This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

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The authors have nothing to disclose.

השלבים הקריטיים ביותר בפרוטוקול הם 1) בהצלחה שמירה על tumorigenicity של הקו הסלולרי; 2) להבטיח הפרשת PSA למדידה לפני ההשתלה תאים סרטניים בעכברים; 3) יצירת השעית תא יחיד להשתלה כדי להפחית וריאציה בגודל גידולים; ו -4) הקניית תאים בקצב איטי כדי למנוע ריפלוקס vesico-השופכן, וכתוצאה מכך ה...

המטרה של שיטה זו היא ליצור גידולים בשלפוחית ​​שתן orthotopic murine וכן לפקח על הגידולים המושתלים באופן מדויק ככל האפשר, כך שהעכברים ללא השתלת גידול לא מיוחסים נרפאו ב ניתוח נקודת סיום. בסך הכל, השיטה לראות תפחית את צורך כמויות גדולות של עכברים לניתוח הניסיון ולהבטיח דיוק רב יותר בקביעת תוצאות טיפוליות. פיתוח מודל orthotopic לסרטן חשוב, כמו תאים סרטניים להשתיל מתחת לעור לא לשחזר את הסביבה של מחלה קלינית או לאפשר פיתוח של אסטרטגיות טיפוליות. הארכיטקטורה של שלפוחית ​​השתן מאפשרת ההחדרה של טיפולים בסרטן שלפוחית ​​השתן ישירות לתוך שלפוחית ​​השתן עם תופעות מערכתיות מינימאליות. לכן, במודלים של בעלי חיים כי לשחזר בסביבה זו, כגון מודל orthotopic, שחשובים להעריך טיפולים חדשים. מסקנות מכל ניסוי הגדרה הם dependent על מגבלות המודל. טכניקות פותחו מספר לייצור גידולים בשלפוחית ​​השתן orthotopic בעכברים. אלה מסתמכים על הפגיעה בשכבת glycosaminoglycan של שלפוחית ​​השתן, מה שמאפשר לתאים נגועים להיות מושתלים. הטכניקות בשימוש כוללות electrocautery, שתוצאתה נקודה אחת של נזק לדופן השלפוחית, מה שמוביל להתפתחות גידולים באתר אחד בשלפוחית 1,2. עם זאת, שיעור ההצלחה של השתלת גידול באמצעות electrocautery הוא מפעיל תלוי הנע בין 10 - 90%, והיא כוללת את הסכנה כי לדופן השלפוחית ​​יהיה נקב, שמובילה גידולים המתפתחים חלל הצפק. כווייה כימית מתבצעת באמצעות כסף חנק, אשר פוגע בקיר השלפוחית 3. באופן דומה, חומצה נעשה שימוש כדי לפגוע בקיר השלפוחית 4. טריפסין גם נעשה שימוש כדי לפגוע השלפוחית כמו גם 5. שיטות אלה עלולות לגרום להתפתחות של יותר מ גידול אחד בשלפוחית.יתר על כן, קיימת סכנה של נזק חמור השלפוחית ​​אם הכימיקלים נשארים במגע עם דופן השלפוחית ​​במשך זמן רב מדי. השיטה שפותחה על ידי Ninalga et al. משתמש פולי- L- ליזין בעלי המטען החשמלי החיובי (PLL) 6 מולקולות כדי לצפות את קיר שלפוחית השתן; זה מאפשר לתאי הגידול טעונים שלילית להיצמד שכבת glycosaminoglycan של שלפוחית ​​השתן. שיטה זו בדרך כלל תוצאות יותר גידול אחד לפתח בשלפוחית, אך השתלת גידול היא 80 - 100% 4,7. מבחינה טכנית, זה גם ההליך הקל ביותר לבצע. כדי להבטיח כי הגידולים המתפתחים הם די אפילו בגודל, חשוב כי התאים הסרטניים אינם מקובצי גושים גדולים לפני ההשתלה. על מנת להעריך יעילות טיפולית, עדיף לבצע מחקר זה על עכברים עם גידולים בגודל דומים למדי. לפיכך, מערכת זיהוי טובה שיכול לכמת גודל גידול קצר לאחר ההשתלה היא חשובה. יש מספר אסטרטגיות דבורהn המשמש להערכת גידולים. אלה כוללים הדמיה בתהודה מגנטית (MRI) 8-10, קרינת 11, פליטת אור 12,13, אולטרסאונד 14, ו assay immunosorbent enzyme-linked (ELISA) 15,16. בעוד MRI ואולטרסאונד אינו דורשים שינויים של התאים הסרטניים, יש צורך סוכני ציוד והניגודיות רגישים עבור MRI. Fluorescence-, luminescence-, המבחנים המבוססים ELISA דורשים שינוי של התאים הסרטניים לבטא חלבוני סמן שיכול להיות מזוהה על ידי שיטות אלה. לקבלת הארה, מצע נדרש לצורך זיהוי של פעילות בלוציפראז; ובכך, יש שלב מוסף התייקרות. שניהם הארת הקרינה דורשות ציוד מיוחד. כדי לייצר קרינה, חלבון פלואורסצנטי ירוק (GFP) cyclization, אשר מזורז על ידי חמצן מולקולרי, נדרש. לפיכך, ביטוי של GFP עשוי להיות משתנה בתוך מסת גידול תלוי גישת החמצן, מה שהופך את סמן למדי אמין <sup&gt; 17. שינוי של שורת התאים בעכברי שלפוחית שתן סרטן MB49 להפריש אנטיגן ספציפי לערמונית אדם (PSA) 15,16 כסמן פונדקאי הוא אסטרטגיה אחרת. סמנים אלה גם לספק אמצעים חלופיים המאשר נוכחות גידול על סיום הניסוי, מה שהופך אותם כחלופה אימונוהיסטוכימיה. מחקר זה מדווח על שיטת PLL של השתלת גידול orthotopic ומציג השוואה של מערכות גילוי גידול, כלומר ELISA, קרינה, והדמית אולטרסאונד.

