Humanen multiplen Myelom (MM) Zellen erfordern die Mikroumgebung, die von mesenchymalen Zellen und extrazellulären Matrixkomponenten für das Überleben und die Proliferation. Wir gründeten eine in vivo Hühnerembryo-Modell mit eingepflanzten menschlichen Myelom und mesenchymalen Zellen zu Wirkungen von Krebsmedikamenten auf das Tumorwachstum, Invasion und Angiogenese zu untersuchen.
Das Multiple Myelom (MM), einem bösartigen Plasmazellerkrankung, nicht heilbar und neuartige Medikamente sind erforderlich, um die Prognose von Patienten zu verbessern. Durch das Fehlen der Knochenmikroumgebung und auto / parakrine Wachstumsfaktoren menschlichen MM-Zellen sind schwierig zu kultivieren. Es besteht daher ein dringender Bedarf in vitro und in vivo-Kultursystemen richtige, um festzustellen, um die Aktion neuartiger Therapeutika auf die menschliche MM-Zellen zu untersuchen. Hier präsentieren wir ein Modell für die menschliche multiple Myelomzellen in einem komplexen 3D-Umgebung in vitro und in vivo zu wachsen. MM-Zelllinien OPM-2 und RPMI-8226 transfiziert wurden, um die GFP-Transgen exprimieren und wurden in Gegenwart von humanen mesenchymalen Zellen und Kollagen kultivierten Typ-I-Matrix als dreidimensionale Sphäroide. Zusätzlich wurden Sphäroide auf der Chorioallantoismembran (CAM) des Hühnerembryos gepfropft und das Tumorwachstum wurde durch Stereofluoreszenzmikroskopie überwacht. Beide Modelle ermöglichen die Untersuchung von neuartigen therapeutischen drugs in einer komplexen 3D-Umgebung und die Quantifizierung der Tumorzellmasse nach der Homogenisierung der Transplantate in einer Transgen-spezifischen GFP-ELISA. Außerdem kann angiogene Reaktionen des Wirtes und Invasion von Tumorzellen in das darunterliegende Wirtsgewebe täglich durch ein Stereomikroskop beobachtet und durch immunhistochemische Färbung gegen menschliche Tumorzellen (Ki-67, CD138, Vimentin) oder Host mural Zellen abdeckt Blutgefße analysierende (Desmin / ASMA).
Abschließend kann der Onplantat System studieren MM Zellwachstum und die Angiogenese in einem komplexen 3D-Umgebung und ermöglicht Screening nach neuen therapeutischen Verbindungen Targeting Überleben und die Proliferation von MM-Zellen.
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.
Die Entwicklung neuer therapeutischer Mittel zur feuerfesten MM erfordert weniger zeitaufwendig und teuer in-vivo-Systemen, um die Empfindlichkeit des menschlichen Myelomzellen, um Medikamente zu bewerten. Bisher sind nur wenige in-vivo-Systeme für die präklinische Evaluierung neuer anti-Myelom-Therapien zur Verfügung. Alle von ihnen haben ihre Grenzen für große Screening von Substanzbibliotheken 29.
Die derzeit besten Modelle für menschliche MM-Zellen sind sehr immun-defizienten Mäusen <s…
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 |