הקמת מודל xenograft מיאלומה נפוצה אדם בעוף לחקר צמיחת גידול, פלישה ואנגיוגנזה
Summary
תאים אנושיים מסוג מיאלומה נפוצה (MM) דורשים מייקרו-הסביבה התומכת של תאי mesenchymal ורכיבי מטריצה תאיים להישרדות ושגשוג. הקמנו in vivo מודל עובר עוף עם תאי מיאלומה וmesenchymal אדם engrafted ללמוד השפעות של תרופות לסרטן על צמיחת גידול, פלישה ואנגיוגנזה.
Abstract
מיאלומה נפוצה (MM), מחלה ממארת של תאי פלזמה, נותרת חשוכות מרפא ותרופות חדשניות נדרשות כדי לשפר את הפרוגנוזה של חולים. בשל חוסר microenvironment העצם וגורמי גדילה אוטומטי / אוטוקריני תאי MM אנושיים קשים לטפח. לכן, יש צורך דחוף בהקמה נכונה במבחנה ובמערכות תרבות vivo ללמוד את הפעולה של תרופות חדשניות בתאי MM אנושיים. כאן אנו מציגים מודל לגדל תאי מיאלומה נפוצים אנושיים בסביבת 3D מורכבת במבחנת in vivo. שורות תאי MM OPM-2 וRPMI-8226 היו transfected להביע GFP transgene ועובדו בנוכחות של תאי mesenchymal אנושיים וקולגן סוג אני מטריקס כspheroids תלת-ממדי. בנוסף, spheroids היה מורכב על קרום chorioallantoic (רמ"א) של עוברי עופות וגידול היה פיקוח על ידי מיקרוסקופ פלואורסצנטי סטריאו. שני הדגמים מאפשרים המחקר של DRU הטיפולי החדשניGS בסביבת 3D מורכבת והכימות של מסת התאים הסרטניים לאחר הומוגניזציה של שתלים בGFP-ELISA transgene הספציפי. יתר על כן, תגובות angiogenic של המארח והפלישה של תאי גידול לרקמות מארח subjacent יכולות להיות במעקב יומי על ידי מיקרוסקופ סטריאו ונותחו על ידי מכתים immunohistochemical נגד תאי גידולים בבני אדם (קי-67, CD138, vimentin) או תאי ציור קיר מארח מכסים כלי דם (Desmin / ASMA).
לסיכום, מערכת onplant מאפשרת לימוד צמיחת תאי MM ואנגיוגנזה בסביבת 3D מורכבת ומאפשרת הקרנה לתרכובות טיפוליות חדשניות מיקוד הישרדות והתרבות של תאי 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
הפיתוח של תרופות חדשות לMM עקשן דורש פחות זמן רב ומערכות ב- vivo יקרים להעריך רגישות של תאי MM אנושיים לתרופות. עד כה, רק מעטים מערכות ב- vivo זמינות להערכה פרה-קלינית של טיפולים אנטי-מיאלומה חדשות. לכולם יש את המגבלות שלהם להקרנה בקנה מידה גדולה של ספריות מתחם 29.
…
Disclosures
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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