Kombineret brug af hale vene metastase assays og Real-Time in vivo billeddannelse at kvantificere brystkræft metastatisk kolonisering og byrde i lungerne
Summary
Den beskrevne fremgangsmåde kombinerer eksperimentelle hale vene metastase assays med in vivo levende dyr billeddannelse at tillade real-time overvågning af brystkræft metastase dannelse og vækst i tillæg til kvantificering af metastase antal og størrelse i lungerne.
Abstract
Metastase er hovedårsagen til kræftrelaterede dødsfald, og der er begrænsede terapeutiske muligheder for patienter med metastatisk sygdom. Identifikation og afprøvning af nye terapeutiske mål, der vil lette udviklingen af bedre behandlinger for metastatisk sygdom, kræver prækliniske in vivo-modeller. Demonstreret her er en og musemodel for bestemmelse brystkræft metastatisk kolonisering og efterfølgende vækst. Metastatiske kræftceller er stabilt transduceret med virale vektorer kodning Firefly der og zsgreen proteiner. Kandidat gener er derefter stabilt manipuleret i luciferase/ZsGreen-udtrykker kræftceller og derefter cellerne injiceres i mus via den laterale hale vene til at assay metastatisk kolonisering og vækst. En in vivo Billeddannende anordning anvendes derefter til at måle tumor cellernes bioluminescens eller fluorescens i de levende dyr for at kvantificere ændringer i metastatisk byrde over tid. Udtrykket af det fluorescerende protein gør det muligt at kvantificere antallet og størrelsen af metastaser i lungerne ved afslutningen af forsøget uden behov for skæring eller histologisk farvning. Denne tilgang giver en relativt hurtig og nem måde at teste rollen af kandidat gener i metastatisk kolonisering og vækst, og giver en hel del mere information end traditionelle hale vene metastase assays. Ved hjælp af denne tilgang viser vi, at samtidig Knockdown af ja associeret protein (YAP) og transkriptional Co-aktivator med et PDZ-bindende motiv (TAZ) i brystkræft celler fører til reduceret metastatisk byrde i lungerne, og at denne reducerede byrde er resultatet af signifikant forringet metastatisk kolonisering og reduceret vækst af metastaser.
Introduction
Kræft er fortsat den anden førende dødsårsag verdensomspændende1 og metastase er ansvarlig for de fleste af disse dødsfald2,3. En begrænset forståelse af de molekylære mekanismer, der regulerer metastatisk kolonisering og efterfølgende vækst, har imidlertid hindret udviklingen af effektive behandlinger for metastatisk sygdom. Identifikationen af nye terapeutiske mål kræver en analyse for at teste, hvordan rystet udtryk eller funktion af et kandidat-gen påvirker metastase dannelse og vækst. Mens autoktont musemodeller har deres fordele, er de tidskrævende og dyre at generere, hvilket gør dem mere velegnede til målvalidering i stedet for Målsøgning. Transplantations modelsystemer, hvor kandidat genet er rystet i kræftceller in vitro og derefter virkninger på metastatisk potentiale vurderes in vivo, er billigere og højere gennemløb end autoktone modeller. Desuden er virale vektorer for stabil levering af RNAi, crispr/CAS9, og Trans Genes er bredt tilgængelige, hvilket gør det relativt let at forstyrrer næsten ethvert gen eller gener af interesse i en Cancer cellelinjer. Denne fremgangsmåde kan også bruges til at assay rollen af kandidat gener i metastatisk kolonisering og vækst i humane Cancer cellelinjer ved at transplanteres cellerne til immunkompromitterede eller humaniserede mus.
De to typer af assays, der anvendes til at teste metastase dannelse ved transplanterede cancerceller in vivo er spontane metastase assays og eksperimentelle metastase assays. I spontane metastase assays4,5, kræftceller injiceres i mus, lov til at danne en primær tumor, og derefter spontan metastase dannelse og efterfølgende vækst er analyseret. Styrken af denne model er, at cellerne skal fuldføre alle trin i den metastatiske proces for at danne metastatiske tumorer. Men, mange kræft cellelinjer ikke metastaserer effektivt i spontane metastase modeller, og enhver manipulation af de celler, der påvirker primær tumorvækst kan tolerere resultaterne af metastase assay. Eksperimentelle metastase assays, hvor kræftceller injiceres direkte i omløb, anvendes til at undgå disse faldgruber. Almindeligt eksperimentelle metastase assays omfatter hale vene injektion6,7,8 (og påvist her), intrakardiel injektion9, og Portal vene injektion10.
