Name: development and maintenance of a preclinical patient derived tumor xenograft model for the investigation of novel anti-cancer therapies
Uploaded: 2016-09-30T13:00:00
Description: Watch this Scientific Journal Video about Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies at JoVE.com

This work was supported by grant 1R01CA152303-01.

משפט ואתיקה: דגימות גידול אדנוקרצינומה גס נגזרות חולה התקבלו בהסכמה חולה באונ&#39; קולורדו החולים בהתאם לפרוטוקול שאושר על ידי דירקטוריון הסקירה המוסדי מרובה קולורדו (08-0439). כל עבודת החיה בוצעה תחת פרוטוקולי חיה אושרו על ידי האוניברסיטה של ​​טיפול בבעלי החיים המוסדי ...

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	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.+2015.">Cancer statistics. 2015.</a> CA Cancer J Clin. 65 (1), 5-29 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+characterization+of+human+colon+and+rectal+cancer.">Comprehensive molecular characterization of human colon and rectal cancer.</a> Nature. 487 (7407), 330-337 (2012).</li><li>Arcaroli, J. 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=Tumours+with+elevated+levels+of+the+Notch+and+Wnt+pathways+exhibit+efficacy+to+PF-03084014,+a+gamma-secretase+inhibitor,+in+a+preclinical+colorectal+explant+model.">Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a gamma-secretase inhibitor, in a preclinical colorectal explant model.</a> Br J Cancer. 109 (3), 667-675 (2013).</li><li>Hubbard, J., Grothey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenesis+agents+in+colorectal+cancer.">Antiangiogenesis agents in colorectal cancer.</a> Curr Opin Oncol. 22 (4), 374-380 (2010).</li><li>van Es, J. H., 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=Notch/gamma-secretase+inhibition+turns+proliferative+cells+in+intestinal+crypts+and+adenomas+into+goblet+cells.">Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells.</a> Nature. 435 (7044), 959-963 (2005).</li><li>Cassidy, J. W., Caldas, C., Bruna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+Tumor+Heterogeneity+in+Patient-Derived+Tumor+Xenografts.">Maintaining Tumor Heterogeneity in Patient-Derived Tumor Xenografts.</a> Cancer Res. 75 (15), 2963-2968 (2015).</li><li>Jin, K., 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=Patient-derived+human+tumour+tissue+xenografts+in+immunodeficient+mice:+a+systematic+review.">Patient-derived human tumour tissue xenografts in immunodeficient mice: a systematic review.</a> Clin Transl Oncol. 12 (7), 473-480 (2010).</li><li>Julien, 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=Characterization+of+a+large+panel+of+patient-derived+tumor+xenografts+representing+the+clinical+heterogeneity+of+human+colorectal+cancer.">Characterization of a large panel of patient-derived tumor xenografts representing the clinical heterogeneity of human colorectal cancer.</a> Clin Cancer Res. 18 (19), 5314-5328 (2012).</li><li>Siolas, D., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer Res. 73 (17), 5315-5319 (2013).</li><li>Tentler, J. 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=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nat Rev Clin Oncol. 9 (6), 338-350 (2012).</li><li>Carson, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histotechnology: A Self-Assessment Workbook. , (1996).</li><li>Arcaroli, J. 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=Common+PIK3CA+mutants+and+a+novel+3'+UTR+mutation+are+associated+with+increased+sensitivity+to+saracatinib.">Common PIK3CA mutants and a novel 3' UTR mutation are associated with increased sensitivity to saracatinib.</a> Clin Cancer Res. 18 (9), 2704-2714 (2012).</li><li>Arcaroli, J. 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+NOTCH1+gene+copy+number+gain+is+a+prognostic+indicator+of+worse+survival+and+a+predictive+biomarker+to+a+Notch1+targeting+antibody+in+colorectal+cancer.">A NOTCH1 gene copy number gain is a prognostic indicator of worse survival and a predictive biomarker to a Notch1 targeting antibody in colorectal cancer.</a> Int J Cancer. 138 (1), 195-205 (2016).</li><li>Arcaroli, J. 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=Gene+array+and+fluorescence+in+situ+hybridization+biomarkers+of+activity+of+saracatinib+(AZD0530),+a+Src+inhibitor,+in+a+preclinical+model+of+colorectal+cancer.">Gene array and fluorescence in situ hybridization biomarkers of activity of saracatinib (AZD0530), a Src inhibitor, in a preclinical model of colorectal cancer.</a> Clin Cancer Res. 16 (16), 4165-4177 (2010).</li><li>Lieu, C. H., 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=Antitumor+activity+of+a+potent+MEK+inhibitor,+TAK-733,+against+colorectal+cancer+cell+lines+and+patient+derived+xenografts.">Antitumor activity of a potent MEK inhibitor, TAK-733, against colorectal cancer cell lines and patient derived xenografts.