En kombineret 3D manipuleret væv<em> In vitro</em> /<em> I Silico</em> Lunge Tumor Model for Forudsigelse Drug Effektivitet i specifikke mutations baggrunde
Summary
We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.
Abstract
I den foreliggende undersøgelse, vi kombinerede en in vitro 3D lungetumor model med en in silico model til at optimere forudsigelser af lægemiddel respons baseret på en specifik mutationel baggrund. Modellen er genereret på en decellulariseret porcint stillads, der gengiver vævsspecifikke karakteristika med hensyn ekstracellulære matrix sammensætning og arkitektur, herunder basalmembranen. Vi standardiseret en protokol, der tillader kunstig tumorvæv generation inden for 14 dage, herunder tre dages medicinsk behandling. Vores artikel indeholder flere detaljerede beskrivelser af 3D udlæsning screening teknikker som bestemmelse af spredning indeks Ki67 farvning s, apoptose fra supernatanter af M30-ELISA og vurdering af epitelial til mesenkymale overgang (EMT), som er nyttige redskaber til evaluering af effektiviteten af terapeutiske forbindelser. Vi kunne vise i forhold til 2D kultur en reduktion af spredning i vores 3D-tumor model, der er vedrøed til den kliniske situation. Trods af denne lavere proliferation, modellen forudsagte EGFR -targeted drug reaktioner korrekt i overensstemmelse med biomarkør status som vist ved sammenligning af lunge carcinoma cellelinier HCC827 (EGFR -mutated, KRAS vildtype) og A549 (EGFR vildtype, KRAS – muteret) behandlet med tyrosin-kinase-inhibitor (TKI) gefitinib. For at undersøge drug reaktioner af mere avancerede tumorceller, vi induceret EMT ved langtidsbehandling med TGF-beta-1 som vurderet ved vimentin / pan-cytokeratin immunfluorescensfarvning. En strøm-bioreaktor blev anvendt til at justere kultur til fysiologiske betingelser, hvilket forbedrede væv generation. Desuden viser vi integrationen af narkotika reaktioner på gefitinib behandling eller TGF-beta-1 stimulation – apoptose, spredning indeks og EMT – ind i en boolesk i silico model. Desuden har vi forklare, hvordan narkotika reaktioner af tumorceller med en bestemt mutations baggrund og tællererstrategies mod modstand kan forudsiges. Vi er overbeviste om, at vores 3D in vitro tilgang især med sin i silico udvidelse giver en ekstra værdi for præklinisk narkotika-test i mere realistiske forhold end i 2D cellekultur.
Introduction
Den farmaceutiske industri står over for høje opgivelsesprocent på op til 95% inden for kræftbehandling i det kliniske fase forårsager enorme omkostninger 1-5. En af grundene til dette underskud er, at i øjeblikket effekten af potentielle nye forbindelser vurderes i store screeninger på 2D cellekulturer af kræft cellelinjer eller i dyremodeller. Dyremodeller har en højere kompleksitet, men der er afgørende forskelle mellem mus og mænd 6,7. I det sidste årti, har 3D kræftmodeller ved hjælp af forskellige metoder er genereret til at bygge bro mellem 2D kultur cancer cellelinjer og en kompleks in vivo tumor 6,8,9. Virkningen af 3D-miljø på celledifferentiering og også på signalering er blevet vist i adskillige undersøgelser år siden (f.eks. Ved Mina Bissell) 10,11. I dag, mange 3D-cellekultur modeller er til rådighed, såsom kugleformede kulturer, hydrogeler eller mikrofluide chips 12-16. Selvom THESe modeller øge kompleksiteten sammenlignet med konventionelle 2D dyrkningssystemer, de for det meste mangler et væv mikromiljø, der vides at have tumor-understøttende påvirkninger samt påvirkninger lægemiddeleffektivitet.
