Isolering af cirkulerende tumorceller i en ortotopisk musemodel af kolorektal cancer
Summary
Vi beskriver etableringen af ortotopiske kolorektale tumorer via injektion af tumorceller eller organoider i musens cecum og den efterfølgende isolering af cirkulerende tumorceller (CTC'er) fra denne model.
Abstract
På trods af fordelene ved let anvendelighed og omkostningseffektivitet har subkutane musemodeller alvorlige begrænsninger og simulerer ikke nøjagtigt tumorbiologi og tumorcelleformidling. Ortotopiske musemodeller er blevet introduceret for at overvinde disse begrænsninger; Imidlertid er sådanne modeller teknisk krævende, især i hule organer som tyktarmen. For at producere ensartede tumorer, som pålideligt vokser og metastaserer, er standardiserede teknikker til fremstilling af tumorceller og injektion kritiske.
Vi har udviklet en orthotopisk musemodel af kolorektal cancer (CRC), der udvikler stærkt ensartede tumorer og kan anvendes til tumorbiologi studier samt terapeutiske forsøg. Tumorceller fra enten primære tumorer, 2 -dimensionelle (2D) cellelinier eller 3-dimensionelle (3D) organoider injiceres i cecum og afhænger af metastatiske potentialer af de injicerede tumorceller danner stærkt metastaserede tumorer. Desuden,CTC'er findes regelmæssigt. Her beskriver vi teknikken for tumorcellepræparation fra både 2D-cellelinier og 3D-organoider såvel som primær tumorvæv, kirurgiske og injektionsteknikker samt isolering af CTC'er fra de tumorbærende mus og aktuelle tips til fejlfinding.
Introduction
Kolorektal cancer (CRC) er en af de mest almindelige årsager til kræftdød i de vestlige lande. 1 Mens den primære tumor ofte kan resekteres, forværrer fjerntliggende metastaser dramatisk prognosen og fører ofte til døden. 2 , 3 Den biologiske korrelat af metastase er cirkulerende tumorceller (CTC'er), som løsner fra tumoren, overlever i omløb, fastgøres til epitelet i målorganet, invaderer orgelet og i sidste ende vokser ud til nye læsioner. 4 Selvom CTCs er kendt for at være af prognostisk relevans, er deres biologi kun delvis forstået som følge af deres ekstreme sjældenhed i CRC, 5 , 6 , 7 , 8 , 9 . 10
Musemodeller er en kraftig tOol at studere forskellige aspekter af kræftbiologi. Klassiske subkutane tumormodeller fremstilles ved subkutan injektion af tumorceller i modtagermus, som kan være enten immunokompetente (hvis syngene murine tumorceller anvendes) eller immunodeficient. Subkutane tumormodeller er billige og producerer data hurtigt; Deres slutpunkt tumor vækst kan let og ikke-invasivt måles. Imidlertid mislykkes 88% af de nye forbindelser, der har påvist antitumoraktivitet i sådanne modeller i kliniske forsøg. 11 Dette skyldes dels forskelle mellem mennesker og mus; En stor del af dette svigt skyldes imidlertid den lave prædiktive værdi af subkutane musemodeller.