</a> Oncotarget. 6 (33), 34561-34572 (2015).</li><li>Pitts, T. 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=Association+of+the+epithelial-to-mesenchymal+transition+phenotype+with+responsiveness+to+the+p21-activated+kinase+inhibitor,+PF-3758309,+in+colon+cancer+models.">Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models.</a> Front Pharmacol. 4, 35 (2013).</li><li>Song, E. K., 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=Potent+antitumor+activity+of+cabozantinib,+a+c-MET+and+VEGFR2+inhibitor,+in+a+colorectal+cancer+patient-derived+tumor+explant+model.">Potent antitumor activity of cabozantinib, a c-MET and VEGFR2 inhibitor, in a colorectal cancer patient-derived tumor explant model.</a> Int J Cancer. 136 (8), 1967-1975 (2015).</li><li>Tentler, J. 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=Identification+of+predictive+markers+of+response+to+the+MEK1/2+inhibitor+selumetinib+(AZD6244)+in+K-ras-mutated+colorectal+cancer.">Identification of predictive markers of response to the MEK1/2 inhibitor selumetinib (AZD6244) in K-ras-mutated colorectal cancer.</a> Mol Cancer Ther. 9 (12), 3351-3362 (2010).</li><li>Bardelli, 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=Amplification+of+the+MET+receptor+drives+resistance+to+anti-EGFR+therapies+in+colorectal+cancer.">Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer.</a> Cancer Discov. 3 (6), 658-673 (2013).</li><li>Bertotti, 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=A+molecularly+annotated+platform+of+patient-derived+xenografts+("xenopatients")+identifies+HER2+as+an+effective+therapeutic+target+in+cetuximab-resistant+colorectal+cancer.">A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.</a> Cancer Discov. 1 (6), 508-523 (2011).</li><li>Davis, 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=Combined+inhibition+of+MEK+and+Aurora+A+kinase+in+KRAS/PIK3CA+double-mutant+colorectal+cancer+models.">Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models.</a> Front Pharmacol. 6, 120 (2015).</li><li>Morelli, M. P., 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=Preclinical+activity+of+the+rational+combination+of+selumetinib+(AZD6244)+in+combination+with+vorinostat+in+KRAS-mutant+colorectal+cancer+models.">Preclinical activity of the rational combination of selumetinib (AZD6244) in combination with vorinostat in KRAS-mutant colorectal cancer models.</a> Clin Cancer Res. 18 (4), 1051-1062 (2012).</li><li>Pitts, T. 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=Dual+pharmacological+targeting+of+the+MAP+kinase+and+PI3K/mTOR+pathway+in+preclinical+models+of+colorectal+cancer.">Dual pharmacological targeting of the MAP kinase and PI3K/mTOR pathway in preclinical models of colorectal cancer.</a> PLoS One. 9 (11), e113037 (2014).</li><li>Spreafico, 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=Rational+combination+of+a+MEK+inhibitor,+selumetinib,+and+the+Wnt/calcium+pathway+modulator,+cyclosporin+A,+in+preclinical+models+of+colorectal+cancer.">Rational combination of a MEK inhibitor, selumetinib, and the Wnt/calcium pathway modulator, cyclosporin A, in preclinical models of colorectal cancer.</a> Clin Cancer Res. 19 (15), 4149-4162 (2013).</li><li>Arcaroli, J. 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=ALDH++tumor-initiating+cells+exhibiting+gain+in+NOTCH1+gene+copy+number+have+enhanced+regrowth+sensitivity+to+a+gamma-secretase+inhibitor+and+irinotecan+in+colorectal+cancer.">ALDH+ tumor-initiating cells exhibiting gain in NOTCH1 gene copy number have enhanced regrowth sensitivity to a gamma-secretase inhibitor and irinotecan in colorectal cancer.</a> Mol Oncol. 6 (3), 370-381 (2012).</li><li>Hoey, 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=DLL4+blockade+inhibits+tumor+growth+and+reduces+tumor-initiating+cell+frequency.">DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency.</a> Cell Stem Cell. 5 (2), 168-177 (2009).</li><li>Ikebuchi, F., 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=Dissociation+of+c-Met+phosphotyrosine+sites+in+human+cells+in+response+to+mouse+hepatocyte+growth+factor+but+not+human+hepatocyte+growth+factor:+the+possible+roles+of+different+amino+acids+in+different+species.">Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.</a> Cell Biochem Funct. 31 (4), 298-304 (2013).</li><li>Zhang, Y. W., 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=Enhanced+growth+of+human+met-expressing+xenografts+in+a+new+strain+of+immunocompromised+mice+transgenic+for+human+hepatocyte+growth+factor/scatter+factor.">Enhanced growth of human met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor.</a> Oncogene. 24 (1), 101-106 (2005).</li></ol>