For at løse dette problem, vi genereret en 3D-tumor model baseret på en biologisk stillads kaldet SISmuc (lille-tarm-submucosa + slimhinde), der er afledt af en decellulariseret porcin jejunum. Derved vævsarkitekturen og vigtige bestanddele af ECM såsom forskellige kollagener samt basalmembranen struktur bevares 17. Denne unikke egenskab er afgørende for tumormodel generation af karcinomer, der opstår fra epitel og omfatter omkring 80% af faste tumorer. Endvidere er proliferationshastighed i vores væv-manipuleret tumormodel reduceret i forhold til de kunstigt høje opnået i 2D kultur. Som spredning er en vigtig parameter i vurderingen af narkotika effekt, er narkotika testning aktiveret i vores model i mere ensbetingelser til in vivo tumorer 17.
For at vurdere potentialet i vores model til at forudsige biomarkør-afhængige narkotika effekt korrekt, vi her præsentere data for to forskellige lungekræft cellelinier, der adskiller sig i deres EGFR -biomarker status. Denne mutationsstatus er begyndt at blive bestemt rutinemæssigt i NSCLC patienter. Målrettede behandlinger med TKI'er såsom EGFR inhibitor gefitinib mod tumorer, der bærer en aktiverende EGFR mutation viser overlegne resultater sammenlignet med dem med platinbaseret kemoterapi 18-21.
Vi har etableret flere read-out teknikker, der er relevante for at vurdere sammensatte effekt. Desuden efter TGF-beta-1 stimulation er vi i stand til at undersøge sammensatte handlinger i tumorceller, der startede EMT proces, der menes at være et vigtigt skridt i malign transformation 22,23, og som er forbundet til narkotika resistance 24.
3D tumor model tillader overvågning celle-specifikke reaktioner på målrettede behandlinger, kemoterapi eller narkotika kombinationer med gode kontraster. For yderligere at styrke og fremskynde lægemiddel-screening og at møde modstand, dette suppleret med en i silico simulering. Baseret på et par eksperimenter, kan tumorrespons forudsiges i silico om resultatet for en bred vifte af lægemidler og deres kombinationer.
Protocol
Representative Results
Discussion
Vi har etableret en kombineret in vitro / in silico tumor testsystem til biomarkør-styret behandling forudsigelser. In vitro model evaluerer forskellige vigtige aspekter af sammensatte handlinger såsom ændringer af tumorcelleproliferation og apoptose på en specifik mutationel baggrund, der også kan simuleres i silico 17. Her præsenteres den standardiserede protokol for 3D tumor model generation og sammensatte test herunder kvantificering af proliferation og apoptose og etabler…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev sponsoreret af Center for Tværgående Clinical Research (IZKF, tilskud BD247) fra University Hospital i Würzburg og Bayern Fit-programmet (tildelt Heike Walles).
Materials
| Bioreactors | Chair of Tissue Engineering and Regenerative Medicine, Würzburg (GER) | Bioreactor setup | |
| BioVoxxel Toolbox (ImageJ / Fiji) | Jan Brocher, Thorsten Wagner, https://github.com/biovoxxel/BioVoxxel_Toolbox | ||
| Cell crowns | Chair of Tissue Engineering and Regenerative Medicine, Würzburg (GER) | for static 3D culture | |
| CellDesigner | http://www.celldesigner.org/ | – | This software was used for drawing the network. |
| citrate buffer stock solution (10x) | in house production | 42 g/l Citric acid monohydrate, 17.,6 g/l Sodium hydroxide pellets in deionized water, pH 6,.0, stored at RT. | |
| citrate buffer working solution | in house production | 10 % Citrate buffer stock solution in demineralized water, stored at RT. | |
