Isolering og dyrkning Udvidelse af Tumorspecifikke endotelceller
Summary
We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.
Abstract
Frisk isolerede tumorspecifikke endotelceller (TEC) kan bruges til at udforske molekylære mekanismer af tumorangiogenese og tjene som en in vitro model for udvikling af nye angiogeneseinhibitorer for kræft. , Langsigtet in vitro-udvidelse af murine endothelceller (EF) er imidlertid en udfordring på grund af fænotypisk drift i kultur (endotel-til-mesenchymale overgang) og forurening med ikke-EF. Dette gælder især for TEC som let udkonkurreret af co-oprenset fibroblaster eller tumorceller i kultur. Her er en high fidelity isolation metode, der drager fordel af immunmagnetisk berigelse kombineret med koloniselektion og in vitro ekspansion beskrevet. Denne tilgang skaber rene EF fraktioner, der er helt fri for forurenende stromale eller tumorceller. Det er også vist, at slægt-spores Cdh5 CRE: ZsGreen l / s / l reporter mus, der anvendes sammen med protokollen beskrevet heri, er et værdifuldt redskab til at kontrollere cellerenhed som de isolerede EF kolonier fra disse mus viser holdbare og strålende ZsGreen fluorescens i kultur.
Introduction
Endotelceller (EF) er afgørende under udviklingen af solide tumorer. Fra indledningen af angiogene kontakten i hvilende tumorer til formidling og såning af metastaser på fjerne steder, EF udgør ledningerne, der giver blod, ilt og næringsstoffer til at opretholde væksten tumor 1. Så sent antydet, EF har også perfusion-uafhængige funktioner og danner en niche, som understøtter væksten af kræft stamceller og andre stromale tumorceller 2-5. Således højt oprenset tumor-specifikke (TEC), EF for in vitro-dyrkning muliggør rutinemæssige funktionelle studier, som vil belyse nye molekylære mekanismer medierer tumor angiogenese og krydstale med tumorceller.
EF er højt specialiserede afhængigt af vævet oprindelseslandet 6. På grund af den heterogene karakter af de forskellige tumortyper og tumor mikromiljø kan TEC også vise unikke funktioner, der afspejler en tumor-specifik specialisering of vaskulaturen. For eksempel er der slående variabilitet i genekspression signaturer i TEC isoleret fra forskellige typer eller kvaliteter af tumorer 7,8. Hyppige co-oprensning af ikke-EF, især tumorassocierede fibroblaster og tumorceller, med TEC, kan dog forvirre hele genomet ekspression analyser. Disse uønskede celletyper er særligt problematisk i undersøgelser, der er afhængige af langsigtede in vitro-udvidelse af TEC kulturer.
Beskrevet her er der en high-fidelity metode, konsekvent producerer rene kulturer fra EF tumorer og andre væv. Efter immunomagnetisk kolonne berigelse af EF fraktioner og fjernelse af co-oprensede ikke-EF at en yderligere kloning-ring skridt indfange rene EF kolonier anvendes 9. Hver koloni kan udvides i kultur til flere passager uden fremkomsten af kontaminerende ikke-EF. Denne metode giver også flere EF-kloner fra et enkelt isolation procedure, som er ideel til studiet af endothelial heterogenitet. Desuden er det vist, at Cdh5 CRE: ZsGreen l / s / l reporter mus er et værdifuldt redskab til at generere "skæbne-kortlagt" og uudsletteligt mærket EF, som opretholder ZsGreen fluorescens i kultur 10. Med mindre justeringer af protokollen, bør denne metode kunne tilpasses forskellige tumortyper eller normale væv.
Protocol
Representative Results
Discussion
På grund af de problemer med at opnå rene primære TEC kulturer, mange in vitro-undersøgelser erstatning TEC med kommercielt tilgængelige EF linjer eller primær EF såsom menneskelig navlestrengen vene EF (HUVEC) 13. Dog kan disse EF befolkninger mod normalt væv kun tjene som en proxy for TEC, der markant adskiller sig fra deres normale modstykker. For eksempel TEC er fænotypisk og funktionelt unormale in vivo og nogle af disse abnormaliteter kan være smitsomme in vitro <sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.