פלטפורמת גילוי תרופות PDTX מציעה מודל משופר לחסרונות של במודלים פרה אחרים כי הם אמינים בחיזוי עשייה קלינית של תרכובות חדשות. חשוב לציין, גידולים במודל זה מהבחינה ביולוגית יציבים, שומר על פוטנציאל גרורתי, ולהציג היענות תרופה דומה מדור לדור. במודל זה, גידולים שמקורם בחול...

סרטן מעי גס (CRC) הוא תורם משמעותי מקרי מוות מסרטן בארצות הברית. בשנת 2015, היו כ 132,700 מקרים חדשים של CRC עם 49,700 מקרי מוות 1. למרות הפרוגנוזה בחולים עם מחלה מקומית מצוין, חולים עם מחלה מתקדמת יש תוצאות עניות, מה שהופך בעדיפות עליונה זו בפיתוח טיפולים חדשניים. למרות רמת משטר כימותרפיות טיפול ביולוגיות חדשות יותר שנפרסות נגד מחלה זו, חל רק גידול מצטבר בהישרדות כוללת. בהתאם לכך, ישנו מאמץ משמעותי בהבנת מסלולי הנהג המעורב בקידום צמיחת גידול במחלה זו. רשת אטלס הגנום הסרטן זיהתה לאחרונה מסלולים עיקריים רבים מעורבים dysregulation CRC וכוללים: Wnt, phosphoinositide 3-kinase (PI3K), RAS,-β גורם גדילה ההפיכה (β TGF-) ו TP53 2. יחד, עם חקירות מתאר otמסלולים לה להגביר הצמיחה CRC היה להצית את הפיתוח של טיפולים חדשים שנועדו לשפר את ההישרדות משמעותית באוכלוסיית חולים זו 3-5. במודלים פרה ניצול בפיתוח התרופה האונקולוגית היו חיוניים בתהליך זה בחיזוי העשייה הקלינית של תרכובות חדשות אלה. במודלים פרה שונים כבר נוצלו בתהליך פיתוח תרופות. בהתחשב בכך במודלים של בעלי חיים מהונדסים פרה הנציחו שורות תאים לא הצליחו בקביעת הפעילות הקלינית של טיפולים אונקולוגיים רומן, בעיקר בשל החוסר יכל על מנת לשקף את המורכבות של גידולים אנושיים, xenograft גידול נגזר חולה (PDTX) מודלים הוקמו. היתרון הגדול של מודל זה הוא כי ההטרוגניות גידול נשארו שלמות וללא משקפות היטב את המאפיינים המולקולריים clonality של שמקורם בחולת גידול 6-9. מודלים PDTX לספק מעולה in vivoפלטפורמה פרה ללמוד סוכני רומן, שבילים עמידים לתרופות, אסטרטגיות combinational, ביולוגיה של תא גזע סרטני 10. סקירה כללית של תהליך PDTX מתוארת באיור 1. זה מתחיל במרפאת, הסכמה חולה כדי לאפשר כמה רקמת הגידול העודפת שלהם כדי לשמש למחקר זה. לאחר מכן, בעת הניתוח, פיסת הגידול גולם על ידי פתולוג והכניסה תקשורת להיות מועברת אנשי מחקר. מיד אחרי זה, קטע של הגידול הוא חתך לחתיכות קטנות מושתלים לעכברים immunodeficient תת עורי. לאחר הגידול גדל, הוא passaged לתוך דורות שונים של עכברים כדי לשמור על גידול 10. בדרך כלל, לאחר שדור F3 הגידול ניתן להרחיב לתוך מחקר טיפול שבו תרכובות חדשות ו / או טיפולים קומבינטוריים מוערכים. Next Gen Seq (Exome Seq, RNA Seq ו SNP מערך) ניצול סמנים חזוי פוטנציאליים לגלותed המסייעים בבחירת חולים שעשויים להפיק תועלת מטיפול מסוים. מטרות העל של שימוש במודלי PDTX הן: 1) להעריך את היעילות של טיפולים חדשניים כסוכן יחיד או בשילוב ו -2) לזהות סמנים חזויים של רגישות או התנגדות לפני למחקר קליני. בכתב היד הזה, אנו מספקים את המתודולוגיה בייזום ואחזקה של בנק CRC PDTX ולספק את היתרונות והמגבלות של מודל זה גילוי ופיתוח תרופות. <img alt="איור 1" src="/files/ftp_upload/54393/54393fig1.jpg" /> סקירה כללית באיור 1. לפרוטוקול דגם CRC PDTX. גידול נגזר חולה מתקבלת מניתוח והזריק מייד לעכברים בעירום athymic תת עורי. לאחר הגידול גדל היא מורחבת לתוך הדורות הבאים, ובסופו של דבר הרחיב ללימודי טיפול. respo טיפולnses מוערך וביומרקרים חזויים מזוהים שעשויים לסייע בבחירה חולה. <a href="https://www.jove.com/files/ftp_upload/54393/54393fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a>