Formålet med protokollen præsenteres her er at give en in vivo eksperimentel metastase assay, der giver en forsker til at overvåge metastase dannelse og vækst i realtid, samt at kvantificere slutpunktet metastase nummer og størrelse i lungerne af samme mus. For at opnå dette, traditionelle eksperimentelle hale vene metastaser assays6,7,8 kombineres med levende dyr billeddannelse, ved hjælp af en in vivo billedenhed9,11,12,13,14. Tumor celler, der stabilt udtrykker både der og et fluorescerende protein injiceres i mus via den laterale hale vene, og derefter anvendes in vivo billedenheden til at måle ændringer i metastatisk belastning i lungerne over tid (figur 1). In vivo Live Animal Imaging-enheden kan dog ikke skelne mellem eller måle størrelsen af individuelle metastaser. Ved afslutningen af forsøget anvendes et fluorescerende stereomicroskop til at tælle antallet og måle størrelsen af de fluorescerende metastaser i lungerne uden behov for skæring og histologi eller Immunhistokemi (figur 1). Denne protokol kan bruges til at teste, hvordan ændring af udtryk eller funktion af et kandidat-gen påvirker metastase dannelse og vækst. Potentielle terapeutiske forbindelser såsom små molekyler eller funktions blokerende antistoffer kan også testes.
For at demonstrere denne tilgang, vi først udført et bevis for koncept eksperiment, hvor den væsentlige replikation faktor, replikation protein a3 (RPA3) er slået ned i metastatisk mus brystkræft celler. Vi viser, at mus injiceret med RPA3 Knockdown celler har betydeligt mindre metastatisk byrde på hvert tidspunkt i forhold til mus injiceres med kontrol celler. Analyse af de metastase-holdige lunger viser, at denne reducerede metastatiske byrde er resultatet af signifikant reduceret metastatisk kolonisering og nedsat vækst af de metastaser, der dannes. For yderligere at demonstrere denne teknik, vi testede, om samtidige knock down af ja associerede protein (YAP) og transkriptional Co-aktivator med en PDZ-bindende motiv (TAZ) hæmmer metastatisk kolonisering eller efterfølgende vækst. YAP og TAZ er to relaterede transkriptionelle Co-aktivatorer, der er de kritiske downstream effektorer af Hippo pathway. Vi15,16 og andre har impliceret Yap og Taz i metastase (revideret i17,18,19), tyder på, at disse proteiner er gode terapeutiske mål. Konsekvent, vi fandt, at mus injiceres med YAP/TAZ Knockdown celler havde signifikant reduceret metastatisk byrde. Analyse af lungerne viste, at YAP/TAZ Knockdown celler dannede mange færre metastaser, og at de metastaser, der gjorde form var mindre. Disse eksperimenter viser, hvordan eksperimentelle metastase assays giver en forsker til hurtigt og billigt at teste rollen af et kandidat-gen i metastase dannelse og vækst. De viser yderligere, hvordan den kombinerede anvendelse af levende animalsk billeddannelse og fluorescerende kvantificering af metastaser i hele lunger gør det muligt for forskeren bedre at forstå trinene under metastatisk kolonisering.
Protocol
Representative Results
Discussion
Kritiske trin i metoden
Det er afgørende at optimere antallet af injicerede celler (trin 3) for en given cellelinje og musestamme, da dette i høj grad kan påvirke antallet af metastaser, der dannes, og længden af eksperimentet. Hvis der injiceres for mange celler, eller hvis metastaser vokser for længe, kan det være svært at tælle metastaser, hvilket gør det vanskeligt at vurdere virkningerne af den genetiske manipulation. Men, hvis for få celler injiceres, få eller ingen metastaser kan dan…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Emily Norton for at hjælpe med virusinfektioner og kritisk læsning af manuskriptet. Vi takker også Ryan Kanai for hjælp til at erhverve billeder af lungerne og Kate E. Tubbesing for at få hjælp til billedanalyse af de grønne metastaser i lungerne. Vi takker dyreforsknings facilitetens personale for deres støtte og for hjælp til forberedelsen af denne video. Dette arbejde blev støttet af en Susan G. Komen Career Catalyst Grant, der blev tildelt J.M.L. (#CCR17477184).
