Mieloma multiplo (MM), le cellule umane richiedono il microambiente favorevole cellule mesenchimali e componenti della matrice extracellulare per la sopravvivenza e la proliferazione. Abbiamo stabilito un vivo pollo modello embrione con cellule di mieloma e mesenchimali umane trapiantate per studiare gli effetti dei farmaci anti-tumorali sulla crescita del tumore, l'invasione e l'angiogenesi.
Il mieloma multiplo (MM), una malattia delle cellule del plasma maligne, resta incurabile e nuovi farmaci sono necessari per migliorare la prognosi dei pazienti. A causa della mancanza del microambiente dell'osso e fattori di crescita auto / paracrino cellule MM umane sono difficili da coltivare. Pertanto, non vi è un urgente bisogno di stabilire una corretta in vitro e in sistemi di coltura in vivo per studiare l'azione di nuove terapie sulle cellule umane di MM. Presentiamo qui un modello di crescere cellule di mieloma multiplo umano in un ambiente 3D complesso in vitro e in vivo. Linee cellulari di MM OPM-2 e RPMI-8226 sono state trasfettate per esprimere la GFP transgene e sono stati coltivati in presenza di cellule mesenchimali umane e collagene di tipo I matrice come sferoidi tridimensionali. Inoltre, sferoidi sono stati innestati sulla membrana corioallantoidea (CAM) di embrioni di pollo e la crescita del tumore è stata monitorata mediante microscopia a fluorescenza stereo. Entrambi i modelli consentono lo studio del romanzo dru terapeuticogs in un ambiente 3D complesso e la quantificazione della massa tumorale dopo omogeneizzazione di innesti in un GFP-ELISA specifico transgene. Inoltre, le risposte angiogenici dell'ospite e l'invasione delle cellule tumorali nel tessuto ospite soggiacente possono essere monitorate quotidianamente da un microscopio stereo e analizzati mediante immunoistochimica contro le cellule tumorali umane (Ki-67, CD138, Vimentin) o cellule murali ospiti che coprono i vasi sanguigni (desmin / ASMA).
In conclusione, il sistema permette onplant studiare crescita cellulare MM e l'angiogenesi in un ambiente 3D complesso e permette lo screening per composti terapeutici mirati sopravvivenza e la proliferazione delle cellule di MM.
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.
Lo sviluppo di nuovi agenti terapeutici per refrattario MM richiede meno tempo e costosi sistemi in vivo per valutare la sensibilità di cellule MM umane ai farmaci. Finora, solo pochi sistemi in vivo sono a disposizione per la valutazione preclinica di nuove terapie anti-mieloma. Tutti loro hanno i loro limiti per lo screening su larga scala di librerie di composti 29.
I migliori modelli attuali di cellule di MM umane sono altamente immuni topi con deficit 7,13,30 ed embrioni di tacch…
The authors have nothing to disclose.
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).
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 |