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI or DMEM</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:499px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Non-essential Amino Acids</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">35-010-CV</td>
			<td>Thaw in -4 &#176;C, then activate for 30 minutes at 60 &#176;C water bath</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CPT blood tube</td>
			<td style="width:81px;">BD vacutainer</td>
			<td style="width:119px;">362761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube</td>
			<td style="width:81px;">Surelock</td>
			<td style="width:119px;">A-7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phospate-Buffered Saline</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cyrogenic vials</td>
			<td style="width:81px;">Cyroking</td>
			<td style="width:119px;">C0732901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic tumor cutting dish</td>
			<td style="width:81px;">Trueline</td>
			<td style="width:119px;">TR4001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:81px;">Roboz</td>
			<td style="width:119px;">RS-5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:81px;">Roboz</td>
			<td style="width:119px;">RS-5135</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Matrigel (gelatinous protein mixture)</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">354234</td>
			<td>Store at -20 or -80 &#176;C, then thaw on ice, do not leave at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Formalin cups</td>
			<td style="width:81px;">Protocol</td>
			<td style="width:119px;">032-059</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Liquid Nitrogen Dewar Storage</td>
			<td style="width:81px;">Thermolyne</td>
			<td style="width:119px;">CY50900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable liquid nitrogen dewar</td>
			<td style="width:81px;">Nalgene</td>
			<td style="width:119px;">4150-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:81px;">Fischer</td>
			<td style="width:119px;">67-68-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Freezing container: Mr Frosty</td>
			<td style="width:81px;">Nalgene</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropyl Alcohol</td>
			<td style="width:81px;">Decon</td>
			<td style="width:119px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Trocars</td>
			<td style="width:81px;">Innovative Research of America</td>
			<td style="width:119px;">MP-182</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anesthesia machine</td>
			<td style="width:81px;">Patterson Veterinary</td>
			<td style="width:119px;">none</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anesthesia box</td>
			<td style="width:81px;">Patterson Veterinary</td>
			<td style="width:119px;">none</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:81px;">Vet one</td>
			<td style="width:119px;">1038005</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">F-Air Canister</td>
			<td style="width:81px;">Bickford Omnicon</td>
			<td style="width:119px;">80120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Meloxicam</td>
			<td style="width:81px;">Vet one</td>
			<td style="width:119px;">5182-90C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calipers</td>
			<td style="width:81px;">Fowler</td>
			<td style="width:119px;">54-100-167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Weight scale</td>
			<td style="width:81px;">Ohaus</td>
			<td style="width:119px;">Scout Pro SP601</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and maintenance of a preclinical patient derived tumor xenograft model for the investigation of novel anti-cancer therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

