This research is supported by funding from CBCF (to ZW).

1. הכנת תאים עבור הדמיה חיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות זריעת תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש תאי סרטן השד MCF-7 נבחרו להתנגדות docetaxel (MCF-7 TXT) וקו תא ההורים שאינם העמיד שלהם (CC MCF-7). תהליך הבחירה מפורט לאפיון שורות תאים שנבחרו אלה תוארו בעבר 24. </li><li style=";text-align:right;direction:rtl"> לגדול כל תאים בצלחות תרבות 10 ס&quot;מ על 37 מעלות צלזיוס במדיום מורכב 90% של המדיום של הנשר שונה של Dulbecco (DMEM) ו -10% בסרום שור העובר (FBS) שיושלם עם חומצות אמינו לא חיוניות. לשמור על המדיום באווירה 2 5% CO. שמור על תאי TXT MCF-7 ב 5 docetaxel ננומטר. </li><li style=";text-align:right;direction:rtl"> תאי זרע על coverslips הדמיה לחיות. לעקר 24 מ&quot;מ coverslips זכוכית מצופה פולי- L- ליזין על ידי שטיפה אותם עם אלכוהול 70% ולאחר מכן לחשוף אותם UV למשך הלילה. מניחים את coverslips לתוך צלחת תרבות 6-גם של 35 מ&quot;מ. Plאס coverslip אחד בכל טוב. </li><li style=";text-align:right;direction:rtl"> הסר את המדיום מצלחת התרבות של 10 ס&quot;מ לשטוף את התאים עם PBS. הוסף 0.5 מ&quot;ל של טריפסין-EDTA כדי בצלחת כדי לנתק את התאים מצלחת. הוסף 5 מ&quot;ל של מדיום תרבות צלחת לנטרל את טריפסין בעקבות ניתוק של התאים. </li><li style=";text-align:right;direction:rtl"> ספירת התאים עם hemocytometer כדי לקבוע את מספר הסלולרי ליחידת נפח. </li><li style=";text-align:right;direction:rtl"> להוסיף כ 10 5 תאים לכל מבוסס היטב על המספר הנייד ליחידת נפח. אחרי שלחצתי אותו בעדינות, לשים צלחת בחזרה לתוך החממה כדי לאפשר לתאים לצרף את coverslips. </li></ol></li><li style=";text-align:right;direction:rtl"> הביטוי של טובולין α GFP-tagged <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי assay דינמיקת microtubule, transduce התאים עם GFP-tagged α-טובולין באמצעות שלט תמרת GFP המסחרי (למשל, BacMam) על פי הפרוטוקול של היצרן. </li><li style=";text-align:right;direction:rtl"> תרבות תאי הבאר במשך 36 שעות בתוך חממת תרבית תאי 37 ° C כדי לאפשר comהידבקות plete ו מפגש 60%. </li><li style=";text-align:right;direction:rtl"> חשב את הנפח המתאים של GFP-tagged α-טובולין להוסיף כל טוב, על פי המשוואה הבאה: <img alt="משוואה" src="/files/ftp_upload/55027/55027eq1.jpg" /> מספר התאים הוא המספר הכולל המשוער של תאים היטב בעת תיוג ואת PPC הרצוי הוא מספר חלקיקים לכל תא. הערה: מתזמנת את תאי MCF-7 צריכה הכפילה את ספירת התאים מ 10 5 2 x 10 5 עם PPC רצוי של 20, נפח דרושים כל coverslip לכן 40 μL. עבור תאים שונים, היקף מחושב על בסיס הנוסחה לעיל לא יכול להיות הטוב ביותר. ההיקף המדויק צריך להיות מותאם בהתאם לרמת הביטוי בפועל של טובולין GFP. </li><li style=";text-align:right;direction:rtl"> מערבבים את פעמים טובולין-α GFP-tagged כמה על ידי היפוך כדי להבטיח פתרון הומוגני. לא מערבולת. הערה: לקבלת השליטה, Null (שליטה) מגיב חסר כל גנטי יונקיםאלמנטים