Mise en place d'un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l'invasion et l'angiogenèse
Summary
Les cellules humaines myélome multiple (MM) nécessitent le micro-environnement favorable des cellules mésenchymateuses et des composants de la matrice extracellulaire pour la survie et la prolifération. Nous avons établi un modèle in vivo d'embryons de poulet avec des cellules de myélome et mésenchymateuses humaines greffées à étudier les effets des médicaments contre le cancer sur la croissance tumorale, l'invasion et l'angiogenèse.
Abstract
Le myélome multiple (MM), une maladie des cellules plasmatiques malignes, reste incurable et de nouveaux médicaments sont nécessaires pour améliorer le pronostic des patients. En raison de l'absence du microenvironnement osseux et des facteurs de croissance auto / paracrine des cellules MM humaines sont difficiles à cultiver. Par conséquent, il ya un besoin urgent d'établir une bonne in vitro et dans les systèmes de culture in vivo pour étudier l'action de nouveaux produits thérapeutiques sur les cellules de MM humaines. Ici, nous présentons un modèle à cultiver des cellules du myélome multiple humain dans un environnement 3D complexe in vitro et in vivo. Des lignées cellulaires MM OPM-2 et le milieu RPMI-8226 ont été transfectées pour exprimer le transgène GFP et ont été cultivées en présence de cellules mésenchymateuses humaines et de collagène de type I Matrice sphéroïdes tridimensionnels. En outre, les sphéroïdes ont été greffés sur la membrane chorio-allantoïde (CAM) d'embryons de poulet et la croissance tumorale a été contrôlée par microscopie à fluorescence stéréo. Les deux modèles permettent l'étude de roman dru thérapeutiquegs dans un environnement 3D complexe et la quantification de la masse des cellules tumorales après des greffes homogénéisation dans un transgène GFP spécifique au test ELISA. En outre, les réponses angiogéniques de l'hôte et l'invasion des cellules tumorales dans le tissu de l'hôte sous-jacent peuvent être surveillées quotidiennement par un microscope stéréoscopique et analysés par coloration immunohistochimique à l'encontre des cellules humaines tumorales (Ki-67, CD138, Vimentin) ou des cellules murales hôtes couvrant vaisseaux sanguins (desmine / ASMA).
En conclusion, le système onplant permet d'étudier la croissance des cellules MM et l'angiogenèse dans un environnement 3D complexe et permet le criblage de composés thérapeutiques ciblant la survie et la prolifération des cellules MM.
Introduction
Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency 1. Although new treatment options such as proteasome inhibitors (bortezomib) and immune modulatory drugs (pomalidomide and lenalidomide) are available, MM still remains an incurable malignancy with a grim prognosis 2. The bad prognosis might be explained by the extraordinary heterogeneity of MM cell clones that contributes to variable responses to therapy, in particular under long time treatment and selection pressure of MM clones 3.
Preclinical testing of new drugs and their combinations in vitro and in vivo is a critical and time-consuming step for future drug development. Thus, useful in-vivo models of MM are required to gain a better understanding of the biology of the disease and to enable the discovery of new drugs. Actually, the best xenotransplantation models for hematological malignancies and therapeutics are immune-deficient mice, such as the severe-combined immunodeficient (SCID) mice 4-7, the non-obese diabetic/SCID (NOD/SCID) mice 8,9 or the β-microglobulin-knockout NOD/SCID mice 10,11.
Although murine models of human MM in some aspects can resemble the phenotype of human disease, immune-deficient mice are inbred, therefore simulate only one individual response to a drug and costs are very high. Due to immunosuppression animals require special maintenance conditions and the engraftment of human MM in mice requires 6 weeks to 2 months 9,12, unless cells are grafted directly to the bone marrow using a technically demanding procedure with lower rates of animal survival 7,13. Therefore, new methods using stem-cell based organoid models 14, tissue engineering 15 or sophisticated 3D cell culture models 16 have been established. They will compete in the near future with classical animal experiments for preclinical drug testing, but cannot replace systemic toxicity tests in living organisms.
The chicken embryo has been demonstrated before to be a suitable organism for xenotransplantation of human cells and tissues due to lack of adaptive immune response until hatching 17-19. Moreover, each chicken embryo reflects an individual reaction to applied drugs or tumor cells due to genetic diversity within the chicken population. The chorioallantoic membrane (CAM) is a well-established system to study tumor-dependent angiogenesis 20-22. When solid tumors are grafted to the CAM, they display many characteristics of cancers in vivo, including proliferation, invasion, angiogenesis and metastasis 23-27.
Based on the previous experience of our group with CAM xenograft models20,26,27, a human MM model was established that combines the advantage of a human 3D culture system with the model of ex ovo developing chicken embryos. This MM model system allows real time monitoring of MM growth progression, quantification of cell mass and preclinical drug testing.