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:404px;">MB49-PSA cells</td>
			<td style="width:188px;">N/A</td>
			<td style="width:188px;">N/A</td>
			<td style="width:113px;">ref (Wu QH, 2004)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640 media</td>
			<td>HyClone</td>
			<td>SH30027.01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media</td>
			<td>Biowest</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American, Premium</td>
			<td>Biowest</td>
			<td>S181B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30088.03&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine</td>
			<td>Biowest</td>
			<td>X0550</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin-Streptomycin</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hygromycin B</td>
			<td>Invitrogen</td>
			<td>10687-010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">free PSA (Human) ELISA kit</td>
			<td>Abnova</td>
			<td>KA0209</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TRIzol Reagent for RNA extraction&#160;</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>4374967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Mouse Actb</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Mm00607939_s1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Human KLK3</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Hs00426859_g1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C57BL/6J female mice</td>
			<td>In Vivos</td>
			<td></td>
			<td>4-6 wk old</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reversal drug (1mg/kg Atipamezole)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ear punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>72893-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hartmann&#39;s solution or Compound sodium lactate</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat pack - HotHands handwarmers</td>
			<td>Heatmax Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Introcan Certo IV catheter</td>
			<td>B Braun</td>
			<td>4251300</td>
			<td>24G x 3/4&#8243;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aquagel Lubricating jelly</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;Poly-L-lysine solution, 0.01%,</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Quantichrom Creatinine Assay Kit</td>
			<td>BioAssay Systems</td>
			<td>DICT-50&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fluorescent dye - VivoTrack 680</td>
			<td>Perkin Elmer</td>
			<td>NEV12000&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNAlater-ICE Frozen Tissue Transition Solution</td>
			<td>Ambion</td>
			<td>4427575</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Realtime PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">7500 Software v2.3</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Metabolic Cage</td>
			<td>Tecniplast&#160;</td>
			<td>Vertical type rack for 12 cages</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSCanto I system&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSDiva software v7</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IVIS SpectrumCT in vivo imaging system&#160;</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Living Image Software v3.1</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vevo 2100 imaging system&#160;</td>
			<td>VisualSonics&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine orthotopic bladder tumor model and tumor detection system