הדמיון של מוטציות נפוצות במודלים CRC PDTX ואת TCGA חקרנו אם אחוז המוטציות השכיחות (KRAS, NRAS, BRAF, PIK3CA, APC, CTNNB1 ו TP53) בבנק CRC PDTX היה נציג לתדר המוטציה לראות באוכלוסיית חולי CRC. כפי שניתן לראות <str...

Watch this Scientific Journal Video about Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies at JoVE.com

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

ניצול גידולים שמקורם המטופל במודל פרה-קליני תת עורית הוא דרך מצוינת ללמוד את היעילות של טיפולים חדשניים, גילוי סמן ביולוגי חזוי, ושבילי סמים עמידים. מודל זה, בתהליך פיתוח התרופות, יש חשיבות רבה לקביעת גורלו של טיפולים חדשניים נגד סרטן רבים לפני למחקר קליני.

פיתוח ותחזוקה של גידול נגזר החולה פרה xenograft דגם לחקירת Novel טיפולים נגד סרטן

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

The overall goal of this subcutaneous Patient Derived Tumor Xenograft procedure is to the study the efficacy of novel therapies predictive biomarker discovery and drug resistant pathways in tumors. This method can help answer key questions in the oncology field about whether a particular drug exhibits activity against a specific tumor type. The main advantage of this technique is that Patient Derived Tumor Xenographs maintain the molecular, genetic and histologist heterogeneity typical of the tumors of origin, making the model more clinically relevant.Demonstrating the procedure will be Stacey Bagby, a professional research assistant and certified veterinary technician from our laboratory. After collecting one to two milliliters of blood in a blood cell separation tube containing sodium citrate, centrifuge the samples and transfer the peripheral blood mononuclear cell layer into a 1.5 milliliter micro-centrifuge tube. Fill the tube to the top with sterile PBS.Then, spin down the cells again. Followed by a quick wash with one milliliter of PBS, being careful not to disturb the PMBC pellet. Then, use a pipette to transfer the plasma from the blood cell collection tube to a 1.5 milliliter sterile cryogenic vial, and store the plasma and the PBMC pellet at negative 80 degrees Celsius.Transfer the human tumor tissue to the animal facility in a sterile cup containing complete medium and place the cup in a laminar flow hood. As soon as possible, after harvesting, transfer the tissue into a sterile plastic cutting dish in a small amount of the retrieval medium. Then, using autoclave sterilized small scissors and forceps, cut the tissue into approximately three by three by three milliliter pieces and place 10 to 12 pieces into an autoclave sterilized 1.5 milliliter micro-centrifuge tube containing 300 microliters of gelatinous protein mixture solution on ice for the implantation.For flash frozen storage, transfer the appropriate number of tumor pieces into a new 1.5 milliliter micro-centrifuge tube and place the vial in a liquid nitrogen doer. Then, place in a minus 80 degrees Celsius freezer. For formal infixing and paraffin embedding, place three small tumor pieces into a 10 milliliter 10%formalin cup for 24 hours.After the tissues are processed into paraffin embedded blocks, transfer any remaining tissue pieces into a cryogenic tube. Then, add one milliliter of complete medium supplemented with 10%DMSO to the tissue and store the tubes on ice until they can be transferred into an isopropyl alcohol freezing container. And put that container in a minus 80 degrees Celsius freezer.To inject the tumor tissue, load the pieces of tumor stored in the gelatinous protein mixture solution into autoclaved 12-gauge trocars, taking care that each tumor piece is completely inserted into the trocar. Next, confirm a lack of response to toe pinch in a six to eight week old athymic, nude mouse and transfer the mouse onto a clean field in a sterile laminar flow hood. Deliver the F0 tumor subcutaneously to the dorsal neck region and to each side of the mouse's flank, followed by the subcutaneous administration of analgesia distal to one of the flank sites of tumor injection.Then, place the mouse back in its cage with monitoring until it is fully recovered. Monitor the growth and health of the xenograph-implanted mice at least once per week, recording the tumor sizes, tumor generation, date of tumor injection, and health of the mice in an appropriate tracking system. When a tumor reaches approximately 1, 500 to 2, 000 cubic millimeters in size, use autoclaved scissors and forceps to excise the tumor and passage