וניתן להשתמש בו כדי לקבוע אפקטי קרינת רקע baculovirus בתיווך פוטנציאליים. פקד תמרת GFP הלא ממוקדת עשוי לשמש גם, אשר יאיר את התא כולו. </li><li style=";text-align:right;direction:rtl"> החלף את מדיום התרבות עם מדיום חדש. הוסף 2 מ&quot;ל של מדיום חדש ו -40 μL של ה- GFP-tagged α-טובולין היטב כל אחד. </li><li style=";text-align:right;direction:rtl"> לנער את הצלחת בעדינות כדי לאפשר ערבוב מלא של טובולין-α GFP-tagged עם המדיום לתרבות. מחזירים את הצלחת אל האינקובטור תרבות דגירה במשך 24 שעות כדי לאפשר את הביטוי של טובולין GFP. </li></ol></li><li style=";text-align:right;direction:rtl"> תאי Treat עם docetaxel הערה: ניסוי זה היא לבדוק את ההשפעה של docetaxel על חוסר יציבות microtubule דינמי. לפיכך, תאי מטופלים עם הריכוז הרצוי של docetaxel. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן בינוני מורכב 90% של DMEM ו -10% של FBS שיושלם עם חומצות אמינו לא חיוניות. השתמש DMEM ללא פנול אדום בגלל פנול אדום עלולים להפריע fluorescence של טובולין GFP. מוסיפים את הריכוז הרצוי של docetaxel (הנעים בין 10 ננומטר ל -10 מיקרומטר). פתרון המניות של docetaxel הוא 10 מ&quot;מ ב sulfoxide דימתיל (DMSO). </li><li style=";text-align:right;direction:rtl"> טרום לחמם את המדיום המכיל docetaxel עד 37 ° C בחממה בתרבית רקמה. </li><li style=";text-align:right;direction:rtl"> החלף את מדיום התרבות הנורמלי עם המדיום המכיל docetaxel דגירה במשך 30 דקות ב בתרבית רקמת החממה. לא צריך להיות לפחות 3 coverslips לכל טוב עבור כל ריכוז docetaxel. </li></ol></li></ol> 2. הדמיה חיה לבדיקת חוסר יציבות microtubule דינמי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדרת המערכת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע ניסויים בתא שמור על 37 מעלות צלזיוס, 5% CO 2. לרכוש תמונות קרינה על ידי זמן לשגות עם מערכת מיקרוסקופית מצויד בעדשה אובייקטיבית 60X, 1.42 NA שמן על מיקרוסקופ פלואורסצנטי הפוך עם מצלמת CCD. </li><li style=";text-align:right;direction:rtl"> הגדר את תאי הדמיה לחיות. טרום חם הוא לבעל המדגםהמדיום ל -37 מעלות צלזיוס. בחר coverslip לבחון. הר coverslip ריבית על בעל מדגם דגירה זה עם 1 מ&quot;ל של מדיום המכיל-docetaxel מוכן בשלב 1.3.1. </li></ol></li><li style=";text-align:right;direction:rtl"> הדמיה חיה של חוסר יציבות microtubule דינמי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהה את התאים אשר ישמשו הדמיה. הם צריכים להיות שטוחים, יש עוצמת קרינה גבוהה של GFP-טובולין באו לידי ביטוי, ויש מבני microtubule ברורים. </li><li style=";text-align:right;direction:rtl"> בחר תא אחד מהקבוצה של חוליות מזוהות משלבים 2.2.1 לבחון תחילה. דגש על אזור הפריפריה של התא המזוהה. הגדר את התכנית עבור זמן חשיפה ואת תדירות רכישה של תמונה. ככל שזמן חשיפה הוא בדרך כלל 0.5 s, התמונות תירכשנה כל שתי שניות. </li><li style=";text-align:right;direction:rtl"> לאפשר 20 דקות נוספות עבור צעדים מן 1.33 ל 2.2.3. לכן, בדיוק 50 דקות לאחר תוספת של