Protocol
Representative Results
Discussion
Le développement de nouveaux agents thérapeutiques pour MM réfractaire nécessite moins de temps et coûteux systèmes in vivo pour évaluer la sensibilité des cellules MM humains aux médicaments. Jusqu'à présent, seulement peu de systèmes in vivo sont disponibles pour l'évaluation préclinique de nouveaux traitements anti-myélome. Tous ont leurs limites pour le criblage à grande échelle de bibliothèques de composés 29.
Les meilleurs modèles actuels pour les cellules de MM …
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors want to thank Ms. Cornelia Heis for her excellent technical assistance in immunohistochemistry and preparation of chicken embryos. This work was supported by the Austrian Science Fund (FWF Grant No. P19552) and the European Union (EU FP7 project Optatio No: 278570).
Materials
| RPMI-8226 cells | DSMZ | ACC 9 | STR profiled |
| OPM-2 cells | DSMZ | ACC 50 | STR profiled |
| Human mesenchymal stem cells | PromoCell | PC-C-12974 | |
| HEK293FT cells | Invitrogen | R700-07 | |
| RPMI1640 Medium | Sigma Aldrich | R0883 | |
| Fetal Bovine Serum HyClone | ThermoScientific | SH30070.03 | |
| L-Glut- Pen- Strep solution | Sigma | G6784 | |
| DMEM Medium | Gibco | 31966 | |
| NEAA | Sigma Life Sciences | M7145 | |
| Transfection Medium/Opti-MEM | Gibco | 51985 | |
| eGFP lentiviral particles | GeneCopoeia | LPP-EGFP-LV105 | Ready to use viral particles |
| pLenti6/V5Dest6 eGFP vector | Invitrogen | PN 35-1271 | from authors |
| ViralpowerTM packaging mix | Invitrogen | P/N 35-1275 | |
| Transfection reagent/ Lipofectamin 2000 | Invitrogen | 11668-027 | |
| Blasticidin | Invitrogen | R210-01 | |
| Neomycin | Biochrom | A2912 | |
| Collagen-Type1 Rat Tail | BD Biosciences | 354236 | |
| DMEM powder | Life Technologies | Art.Nr. 10338582 | |
| plitidepsin | Pharmamar | ||
| bortezomib | LKT Lab., Inc. | B5871 | |
| SPF-white hen eggs | Charles River | Fertilized white Leghorn chicken eggs | |
| Plastic weighing boats | neoLab | Art.Nr. 1-1125 | for ex-ovo culture |
| Petridish square (Lids) | Simport | D210-16 | for ex-ovo culture |
| RIPA Buffer (10x) | Cell Signaling | #9806 | |
| Protease Inhibitor Tablets | Roche | 11 836 170 001 | |
| Complete Mini EDTA-free | |||
| GFP ELISA | Cell Biolabs, Inc. | AKR-121 | |
| Histocette II | Simport | M493-6 | |
| PFA 37% | Roth | 7398.1 | |
| DPBS | Lonza | BE17-512F | |
| Ethanol absolut | Normapur | 20,821,321 | |
| Roti-Histol | Roth | Art.Nr.6640.4 | |
| Paraplast | Sigma | A6330 | |
| SuperFrost Microscope Slides | R. Langenbrinck | Art.-Nr. | |
| Labor- u. Medizintechnik | 03-0060 | ||
| DakoCytomation Wash Buffer 10x | DakoCytomation | Code-Nr. | |
| S 3006 | |||
| Target Retrieval Solution (10x) pH 6,1 | DAKO | Code-Nr. | |
| S 1699 | |||
| H2O2 | Merck | ||
| m-a-hu ASMA clone 1A4 | DAKO | M0851 | |
| m-a-hu CD138 clone MI15 | DAKO | M7228 | |
| m-a-hu Vimentin clone V9 | DAKO | M0725 | |
| m-a-hu Desmin clone D33 | DAKO | M0760 | |
| m-a-hu Ki67 clone MIB-1 | DAKO | M7240 | |
| biotinylated goat- anti-mouse IgG | Vector Laboratories Inc. | BA-9200 | |
| Vectastain Elite ABC Kit | Vector Laboratories Inc. | # PK-6100 | |
| FAST DAB Tablet Set. | Sigma Biochemicals | # D4293 | |
| Mayer’s haemalaun solution | Merck | 1,092,490,500 | |
| Roti Histokitt | Roth | Art.Nr.6638.2 | |
| Bench top rotary microtome | Thermo Electron, Shandon Finesse ME+ | ||
| Tissue embedding station | Leica, TP1020 | ||
| Egg-Incubator | Grumbach | BSS160 | |
| Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light | Olympus, SZX10 | ||
| Ultra Turrax | IKA T10 | Homogenizer |
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