the F1 tumor into the next generation of five animals, as just demonstrated, taking care to collect flash frozen formalin and viable tumor samples as demonstrated for each tumor.When the remaining mice from the F0 tumor group exhibit tumors approximately 1, 500 to 2, 000 cubic millimeters in size, collect these tumors as just demonstrated for collection. When the stage F15 tumor generation is reached, use the earliest generation of viable tumor pieces available in liquid nitrogen storage for the next series of tumor injections. When the desired tumor is very large, 1, 500 to 2, 000 cubic millimeters, follow the procedures above to collect the tumor to expand it into the desired amount of mice.Dose the mice with the drug and weigh and measure them twice per week. Randomize the mice into treatment groups with 10 tumors per group, and a group tumor volume average of 100 to 300 cubic millimeters within a few standard deviations of each other. Then, sort the mice into the appropriate groups for treatment.Dual color fluorescence in situ hybridization for the human and mouse caught one DNA demonstrates that the human stromal cells in the F0 tumor are replaced by the mouse stromal cells in the F1 tumor. Whereas, all of the human-derived hepatocyte growth factor in the F0 generation is replaced with mouse hepatocyte growth factor in F1 generation, the vascular endothelial growth factor ligand consists of both human and mouse protein in the F1 generation. The treatment of different generations of the same tumor does not change the treatment response.With the resistance or sensitivity of the tumor appearing to be tumor strain rather than tumor generation specific. Further, the frequency of mutations in these commonly mutated genes is very similar between the cancer genome atlas and the colorectal patient-derived tumor xenograph bank. Suggesting that the common mutations observed in the colorectal patient population are well represented in the colorectal patient-derived xenotransplant model.Once mastered, this technique can be completed in one to two hours, if performed properly. While attempting this procedure, it is important to have a cohesive clinical team for identifying and obtaining consent from the cancer patients and for the removal and grossing of the tumor tissue. Following this procedure, other methods, like the investigation of anti-cancer compounds as single or combination agents, can be performed to answer additional questions about the viability of these strategies for patients with a particular tumor type.After its development, this technique paved the way for researchers in the field of oncology to explore novel cancer therapeutics, tumor heterogeneity, treatment resistant mechanisms, cancer stem cell biology, and tumor stromal interactions. After watching this video, you should have a good understanding on how to develop and maintain a patient derived tumor xenograph model for evaluating novel cancer therapeutics.

Research

JoVE Journal

Cancer Research

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

תיוג של xenografts הנגזר חולה סרטן השד עם כתבים לעקיבים עבור צמיחת גידולים וגרורים מחקרים

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

קרצינומה תא syngeneic עכבר הדגם של גרורתי כליות עבור הערכה כמותית אורך ורוחב של טיפולים פרה-קליניים

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

Engraftment גידול במודל של עכברים Xenograft של האדם מנטל לימפומה התא

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

הקמתה של תרבות העיקרי של סרקומה של רקמות רכות, נגזר החולה

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

פולימראז דיגיטלי Assay תגובת שרשרת עבור הווריאציה גנטי לחולה סדיר פוליפוזיס באמצעות התבנית שבב-ב--שפופרת

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

החקירה של תלות גנטית באמצעות מבחני תחרות המבוססות על CRISPR-Cas9

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

מטופל-נגזר אורתוטופית מודלים של תאים האדם Urothelial קרצינומה של סרטן המעי הגס ואת הגידול גרורות ספונטנית

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

מטופל-נגזר הטרוגנית מודל Xenograft של סרטן הלבלב באמצעות הזחלים Zebrafish כפונדקאים עבור הערכת סמים השוואתי

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

מלנומה מטופלת-נגזר מודל Xenograft

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

הקמת החולה סרטן קיבה-מודלים נגזרים הנגזרות וקווי התא הראשי

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...