Ortotopiske musemodeller, hvor tumorcellerne injiceres i oprindelsesorganet og således vokser i deres oprindelige mikromiljø, anvendes derfor i stigende grad i kræftforskning. 11 , 12 , </sUp> 13 , 14 Ortotopiske modeller simulerer ikke kun lokale tumorvækstbetingelser; Som et resultat af det anatomisk korrekte sted for tumorvækst tillader orthotopiske musemodeller også realistisk simulering af metastase og bruges derfor til at studere CTC biologi 8 , 15 , 16 eller deres respons på forskellige behandlinger i CRC. 13 , 17
En stor ulempe ved orthotopiske musemodeller er deres tekniske kompleksitet. Afhængigt af orglet, hvor cellerne skal injiceres, er læringskurven indtil eksperimenten i stand til at inducere reproducerbare tumorer ret lang. Dette gælder især for kolorektale kræftmodeller, da tumorcellerne skal injiceres i tarmvæggen, hvilket ofte resulterer i perforering, tumorcellelækage eller endoluminalt tumorcelletab. DetRticle er beregnet til at beskrive fremgangsmåden til cellepræparation fra primære vævsprøver, 2D cellelinier og 3D organoid kultur og deres injektion i cecum af mus. Teknikken beskrevet her fører til stærkt ensartede tumorer og afhænger af tumorbiologien af den cellelinie, der anvendes til injektion, reproducerbar dannelse af fjerne metastaser og CTC'er i modtagermuserne. 15
Protocol
Representative Results
Discussion
På trods af deres præklinisk dokumenterede aktivitet i subkutane musemodeller fejler det store flertal af nye forbindelser i kliniske forsøg og når aldrig klinikken. 11 Denne åbenlyse mangel på subkutane musemodeller til nøjagtigt at simulere tumorens biologi og vækstmønstre har ført til udviklingen af orthotopiske musemodeller baseret på injektion af tumorceller direkte ind i det oprindelige organ.
Ortotopiske musemodeller er i stand til at simulere …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af den tyske forskningsstiftelse (WE 3548 / 4-1) og Roland-Ernst-Stiftung für Gesundheitswesen (1/14).
Materials
| Cell culture Media and Components | |||
| Advanced DMEM F12 | Invitrogen | 12634010 | DMEM/ F12 +++ medium |
| HEPES (1 M) | Life Technologies GmbH | 15630056 | DMEM/ F12 +++ medium |
| Glutamax-I Supplement (200 mM) | Life Technologies GmbH | 35050038 | DMEM/ F12 +++ medium |
| Penicillin/Streptomycin (PenStrep) | Life Technologies GmbH | 15140122 | DMEM/ F12 +++ medium |
| DMEM | Life Technologies GmbH | 61965026 | basic medium of 2D cell lines (DMEM/10%FCS) |
| Fetal Calf Serum (FCS) | BIOCHROM AG | S 0115 | basic medium of 2D cell lines (DMEM/10%FCS) |
| TrypLE Express enzymatic dissociation buffer | Life Technologies GmbH | 12604021 | |
| Matrigel basement membrane matrix (BMM, phenol red free) | CORNING B.V. Life Sciences | 356231 | |
| Dulbecco's Phosphate Buffered Saline | Life Technologies GmbH | 14190169 | |
| Trypsin-EDTA (0,25%, Phenol-Red) | Life Technologies GmbH | 25200072 | |
| 6-/48-well plates with lid | CORNING | 3516/3548 | |
| cell culture flask 75cm², 250 mL | VWR International GmbH | 734-2066 | |
| cell culture flask 150cm², 600 mL | Corning B.V. Life Sciences | 355001 | |
| Eppendorf tubes 1,5 mL / 2 mL | Sarstedt AG & Co. | 72.706.400/ 72.695.400 | |
| 15 ml, 50 ml centrifuge tubes | Greiner-Bio-One GmbH | 188271/227270 | |
| TC10 Counting Slides (for TC20 Counting Machine) | Bio-Rad Laboratories GmbH | 1450016 | |
| Pasteur pipettes (glass, 150 mm) | Fisher Scientific GmbH | 11546963/ FB50251 | thinly pulled by using a bunsen burner |
| gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | for primary tumor tissue preparation |
| MACSmix Tube Rotator | Miltenyi Biotec | 130-090-753 | for primary tumor tissue preparation |
