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).

על פי החוק האוסטרי, והמשרד של בעלי חיים במעבדת רווחה של עוברי עופות שירות בריאות הציבור האמריקאים אינם נחשבים כבעלי חיים בעלי חוליות חיים עד משרד NIH hatching.The של בעלי חיים במעבדת רווחה סיפק הדרכה נכתבה בתחום זה (http: // www.grants.nih.gov/grants/olaw/references/ilar91.htm ופרסום NIH לא .: 06-4515). 1. תא התרבות וlentiviral Transfection <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שורות תאי MM תרבות OPM-2, RPMI-8226 ותאי גזע mesenchymal אדם ממח עצם במדיום RPMI1640, בתוספת 10% בסרום שור עוברי עגל ו -100 IU / פניצילין מיליליטר, 100 מיקרוגרם / מיליליטר סטרפטומיצין וגלוטמין 2 מ&quot;מ בנוכחות 5% CO 2 על 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> Transfect 5 x 10 6 תאי HEK 293FT עם תערובת נגיפית אריזת DNA (9 מיקרוגרם) ו -3 מיקרוגרם pLenti6 / V5 dest eGFP וקטור על ידי השימוש של מגיב transfection liposomal 30 μl ובינוני transfection (10 מיליליטר). הסר בינוני transfection לאחר 12 שעות ולהוסיף 10 מיליליטר DMEM בינוני עם 10% בסרום שור עגל וחומצות אמינו לא חיוני 1% (NEAA). </li><li style=";text-align:right;direction:rtl"> לאחר 5 ימים, לאסוף supernatants של תאי HEK293FT לאחר צנטריפוגה של תאי שחייה (1000 XG, 5min). לקבוע כייל נגיף על ידי PCR בזמן אמת כפי שתואר במקום אחר 28. </li><li style=";text-align:right;direction:rtl"> Transfect 1 x 10 6 תאי MM עם חלקיקי eGFP lentiviral (1 x 10 5 חלקיקים) בצלחת גם 24 במדיום גידול שלם. לאחר 3 ימים, להתחיל תהליך בחירה על ידי הוספת 2 מיקרוגרם / מיליליטר blasticidin עד בינוני התרבות. לlentivirus eGFP הזמינים המסחרית, השתמש 500 מיקרוגרם / מיליליטר נאומיצין. </li><li style=";text-align:right;direction:rtl"> לאחר 2 שבועות של בחירה, אשכולות של eGFP להביע תאי MM יופיעו; לאסוף תאים על ידי צנטריפוגה (1000 XG, 5 דק &#39;) ולהרחיב אותם כOPM-2 eGFP וRPMI-8226 sublines eGFP לניסויים (סעיף 2 ו -3). </li></ol> דגם מיאלומה ספרואיד 2. 3D-מרובה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> סוג אני קולגן צ&#39;יל פתרון ו -10 x o DMEMקרח n. </li><li style=";text-align:right;direction:rtl"> מערבבים 1/10 נפח של 10 DMEM בינוני x למטריצת קולגן; להוסיף NaOH (0.2 N) לנטרל פתרון קולגן חומצי לערך ה- pH של 7.4; קולגן חנות / פתרון בינוני על קרח. </li><li style=";text-align:right;direction:rtl"> מערבבים שורות תאים מהונדסים MM (OPM-2 eGFP או RPMI-8226 eGFP; 250,000 לאליפטית,) עם תאים אנושיים mesenchymal (50,000 תאים / אליפטית, כלומר, ירידה של 30 μl). </li><li style=";text-align:right;direction:rtl"> תערובת תא צנטריפוגה ב 15 מיליליטר צינורות (1000 XG, 5min), להוסיף תערובת קרה מוכן קולגן (1mL) לתא גלולה ומערבבים היטב (עם 1,000 קצה μl). </li><li style=";text-align:right;direction:rtl"> מייד פיפטה 30 μl של תערובת קולגן / תא (עם 100 טיפ μl) בצלחת גם 24 על סרט פרפין סטרילי ולאפשר תערובת התא / קולגן לפלמר במשך 30 דקות ב 37 מעלות צלזיוס (ראה איור 1 א). </li><li style=";text-align:right;direction:rtl"> spheroids MM שכבה עם מדיום תרבות 1mL המכיל 1, 10 ו 100 ננומטר בורטזומיב (ראה איור 1). </li><li style=";text-align:right;direction:rtl"> לאחר 72 שעות של דגירה על 37 מעלות צלזיוס, spheroids מסמך על ידיstereomicroscopy הקרינה (ראה איור 1 ג &#39;). </li><li style=";text-align:right;direction:rtl"> העבר כל אליפטית על ידי השימוש במלקחיים עם לסתות שטוחות רחבות בצינור תגובה למדידה של ה- GFP (ראה איור 1D). </li></ol> 3. 3D מודל xenograft מיאלומה נפוצה ברפואה המשלימה והאלטרנטיבית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה ביצי עוף בחממה מיוחדת לביצים עופות על 37 מעלות צלזיוס ולחות 70% לשלושה ימים. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, ביצים פתוחות ועוברי העברה לעיקור עם סירות במשקל פלסטיק אתנול,, 10 סנטימטרים מרובעים עם מכסה צלחת תרבית תאי דגירה &quot;לשעבר אובו&quot; לעוד שישה ימים, כך שCAM הוא מסוגל לפתח (ראה איור 2 א). </li><li style=";text-align:right;direction:rtl"> סוג אני קולגן צ&#39;יל פתרון ו -10 DMEM x על קרח, לערבב 1/10 נפח של 10 x DMEM בינוני למטריצת קולגן, להוסיף NaOH (0.2 N) לנטרל פתרון קולגן חומצי לערך ה- pH של 7.4; קולגן חנות / פתרון בינוני על קרח. </li><li style=";text-align:right;direction:rtl"> מערבבים קווי MM המהונדס תא (OPM-2 eGFP אוRPMI-8226 eGFP; 250,000 לאליפטית,) עם תאי mesenchymal אנושיים (50,000 תאים / אליפטית). </li><li style=";text-align:right;direction:rtl"> תאי צנטריפוגה ב 15 מיליליטר צינורות (1000 XG, 5min; לכל מתחם בדיקת בקבוקון 1), להוסיף 1mL תערובת קולגן מוכן קרה עם תרופה (בריכוז רצוי עבודה) לתא גלולה ומערבבים היטב (עם 1,000 קצה μl). </li><li style=";text-align:right;direction:rtl"> הנח טיפות קולגן (30 μl כל אחד) על parafilm ב6 גם פטה למשך 30 דקות כדי לאפשר פילמור של מטריקס על 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> העברה &quot;onplants&quot; מצעד 3.6 עם השימוש במלקחיים על פני השטח לא מטופל של CAM (2 סנטימטרים מהעובר) של עוברי תרנגולת 9 יום בן (4 onplants לכל עובר עוף, ראה איור 2) </li><li style=";text-align:right;direction:rtl"> לאחר 5 ימים של צמיחה בvivo בחממת ביצה על 37 מעלות צלזיוס ולחות 70%, xenografts מסמך על ידי stereomicroscopy הקרינה (ראה איור 2 ג). להרדים עוברי עוף בהיפותרמיה על 4 מעלות צלזיוס במקרר 5 שעות. </li><li style=";text-align:right;direction:rtl"> הסר xenografts עם רקמת CAM subjacent ידי מספריים עיניים ומלקחיים עם לסתות שטוחות רחבות. השתמש בם למדידה של ה- GFP (סעיף 4, ראו איור 2 ד) או לצורך ניתוח immunohistochemical של כלי דם ו / או תאים פולשים גידול (סעיף 5). </li></ol> 4. כימות חלבון eGFP ידי ELISA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העבר כל אליפטית MM או xenograft נכרת ל0.5 מיליליטר Ripa מאגר המכיל 200 מיקרוגרם / מיליליטר מעכבי פרוטאז. </li><li style=";text-align:right;direction:rtl"> Homogenize אליפטית / xenograft עם homogenizer רקמות על קרח. </li><li style=";text-align:right;direction:rtl"> בצע שלוש הקפאה / הפשרה מחזורים בחנקן נוזלי ו -37 ° C אמבט מים. </li><li style=";text-align:right;direction:rtl"> homogenate צנטריפוגה על 4 מעלות צלזיוס למשך 20 דקות (12,000 ז) וsupernatants חנות. </li><li style=";text-align:right;direction:rtl"> לדלל דגימות 01:20 במאגר assay (200 μl) של ערכת ELISA. למדוד GFP-ידי רמות ה- GFP מסחרי ELISA ערכה באמצעות נוגדנים אנטי-GFP biotinylated, על פי הפרוטוקול של היצרן. </li></ol> 5. Immunohiניתוח stochemical של כלי דם ותאי גידול פולשים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תקן xenografts נכרת בשטח CAM ב -4% O paraformaldehyde / N ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> הנח xenografts הקבוע לקלטות הטבעה ולהעביר אותם לתחנת הטבעת רקמות עם סדרת הגדלת מדורגת אלכוהול (50%, 70%, 80%, 95% אתנול, xylol ופרפין; כל דקות שלב 60). </li><li style=";text-align:right;direction:rtl"> xenografts סעיף (5 מיקרומטר) על ידי השימוש בmicrotome הסיבובי benchtop. אופה חלקי פרפין על O שקופיות זכוכית / N ב 56 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> סעיפי Deparaffinize על ידי סדרת אלכוהול מדורגת יורדת למים מזוקקים כפולים (xylol, 95%, 80%, 70%, 50% אתנול, מים מזוקקים כפולים; כל שלב 10 דקות). </li><li style=";text-align:right;direction:rtl"> לבצע אחזור אנטיגן באמבט מים (95 מעלות צלזיוס, 20 דקות) עם פתרון אחזור אנטיגן (חיץ citrate-; pH 7.0; נפח 100 μl). </li><li style=";text-align:right;direction:rtl"> לחסום פעילות peroxidase אנדוגני עם H 100 μl 3% 2 O 2 / מתנול למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> חלקי בלוק בPBS המכיל10% עוברי עגל בסרום במשך 45 דקות (נפח 100 μl). </li><li style=";text-align:right;direction:rtl"> כתם במשך שעה 1 עם של נוגדן ראשוני (1μg / מיליליטר) מדולל PBS המכיל 1% בסרום עגל עוברי ב RT 100 μl. </li><li style=";text-align:right;direction:rtl"> לאחר שטיפת 3 פעמים-ב PBS, דגירה של 1h עם נוגדן biotinylated המשני (0.1 מיקרוגרם / מיליליטר) בPBS המכיל 1% בסרום עגל עוברי ב RT. </li><li style=";text-align:right;direction:rtl"> לאחר שטיפת 3 פעמים-בPBS לבצע תגובת צבע על ידי פתרון avidin מצע / יוטין-מורכב (ABC) וdiaminobenzidine (DAB) על פי הוראות יצרן. </li><li style=";text-align:right;direction:rtl"> 5.11. עצור תגובה על ידי העברת חלקים למים מזוקקים כפולים, counterstain עם hematoxylin והר חלקים עם הרכבה בינונית סינטטי. </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+model+systems+to+study+and+visualize+human+tumor+cell+metastasis.">Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis.</a> Histochem.Cell Biol. 130 (6), 1119-1130 (2008).</li><li>Kern, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GRP-78+secreted+by+tumor+cells+blocks+the+antiangiogenic+activity+of+bortezomib.">GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib.</a> Blood. 114 (18), 3960-3967 (2009).</li><li>Ribatti, D., Nico, B., Vacca, A., Presta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gelatin+sponge-chorioallantoic+membrane+assay.">The gelatin sponge-chorioallantoic membrane assay.</a> Nat.Protoc. 1 (1), 85-91 (2006).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+embryo+chorioallantoic+membrane+as+a+model+for+tumor+biology.">The chick embryo chorioallantoic membrane as a model for tumor biology.</a> Exp.Cell Res. , (2014).</li><li>Dohle, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+how+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: how it really works.</a> J.Vis.Exp. (33), (2009).</li><li>Kobayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chick+embryo+model+for+metastatic+human+prostate+cancer.">A chick embryo model for metastatic human prostate cancer.</a> Eur.Urol. 34 (2), 154-160 (1998).</li><li>Koop, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+melanoma+cells+entering+the+microcirculation:+over+80%+survive+and+extravasate.">Fate of melanoma cells entering the microcirculation: over 80% survive and extravasate.</a> Cancer Res. 55 (12), 2520-2523 (1995).</li><li>Martowicz, A., Spizzo, G., Gastl, G., Untergasser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype-dependent+effects+of+EpCAM+expression+on+growth+and+invasion+of+human+breast+cancer+cell+lines.">Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines.</a> BMC.Cancer. 12, 501 (2012).</li><li>Martowicz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpCAM+overexpression+prolongs+proliferative+capacity+of+primary+human+breast+epithelial+cells+and+supports+hyperplastic+growth.">EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth.</a> Mol.Cancer. 12, 56 (2013).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> 6, 34 (2006).</li><li>Farnoushi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+testing+of+drug+response+in+multiple+myeloma+made+possible+by+xenograft+to+turkey+embryos.">Rapid in vivo testing of drug response in multiple myeloma made possible by xenograft to turkey embryos.</a> Br.J.Cancer. 105 (11), 1708-1718 (2011).</li><li>Wunderlich, M., Krejci, O., Wei, J., Mulloy, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD34++cells+expressing+the+inv(16)+fusion+protein+exhibit+a+myelomonocytic+phenotype+with+greatly+enhanced+proliferative+ability.">Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability.</a> Blood. 108 (5), 1690-1697 (2006).</li><li>Hideshima, T., Mitsiades, C., Tonon, G., Richardson, P. G., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+multiple+myeloma+pathogenesis+in+the+bone+marrow+to+identify+new+therapeutic+targets.">Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets.</a> Nat.Rev.Cancer. 7 (8), 585-598 (2007).</li><li>Parikh, M. R., Belch, A. R., Pilarski, L. M., Kirshner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+tissue+culture+model+to+study+primary+human+bone+marrow+and+its+malignancies.">A three-dimensional tissue culture model to study primary human bone marrow and its malignancies.</a> J.Vis.Exp. (85), (2014).</li><li>Schuler, J., Ewerth, D., Waldschmidt, J., Wasch, R., Engelhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+multiple+myeloma:+a+critical+appraisal.">Preclinical models of multiple myeloma: a critical appraisal.</a> Expert.Opin.Biol.Ther. 13, S111-S123 (2013).</li><li>Kirshner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112 (7), 2935-2945 (2008).</li><li>Jilani, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+binding+of+lectins+to+embryonic+chicken+vasculature.">Selective binding of lectins to embryonic chicken vasculature.</a> J.Histochem.Cytochem. 51 (5), 597-604 (2003).</li></ol>