| Citric acid monohydrate | VWR, Darmstadt (GER) | 1002441000 | used for the citrate buffer |
| Cover slips | VWR, Darmstadt (GER) | 631-1339 | |
| DAPI Fluoromount-GTM | SouthernBiotech, Birmingham (USA) | SBA-0100-20 | |
| Databases such as KEGG, HPRD and QIAGEN (Genes & Pathways) | http://www.genome.jp/kegg/pathway.html; http://www.hprd.org/; https://www.qiagen.com/de/geneglobe/ | – | Different known literature databases were used for generating the network topology. |
| Female Luer Lug Style Tee | Mednet, Münster (GER) | FTLT-1 | Bioreactor setup |
| Female Luer Thread Style with 5/16" Hex to 1/4-28 UNF Thread | Mednet, Münster (GER) | SFTLL-J1A | Bioreactor setup |
| Fetal calf serum | Bio&SELL, Feucht (GER) | FCS.ADD.0500 | not heat-inactivated |
| Gefitinib | Absource Diagnostics GmbH, München (GER) | S1025-100 mg | 100 mM stock solution with DMSO |
| Glas flask (Schott, GER) provided with glas hose connection | Weckert, Kitzingen (GER) | custom made | |
| Histofix 4 % (Paraformaldehyd) | Carl Roth, Karlsruhe (GER) | P087.1 | |
| Hose coupling | Mednet, Münster (GER) | CC-9 | Bioreactor setup |
| Incubator for bioreactors | Chair of Tissue Engineering and Regenerative Medicine, Würzburg (GER) | Bioreactor setup | |
| M30 CytoDeathTM ELISA | Peviva, Bromma (SWE) | 10900 | |
| Male Luer Integral Lock Ring | Mednet, Münster (GER) | MTLL230-J1A | Bioreactor setup |
| Moisture chamber | custom made | ||
| Mouse anti Pan-Cytokeratin | Sigma-Aldrich, Munich (GER) | C2562-2ML | Clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2, used 1/100 for immunofluorescence |
| Needlefree Swabable Valve Female Luer | Mednet, Münster (GER) | NVFMLLPC | Bioreactor setup, for sampling, gamma-sterilized |
| O-Ring MVQ 10 red 37*3 mm | Arcus Dichtelemente, Seevetal (GER) | 21444 | O-ring large, Bioreactor setup |
| O-Ring MVQ 70 red 27*2.5 mm | Arcus Dichtelemente, Seevetal (GER) | 19170 | O-ring small, Bioreactor setup |
| PAP pen | Dako, Hamburg (GER) | S002 | |
| Paraffin | Carl Roth, Karlsruhe (GER) | 6642.6 | |
| Peristaltic pump | Ismatec, Wertheim-Mondfeld (GER) | Bioreactor setup | |
| Phosphate Buffered Saline | Sigma-Aldrich, Munich (GER) | D8537-6x500ml | |
| Pump tubing cassette | Ismatec, Wertheim (GER) | IS 3710 | Bioreactor setup |
| Rabbit anti Ki67 | Abcam, Cambridge (UK) | ab16667 | Clone SP6, used for 1/100 for IF |
| Rabbit anti Vimentin | Abcam, Cambridge (UK) | ab92547 | used 1/100 for IF |
| RPMI-1640 medium | Life technologies, Darmstadt (GER) | 61870-044 | warm in 37°C waterbath before use |
| Silicone tube | Carl Roth GmbH, Karlsruhe (GER) | HC66.1 | Bioreactor setup |
| Sodium Hydroxide | Sigma-Aldrich, München (GER) | 30620-1KG-R | used for the citrate buffer |
| SQUAD | http://sbos.eu/docu/docu/SQUAD/doku.php.htm | – | This software was used for performing the semiquantitative simulations. |
| Sterile air filter, pore size 0.2 µm | Sartorius Stedium Biotech, Göttlingen (GER) | 16596-HYK | Bioreactor setup |
| Syringe Luer Lok 5ml | BD Biosciences, Heidelberg (GER) | 309649 | for bioreactor sampling |
| Tissue culture test plates: 6-, 12-, 24-, 96- well | TPP Techno Plastic Products AG, Trasadingen (GER) | 92006, 92012, 92024, 92048 | |
| Transforming growth factor-beta 1 (TGF-β1) with carrier | Cell Signaling, Frankfurt (GER) | 8915LC | stock solution in sterile citrate buffer pH 3.0 |
| Triton X-100 | Sigma-Aldrich, München (GER) | X100-1L | |
| Tween-20 | Sigma-Aldrich, München (GER) | P7949-500ml | for washing buffer of immunofluorescent staining |
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