docetaxel, כמתואר בשלב 1.3.3, להתחיל הדמיה (זה סטנדרטיזציה). </li><li style=";text-align:right;direction:rtl"> רשום את ד microtubuleynamics למשך 2 דקות, מצלם כל 2 שניות. בסך הכל, לרכוש 60 תמונות עבור התא המזוהה. </li><li style=";text-align:right;direction:rtl"> העבר אל התא הבא מזוהה וחזור על שלבים 2.2.2 עד 2.2.4. חזור על התהליך עד 5 תאים כבר צילמו. זה הכרחי כדי לעשות זאת לפני הזמן מגיע 70 דקות לאחר התוספת של docetaxel, כמתואר בשלב 1.3.3. </li><li style=";text-align:right;direction:rtl"> עבור כל תנאי הטיפול, חזור על שלבים 2.2.1 עד 2.2.5 3 coverslips שונים. בסך הכל, תאי 15 תמונה, וזה מספיק נתונים כדי למדוד חוסר יציבות microtubule דינמי. </li></ol></li></ol> 3. עיבוד תמונה וניתוח נתונים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Deconvolution של תמונות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Deconvolve התמונות רכשו להשיג באיכות גבוהה. פתח את תכנית deconvolution מצוידת במערכת מיקרוסקופ. </li><li style=";text-align:right;direction:rtl"> לחץ על &quot;תהליך&quot; ובחר &quot;deconvolve.&quot; גרור את קובץ התמונה אל החלון &quot;קלט&quot; ולחץ על &quot;Do.&quot; קובץ התמונה יהיה deconvolved, ואתקובץ deconvolved יישמר כקובץ חדש באופן אוטומטי. </li></ol></li><li style=";text-align:right;direction:rtl"> דור של קטעי וידאו <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחץ על קובץ תמונת deconvolved לפתוח עם תוכנה מאובזרת. </li><li style=";text-align:right;direction:rtl"> לחץ על &quot;קובץ&quot; ובחר &quot;שמירה בשם הסרט.&quot; בחר &quot;פורמט הסרט&quot; כמו &quot;AV&quot; ואת &quot;סגנון האנימציה&quot; כמו &quot;קדימה&quot;. בחר את &quot;איכות הדחיסה&quot; ל &quot;100%&quot; ואת &quot;קצב הפריימים&quot; ל &quot;5 / שני.&quot; </li><li style=";text-align:right;direction:rtl"> סמן את התיבה של &quot;הנפשת בזמן&quot; ולחץ על &quot;עשה זאת.&quot; הקליפ יופק. </li></ol></li><li style=";text-align:right;direction:rtl"> מדידת קיצור microtubule ושיעור הצמיחה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהה את microtubules אשר ישמש למדידה. עבור כל תא, רק כמה microtubules תוכל להיות הקיצור או הצמיחה שלהם בעקבות ברור ומלא. </li><li style=";text-align:right;direction:rtl"> לבחון כל מסגרת של וידאו כדי לזהות את המסגרת המציגה את אורך ההתחלה של א microtubulend המסגרת עם microtubule המורחבת ביותר או מקוצר. מודד את השינוי באורך בין שני microtubules ואת הזמן שחלף בין שתי המסגרות. </li><li style=";text-align:right;direction:rtl"> חשב את קצב צמיחה או קיצור ידי חלוקת השינוי באורך ידי זמן (היחיד הוא מיקרומטר / s). עבור כל תנאי הטיפול, אחרי לפחות 10 תאים ולמדוד לפחות 20 microtubules. </li><li style=";text-align:right;direction:rtl"> חשב את הממוצע ואת סטיית התקן עבור כל תנאי הטיפול. נתח את הבדל סטטיסטי בין תנאי שימוש במבחן הסטודנטים. דווח על הנתונים בטבלה ו / או בתרשים. </li></ol></li></ol>