| gentleMACS C Tubes | Miltenyi Biotec | 130-093-237 | for primary tumor tissue preparation |
| Human Tumor Dissociation Kit | Miltenyi Biotec | 130-095-929 | for primary tumor tissue preparation |
| Falcon 70µm Cell Strainer | Corning B.V. Life Sciences | 352350 | for primary tumor tissue preparation |
| Name | Company | Catalog Number | Comments |
| Surgical Equipment | |||
| Sevoflurane | AbbVie Germany GmbH & Co. KG | – | |
| Medical oxygen | Air Liquide Medical GmbH | – | |
| Buprenorphine | Temgesic | – | |
| Bepanthen – opthalmic ointment | Bayer Vital GmbH | 10047757 | |
| Normal saline 0.9% (E154) | Serumwerk Bernburg AG | 10013 | |
| Aqua ad injectabilia | Braun | 235144 | |
| 1 mL Syringe (without dead volume) – Injekt-F SOLO | Braun/neoLab | 194291661 | |
| 30G injection needle | BECTON DICKINSON | 304000 | |
| cellulose swabs | Lohmann & Rauscher Deutschland | 13356 | |
| Micro-Adson Forceps | FST – Fine Science Tools | 11018-12 | |
| Iris Scissor – ToughCut | FST – Fine Science Tools | 14058-11 | |
| Olsen-Hegar Needle Holder | FST – Fine Science Tools | 12002-12 | |
| AutoClip Kit | FST – Fine Science Tools | 12020-00 | |
| PDS Z1012H 6/0 C1 (surgical suture) | Johnson & Johnson Medical GmbH | Z1012H | |
| Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer | Parkland Scientific | V3000PS/PK | |
| UltraMicro Pump with Micro4 Controller | World Precision Instruments | UMP3-4 | equipment for highly controlled orthotopic injection |
| Footswitch for SYS-Micro4 Controller | World Precision Instruments | 15867 | equipment for highly controlled orthotopic injection |
| Three-axis Manual Micromanipulator | World Precision Instruments | M325 | equipment for highly controlled orthotopic injection |
| Magnetic Stand for Micromanipulator | World Precision Instruments | M10 | equipment for highly controlled orthotopic injection |
| Steel Base Plate for M10 Magnetic Stand | World Precision Instruments | 5479 | equipment for highly controlled orthotopic injection |
| Hot Plate 062 | Labotect | 13854 | |
| Isis – Hair shaver | AESCULAP – Braun | – | |
| Binocular Surgical Microscope | Parkland Scientific | VS-2Z | |
| Name | Company | Catalog Number | Comments |
| CTC isolation | |||
| EDTA | Roth | 8040.1 | |
| Density gradient medium – Ficoll | StemCell – Lymphoprep | 7801 | |
| Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4 | BioLegend | 324210 | |
| Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8 | BioLegend | 118210 | |
| Petri Dish, ø 60 x 15 mm, 21 cm², Vent | Greiner bio-one | 628102 | |
| Fluorescence Cell Culture Microscope | Leica | ||
| Transferman 4r Micromanipulator | Eppendorf | ||
| CellTram Air | Eppendorf | aspiration pump connected to the micromanipulator | |
| Dmz Universal Microelectrode Puller | Dagan Corporation | required for the manufacturing of micro capillaries for single cell aspiration | |
| Prism Glass Capillaries | Dagan Corporation | ||
| PAP pen | Abcam | ab2601 | |
| Dulbecco's Phosphate Buffered Saline | Life Technologies GmbH | 14190169 | picking buffer |
| Fetal Calf Serum (FCS) | BIOCHROM AG | S 0115 | picking buffer |
| Penicillin/Streptomycin (PenStrep) | Life Technologies GmbH | 15140122 | picking buffer |
| EDTA | Roth | 8040.1 | picking buffer |
| Name | Company | Catalog Number | Comments |
| Immunohistochemistry | |||
| Purified anti-human CD326 (EpCAM) antibody clone 9C4 | BioLegend | 324201 | EpCAM immunohistochemistry (cf, fig 2C) |
| HRP rabbit anti-mouse IgG | Abcam | ab97046 | EpCAM immunohistochemistry (cf, fig 2C) |
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