יש הסופרים לא אינטרסים כלכליים מתחרים

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. ...

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 &#946;-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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RPMI-8226 cells</td>
			<td>DSMZ</td>
			<td>ACC 9</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>OPM-2 cells</td>
			<td>DSMZ</td>
			<td>ACC 50</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells&#160;</td>
			<td>PromoCell</td>
			<td>PC-C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293FT cells&#160;</td>
			<td>Invitrogen</td>
			<td>R700-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160; HyClone</td>
			<td>ThermoScientific</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut- Pen- Strep solution</td>
			<td>Sigma</td>
			<td>G6784</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>31966</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Sigma Life Sciences</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Medium/Opti-MEM&#160;</td>
			<td>Gibco</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>eGFP lentiviral particles</td>
			<td>GeneCopoeia</td>
			<td>LPP-EGFP-LV105</td>
			<td>Ready to use viral particles</td>
		</tr>
		<tr>
			<td>pLenti6/V5Dest6 eGFP vector</td>
			<td>Invitrogen</td>
			<td>PN 35-1271</td>
			<td>from authors</td>
		</tr>
		<tr>
			<td>ViralpowerTM packaging mix&#160;</td>
			<td>Invitrogen</td>
			<td>P/N 35-1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent/ Lipofectamin 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invitrogen</td>
			<td>R210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Biochrom</td>
			<td>A2912</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-Type1&#160; Rat Tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Life Technologies</td>
			<td>Art.Nr. 10338582</td>
			<td></td>
		</tr>
		<tr>
			<td>plitidepsin</td>
			<td>Pharmamar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bortezomib</td>
			<td>LKT Lab., Inc.</td>
			<td>B5871</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">SPF-white hen eggs</td>
			<td rowspan="3">Charles River</td>
			<td rowspan="3"></td>
			<td rowspan="3">Fertilized&#160; white Leghorn&#160; chicken eggs</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Plastic weighing boats</td>
			<td rowspan="2">neoLab</td>
			<td rowspan="2">Art.Nr. 1-1125</td>
			<td rowspan="2">for ex-ovo culture</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Petridish square (Lids)</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td>for ex-ovo culture</td>
		</tr>
		<tr>
			<td>RIPA Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>#9806</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Tablets</td>
			<td rowspan="2">Roche</td>
			<td rowspan="2">11 836 170 001</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Complete Mini EDTA-free</td>
		</tr>
		<tr>
			<td>GFP ELISA</td>
			<td>Cell Biolabs, Inc.</td>
			<td>AKR-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocette II</td>
			<td>Simport</td>
			<td>M493-6</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160; 37%</td>
			<td>Roth</td>
			<td>7398.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolut</td>
			<td>Normapur</td>
			<td>20,821,321</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histol</td>
			<td>Roth</td>
			<td>Art.Nr.6640.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Sigma</td>
			<td>A6330</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">SuperFrost Microscope Slides</td>
			<td>R. Langenbrinck&#160;</td>
			<td>Art.-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Labor- u. Medizintechnik</td>
			<td>03-0060</td>
		</tr>
		<tr>
			<td rowspan="2">DakoCytomation Wash Buffer 10x</td>
			<td rowspan="2">DakoCytomation</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 3006</td>
		</tr>
		<tr>
			<td rowspan="2">Target Retrieval Solution (10x)&#160; pH 6,1</td>