Materials
| Antibiotic-Antimycotic | Sigma-Aldrich | A5955 | |
| Dulbecco's Modified Eagle's medium (1 g/L D-glucose) (LG-DMEM) | Gibco | 11885-084 | |
| EGM-2 Bullet Kit | Lonza | CC4176 | Not all components used |
| Fetal bovine serum (Hyclone) | Thermo Scientific | SH30071.03 | Heat inactivated at 56°C for 30 min |
| Nu-Serum IV | Corning | CB-51004 | |
| Hank's Balanced Salt Solution (HBBS) | Gibco | 14175-095 | |
| Phosphate-buffered saline (PBS) | Gibco | 14190-144 | |
| FACS buffer | – | – | 0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 μm filter |
| 75% v/v ethanol for disinfection | – | – | |
| Anti-PE microbeads | Miltenyi Biotech | 130-048-801 | |
| Bovine serum albumin (BSA) fraction V, 7.5% | Gibco | 15260-37 | |
| Cell freezing media (Bambanker) | Wako Chemicals | 302-14681 | |
| Collagenase type II | Worthington Biochemical | LS004176 | Make stock concentration 2 mg/ml in HBSS |
| Deoxyribonuclease I (DNase) | Worthington Biochemical | LS002004 | Make stock concentration 1 mg/mL in PBS |
| Dil-Ac-LDL | Biomedical Technologies | BT-902 | |
| EDTA, 0.5M, pH 8.0 | Cellgro | 46-034-CL | |
| Enzymatic cell detachment solution (Accutase) | Sigma-Aldrich | A6964-100ML | |
| Gelatin, 2 % in water, tissue culture grade | Sigma-Aldrich | G1393-100ML | Dilute in PBS to make 0.5 % gelatin solution |
| Mouse FcR Blocking Reagent | Miltenyi Biotech | 130-092-575 | |
| Neutral protease (Dispase) | Worthington Biochemical | LS02104 | Make stock concentration 2.5 U/mL in HBSS |
| PE-rat anti-mouse CD31 antibody | BD Pharmingen | 553373 | |
| RBC lysis buffer (BD Pharm Lyse) | BD Pharmingen | 555899 | |
| Sterile water | – | – | |
| Trypan blue, 0.4 % | Life Technologies | 15250-061 | |
| 10 mm tissue culture dishes | Corning | – | |
| 15 mL conical tubes (sterile) | Corning | – | |
| 50 mL conical tubes (sterile) | Corning | – | |
| 6-well tissue culture plates | Corning | – | |
| Tissue-dissociator tubes (gentleMACS) C tubes) | Miltenyi Biotech | 130-093-237 | |
| Cell Separator (MidiMACS) | Miltenyi Biotech | 130-042-302 | |
| Cell strainer 100 μm | Corning | 352360 | |
| Cloning rings (assorted sizes) | Bel-Art Products | 378470000 | |
| Cryotubes | Thermo Scientific | – | |
| Dissecting board | – | – | Sterilize or disinfect with 75% v/v ethanol before use |
| Dissecting forceps and scissors | – | – | Sterilize before use |
| Dissecting pins 2" | – | – | Sterilize before use |
| FACS tubes with 35 μm filter cap | Corning | 352235 | |
| Filter cup (Stericup, 0.22 μm) | Millipore | SCGPU05RE | |
| Fine-tip marker | – | – | |
| Hemocytometer | – | – | |
| LS Columns | Miltenyi Biotech | 130-042-401 | |
| Magnetic Multistand | Miltenyi Biotech | 130-042-303 | |
| Tissue adhesive (Vetbond) | 3M | 1469SB | |
| Centrifuge | Eppendorf | 5810R | Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation |
| Tissue culture hood | – | – | |
| Tissue dissociator (gentleMACS) | Miltenyi Biotech | 130-093-235 | Preset program "m_impTumor_01" used for tissue dissociation |
| Liquid nitrogen freezer | – | – | |
| Microplate or rotary shaker | – | – | |
| Phase contrast light microscope | – | – |
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