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N., Chowbay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenetics+of+target+genes+across+doxorubicin+disposition+pathway:+a+review.">Pharmacogenetics of target genes across doxorubicin disposition pathway: a review.</a> Curr. Drug Metab. 11 (1), 115-128 (2010).</li><li>Murray, S., Briasoulis, E., Linardou, H., Bafaloukos, D., Papadimitriou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taxane+resistance+in+breast+cancer:+mechanisms,+predictive+biomarkers+and+circumvention+strategies.">Taxane resistance in breast cancer: mechanisms, predictive biomarkers and circumvention strategies.</a> Cancer Treat.Rev. 38 (7), 890-903 (2012).</li><li>Kamath, K., Wilson, L., Cabral, F., Jordan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BetaIII-tubulin+induces+paclitaxel+resistance+in+association+with+reduced+effects+on+microtubule+dynamic+instability.">BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability.</a> J.Biol.Chem. 280 (13), 12902-12907 (2005).</li><li>Banerjee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+levels+of+tyrosinated+alpha-,+beta(III)-,+and+beta(IV)-tubulin+isotypes+in+paclitaxel-resistant+MCF-7+breast+cancer+cells.">Increased levels of tyrosinated alpha-, beta(III)-, and beta(IV)-tubulin isotypes in paclitaxel-resistant MCF-7 breast cancer cells.</a> Biochem.Biophys.Res.Commun. 293 (1), 598-601 (2002).</li><li>Wiesen, K. M., Xia, S., Yang, C. P., Horwitz, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+class+I+beta-tubulin+sensitizes+Taxol-resistant+breast+adenocarcinoma+cells+harboring+a+beta-tubulin+mutation.">Wild-type class I beta-tubulin sensitizes Taxol-resistant breast adenocarcinoma cells harboring a beta-tubulin mutation.</a> Cancer Lett. 257 (2), 227-235 (2007).</li><li>Iseri, O. D., Kars, M. D., Arpaci, F., Gunduz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+of+drug-resistant+MCF-7+cells:+implications+for+relation+to+extracellular+matrix+proteins.">Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins.</a> Cancer Chemother.Pharmacol. 65 (3), 447-455 (2010).</li><li>Hembruff, S. L., 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=Role+of+drug+transporters+and+drug+accumulation+in+the+temporal+acquisition+of+drug+resistance.">Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance.</a> BMC.Cancer. 8, 318 (2008).</li><li>Yenjerla, M., Lopus, M., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+dynamic+instability+of+steady-state+microtubules+in+vitro+by+video-enhanced+differential+interference+contrast+microscopy+with+an+appendix+by+Emin+Oroudjev.">Analysis of dynamic instability of steady-state microtubules in vitro by video-enhanced differential interference contrast microscopy with an appendix by Emin Oroudjev.</a> Methods Cell Biol. 95, 189-206 (2010).</li><li>Sammak, P. J., Gorbsky, G. J., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+in+vivo:+a+test+of+mechanisms+of+turnover.">Microtubule dynamics in vivo: a test of mechanisms of turnover.</a> J Cell Biol. 104 (3), 395-405 (1987).</li><li>Walker, R. 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=Dynamic+instability+of+individual+microtubules+analyzed+by+video+light+microscopy:+rate+constants+and+transition+frequencies.">Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.</a> J Cell Biol. 107 (4), 1437-1448 (1988).</li><li>Desai, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+polymerization+dynamics.">Microtubule polymerization dynamics.</a> Annu Rev Cell Dev Biol. 13, 83-117 (1997).</li><li>Walczak, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+and+tubulin+interacting+proteins.">Microtubule dynamics and tubulin interacting proteins.</a> Curr Opin Cell Biol. 12 (1), 52-56 (2000).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