			<td rowspan="2">DAKO</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 1699</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu ASMA clone 1A4</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu CD138 clone MI15</td>
			<td>DAKO</td>
			<td>M7228</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Vimentin clone V9</td>
			<td>DAKO</td>
			<td>M0725</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Desmin clone D33</td>
			<td>DAKO</td>
			<td>&#160;M0760</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Ki67&#160; clone MIB-1&#160;&#160;</td>
			<td>DAKO</td>
			<td>&#160;M7240</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat- anti-mouse IgG</td>
			<td>Vector Laboratories Inc.</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories Inc.</td>
			<td># PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST DAB Tablet Set.</td>
			<td>Sigma Biochemicals</td>
			<td># D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haemalaun solution</td>
			<td>Merck</td>
			<td>1,092,490,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti Histokitt</td>
			<td>Roth</td>
			<td>Art.Nr.6638.2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top rotary microtome</td>
			<td>Thermo Electron, Shandon Finesse ME+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding station</td>
			<td>Leica, TP1020</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg-Incubator</td>
			<td>Grumbach&#160;</td>
			<td>BSS160</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light&#160;</td>
			<td>Olympus, SZX10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Turrax&#160;</td>
			<td>IKA T10</td>
			<td></td>
			<td>Homogenizer</td>
		</tr>
	</tbody>
</table>

establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis

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&quot;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.

בניתוח מבחנה של תרכובות יעד במבחני אליפטית מיאלומה נפוצה 3D בשל המגבלה של תאי culturing MM אנושיים ראשוניים במבחנה הקמנו מודלים תרבות במבחנה חדשים 3D עבור שורות תאים אנושיות MM עושים שימוש במטריקס צמיחה ותאי mesenchy...

Watch this Scientific Journal Video about Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis at JoVE.com

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

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.

הקמת מודל xenograft מיאלומה נפוצה אדם בעוף לחקר צמיחת גידול, פלישה ואנגיוגנזה

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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Research

JoVE Journal

Medicine

12.9K Views.  Innsbruck Medical University.  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 ואנגיוגנז...

Video: Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

במבחנה מערכת לחקר תרדמת גידול ואת החלף ל גידול גרורתי

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-פוטון הדמיה של הפלישה תאים סרטניים במודל עכבר orthotopic של קרצינומה של תאים קשקשיים אוראלי

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

במודל עכבר של Assay את הקרנית Pocket לחקר אנגיוגנזה

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

האדם הפנימי החלב (הר&quot;י) עורק השתלת סטנט: דגם האדם ללמוד פיתוח restenosis ב-סטנט

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

האדם תאים סרטניים נוירואנדוקריניים קווים כמו מודל תלת ממדי לחקר הטיפול האנושי גידול נוירואנדוקריניים

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

רומן ברזולוציה גבוהה<em&gt; בvivo</em&gt; טכניקת הדמיה כדי לחקור את התגובה דינמית של מבנים תוך גולגולתי לצמיחה גידול וTherapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; באוב</em&gt; CAM-assay כXenograftmodel לסרקומה

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

רשתית דגם ניתוק במכרסמים על ידי הזרקת subretinal של נתרן Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

השתנה<em&gt; במבחנה</em&gt; פלישת Assay כדי לקבוע את התפקיד הפוטנציאלי של הורמונים, ציטוקינים ו / או גורמי גדילה בפלישת תאי הסרטן המתווך

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.