Clorhexidine disinfectant | Durvet | NDC# 30798-624-35 | |
| Red blood cell lysis reagent | Sigma | R7757 | |
| Neuraminidase | Sigma | N7885-1UN | |
| EpCAM (CD326+) microbeads* | Miltenyil Biotec | 130-061-101 | |
| Lineage cell depletion Kit, mouse* | Miltenyil Biotec | 130-090-858 | |
| MiniMACS Separator | Miltenyil Biotec | 130-042-102 | |
| Mini MACS Magnetic Stand | Miltenyil Biotec | 130-042-303 | |
| MS Columns | Miltenyil Biotec | 130-042-201 | MS or LS columns can be used, adjust to number of cells. |
| Illumatool Tunable light system | Lightools research | Various | For in vivo fluorescence imaging |
| Xenogen IVIS200 imaging device | Xenogen | Various | For in vivo luminiscence imaging |
| Human Cytokeratin Clone MNF116 Monoclonal antibody | DAKO | M0821 | Pan-cytokeratin |
| Epidermal Growth factor receptor antibody | Cell signaling | 4267S | EGFR |
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