Materials
| 10% SDS-PAGE Gel | For western blot | ||
| 2.5% Trypsin | Gibco | 15090-046 | Trypsin for tissue culture |
| 96 well flat bottom white assay plate | Corning | 3922 | For measuring luciferase and renilla signal in cultured cells |
| Alcohol wipes | For sterolizing the injection site before tail vein injecitons | ||
| BALB/C mice (female, 6 weeks) | Taconic | BALB-F | For tail vein metastatic colonization and burden assays |
| BSA regular | VWR Ameresco | 97061-416 | For western blot |
| Cell lysis buffer | Cell Signaling | For collecting protien samples | |
| Celltreat Syringe Filters, PES 30mm, 0.45 μm | Celltreat | 40-229749-CS | For filtering viral supernatant |
| CO2 and euthanasia chamber | For euthanasing the mice | ||
| Dual-luciferase reporter assay kit | Promega | E1960 | For measuring luciferase and renilla signal in cultured cells |
| Dulbecco&39;s phosphate buffered saline | Himedia | TS1006 | For PBS |
| EDTA | VWR | 97061-406 | Used to dilute trypsin for tissue culture |
| FBS 100% US origin | VWR | 97068-085 | Component of complete growth media |
| Fujifilm LAS-3000 gel imager | Fujifilm | For western blot | |
| GAPDH(14C10) Rabibit mAb | Cell Signaling | 2118 | For western blot |
| Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugate | Thermo Scientific | 31460 | For western blot |
| Human embryonic kidney cells, HEK-293FT | Invitrogen | R70007 | Cell line used for packging virus |
| HyClone DMEM/High clucose | GE Healthcare life sciences | SH30243.01 | Component of complete growth media |
| Hygromycin B, Ultra Pure Grade | VWR Ameresco | 97064-810 | For antibiotic selection of infected cells |
| I3-P/i3 Multi-Mode Microplate/EA | Molecular devices | For measuring luciferase and renilla signal in cultured cells | |
| Imagej | Used for image analysis of lung metastases: threshold set to 25 & 100 | ||
| Immuno-Blot PVDF Membrane | Biorad | 1620177 | For western blot |
| Isoflurane | For mouse anesthesia | ||
| IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device) | PerkinElmer | CLS136340 | For in vivo imaging of metastatic burden |
| Leica M205 FA & Lecia DCF3000 G (GFP and bright field filters) | Leica Microsystems | Microscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain | |
| L-Glutamine | Gibco | 25030-081 | Component of complete growth media |
| Lipofectamine 3000 | Life technologies | L3000008 | For YAP/TAZ-TEAD reporter transfection |
| Living Image 3.2 (image software program) | PerkinElmer | Software for IVIS | |
| Mouse breast cancer cells, 4T1 | Karmanos Cancer Institute | Aslakson, CJ et al.,1992 | Mouse metastatic breast cance cell line |
| Multi-Gauge version 3.0 | Fujifilm | Software for quantifying western blot band intensity | |
| Opti-MEM (transfection buffer) | Gibco | 31985-062 | For packaging virus and transfection |
| Penicillin Streptomycin | Gibco | 15140-122 | Component of complete growth media |
| Pierce BCA protein assay kit | Thermo Scientific | 23225 | For quantifying protein concentration |
| Pierce Phosphatase Inhibitor Mini Tablets | Thermo Scientific | A32957 | Added to cell lysis buffer |
| Pierce Protease Inhibitor Mini Tablets | Thermo Scientific | A32953 | Added to cell lysis buffer |
| Polybrene (hexadimethrine bromide) | Sigma-Aldrich | 45-H9268 | For infection |
| Puromycin | Sigma-Aldrich | 45-P7255 | For antibiotic selection of infected cells |
| Rodent restrainer | For restraining mice during tail vein injeciton | ||
| SDS-PAGE running buffer | For western blot | ||
| TAZ (V3886) Antibody | Cell Signaling | 4883 | For western blot |
| TBST buffer | For western blot | ||
| TC20 automated cell counter | Bio-Rad | For counting cells | |
| Vectors | See Table 1 for complete list of vectors | ||
| VWR Inverted Fluorescence Microscope | VWR | 89404-464 | For visualizing fluorescence in ZSGreen labeled cells |
| Western transfer buffer | For western blot | ||
| XenoLight D-Luciferin K+ Salt | PerkinElmer | 122799 | Substrate injected into mice for in vivo bioluminescent IVIS images |
| X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection) | Roche | 6365787001 | For packaging virus |
| YAP (D8H1X) XP Rabbit mAb | Cell Signaling | 14074 | For western blot |
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