ישנן שתי שיטות עיקריות למדידת חוסר יציבות microtubule דינמי: במבחנה in vivo. בשיטה במבחנה, טובולין מטוהר משמש למדידת חוסר יציבות microtubule דינמית עם מיקרוסקופיה מחשב משופר זמן לשגות הפרש התערבות ניגודיות. בשיטת in vivo, microinjected טובולין ניאון, או הביע GFP-טובולין, ה?...

<html> הגורם המוביל לתמותה מסרטן השד הוא באמצעות גרורות 1, 2. Taxanes, כגון docetaxel ו- paclitaxel, משמש כיום כקו ראשון משטר לטיפול בסרטן השד גרורתי 2, 3, 4, 5, 6. הם חלק מהקבוצה של סוכני מיקוד-microtubule (MTAs) משבשי דינמיקת microtubule. עם זאת, אחד האתגרים הגדולים ביותר באמצעות taxanes בטיפול מרפא הוא הפיתוח של התנגדות taxane בתאים סרטניים, אשר מובילה למחלות ישנות 7. עמידות לתרופות אחראית ליותר מ -90% מכלל מקרי המוות בקרב חולי סרטן שד גרורתי 7. Microtubules נוצרות על ידי פילמור של heterodimers α- ו β טובוליןclass = &quot;Xref&quot;&gt; 8, 9. ההתקנה המדויקת של דינמיקת microtubule חשובה תפקודים תאיים רבים, כולל קיטוב תא, התקדמות מחזור התא, תחבורה תאית, ואיתות תא. חוסר וויסות של microtubules והדינמיקה שלהם ישבש תפקוד התא לגרום מוות של תאים 10, 11. תלוי איך הם לגרום חוסר וויסות זה, תרופות MTA ניתן לסווג גם כסוכני ייצוב microtubule (כלומר, taxanes) או סוכני microtubule-destabalizing (כלומר, אלקלואידים וינקה או באתר קולכיצין סוכנים מחייב) 20. למרות ההשפעות שלהם הפוכות על מונית microtubule, במינון מספיק, כיתות שניהם יכולות להרוג תאים סרטניים באמצעות השפיע על דינמיקת microtubule 21. Taxanes לתפקד בעיקר על ידי ייצוב microtubule ציר 12, המובילחוסר תיאום כרומוזומליות. ההפעלה התמידית הבאה של מחסום הרכבת ציר (SAC), עוצרת את תא מיטוזה. מעצר mitotic ממושך ואז גורם לאפופטוזיס 13, 14. Taxane אינטראקציה עם microtubules דרך האתר taxane מחייב על β טובולין 8, 15, אשר שוהה רק טובולין התאספו 16. מנגנונים מרובים להתנגדות taxane הוצעו 9, 17. מנגנונים אלה כוללים הן התנגדות multidrug כללית בשל ביטוי יתר של חלבונים-בזרימת סמים taxane ספציפי התנגדות 5, 9, 18, 19. לדוגמא, תאים סרטניים עמיד taxane יש בו כדי לשנות ביטוי ותפקוד של β-גיגית מסוימתulin isotypes 5, 9, 19, 20, 21, 22, 23. באמצעות שיטה in vivo למדוד חוסר יציבות microtubule דינמי, אנו מראים כי, כאשר בהשוואה ללא-עמיד, תאי CC MCF-7 הוריה 17, דינמיקת microtubule של תאי docetaxel העמיד MCF-7 TXT הן רגישה לטיפול docetaxel. כדי להבין טוב יותר את הפונקציה של MTAs ואת המנגנון המדויק של התנגדות taxane בתאים סרטניים, זה הכרחי למדוד דינמיקת microtubule. כאן אנו מדווחים שיטה in vivo לעשות זאת. באמצעות הדמיה חיה בשילוב עם ביטוי של טובולין-tagged GFP בתאים, אנו יכולים למדוד את הדינמיקה microtubule של TXT MCF-7 ותאי CC MCF-7 עם ואינטרנטthout טיפול docetaxel. התוצאות יכולות לעזור לנו לעצב תרופות יעילות יותר שיכול להתגבר על התנגדות taxane.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies, Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco, Invitrogen</td>
			<td>12483</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Anti (100x)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>docetaxel</td>
			<td>Sigma-Aldrich</td>
			<td>01885-5mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM phenol red-free</td>
			<td>Gibco, Invitrogen</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td>
			<td>ThermoFisher Scientific</td>
			<td>C10613</td>
			<td>Key reagent for expressing GFP tubulin in cells</td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP</td>
			<td>ThermoFisher Scientific</td>
			<td>B10383</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich+B9:AA9</td>
			<td>472301</td>
			<td>for dissoving decetaxel</td>
		</tr>
		<tr>
			<td>22-mm glass coveslip</td>
			<td>Fisher Scientifics</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>Greiner Bio-One International</td>
			<td>6 Well Celi Culture Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Microscopy Imaging Systems</td>
			<td>GE Health</td>
			<td></td>
			<td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer Set, Hausser Scientific</td>
			<td>Hausser Scientific, Distributed by VWR</td>
			<td>Supplier No.: 1492 VWR No.:15170-172</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

באמצעות הפרוטוקול המובא כאן, חקרנו את ההשפעות של docetaxel על הדינמיקה microtubule של הנורמה (CC MCF-7) ו- docetaxel עמיד (MCF-7 TXT) תאי סרטן השד. שני סטים של תמונות להראות את ההשפעות של docetaxel (0.5 מיקרומטר) על צמיחה microtubule וקיצור ב CC MCF-7 ו MCF-7 תאים TXT <strong...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

הדמיה חיה לחקר חוסר יציבות microtubule דינמי סרטן השד עמיד taxane

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

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7.5K Views.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

Video: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

ניצול הקרנת Genomics פונקציונלית כדי לזהות מטרות תרופה חדשנית פוטנציאלי תרבויות אליפטית תא סרטני

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

מודל הזרקת Portal Vein לחקר גרור בכבד של סרטן השד

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

הדמיה חיה של נדידת תאים אפיתל השד לאחר דלדול ארעית של TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

לטווח ארוך לחיות תאים הדמיה כדי להעריך את גורל בתגובה פקליטקסל

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

מבחני הדמיה לחיות תאים מחקר המוח גליובלסטומה גידול תאי גזע ההעברה, הפלישה

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

לומדת משולשת סרטן השד שלילי באמצעות אורתובנושא שד סרטן השד מודל

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

שימוש בקרום כוראלאלי ממברנה במודל Vivo כדי ללמוד גינקולוגית ואורולוגיים סרטן

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

שימוש בניתוח תמונה מבוסס מחשב כדי לשפר את כימות גרורות הריאות במודל סרטן השד 4T1

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

דוגמנות סרטן השד ברקמת השד האנושית באמצעות מערכת מיקרופיזיולוגית

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

מודל קסנוגרפט של מורין אומאיה לחקר טיפול ישיר ממוקד במחלת לפטומינגיאל

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...