פרוטוקול זה מתאר את הדור של גידולים בשלפוחית ​​השתן אצל נקבות עכברים C57BL / 6J באמצעות שורת תאים סרטניים בשלפוחית ​​השתן בעכברים MB49, אשר שונתה כדי להפריש אנטיגן ספציפי לערמונית האדם (PSA), ואת ההליך לאישור של ההשתלה הגידול. בקיצור, עכברים מורדמים באמצעות תרופות להזרקה והם עשויים להניח במצב הגבה. שתן מתפנה מן השלפוחית ​​50 μL של פולי- L- ליזין (PLL) הוא החדיר לאט בקצב של 10 μL / 20 שניות באמצעות קטטר 24 G IV. הוא עזב בשלפוחית ​​במשך 20 דקות על ידי וסותם את הקטטר. צנתר מוסר PLL הוא שפונה על ידי לחץ עדין על שלפוחית ​​השתן. זה ואחריו החדרה של הקו הסלולרי בעכברים שלפוחית השתן סרטן (1 x 10 5 תאים / 50 μL) בשיעור של 10 μL / 20 שניות. קטטר פקוק למנוע פינוי מוקדם. לאחר h 1, העכברים הם החיו עם תרופת היפוך, ואת שלפוחית ​​השתן שהתפנה. שיעור ההחדרה האיטי חשוב,כפי שהיא מקטינה ריפלוקס vesico-השופכן, אשר יכול לגרום לגידולים להתרחש בדרכי השתן העליונות בכליות. שורת התא צריך להיות גם מחדש תלוי להפחית clumping של תאים, כמו זה יכול להוביל בגדלי גידול אחידים לאחר השתלה. טכניקה זו גורמת גידולים עם יעילות גבוהה. צמיחת גידול מנוטרת על ידי הפרשת PSA שתן. ניטור סמן PSA הוא יותר אמין יותר מאשר אולטרסאונד או דימות פלואורסצנטי לאיתור נוכחות של גידולים בשלפוחית. גידולים בעכברים בדרך כלל להגיע לגודל מקסימאלי משפיע לרעה בריאות בכ 3 - 4 שבועות אם אינו מטופל. באמצעות מעקב אחר צמיחת הגידול, אפשר להבחין עכברים נרפאו מאלו שלא הושתלו בהצלחה עם גידולים. עם ניתוח נקודת סיום רק, זה האחרון עשוי להניח בטעות נרפא על ידי טיפול.

הפרשת PSA מתאי MB49 נמצאה להשתנות עם תקשורת הצמיחה. MB49-PSA הוא גדל DMEM התקשורת כי זה תוצאות הפרשת PSA מוגבר (איור 1 א). כדי לקבוע את הרגישות של ELISA PSA בזמן אמת PCR, מספרים שונים של תאים מפרישי MB49-PSA היו מעורבים עם תאים הורית MB49. PSA ELISA מזהה מינימום של 1 x 10 5...

Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

פרוטוקול זה מתאר את הדור של גידולים בשלפוחית ​​השתן בעכברים orthotopic אצל נקבות עכברים C57BL / 6J ועל ניטור של הגידול.

מודל גידול Murine Orthotopic שלפוחית ​​השתן סרטן מערכת איתור

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

a-murine-orthotopic-bladder-tumor-model-and-tumor-detection-system

Research

JoVE Journal

Cancer Research

14.5K Views.  National University of Singapore, National University Health System.  פרוטוקול זה מתאר את הדור של גידולים בשלפוחית ​​השתן בעכברים orthotopic אצל נקבות עכברים C57BL / 6J ועל ניטור של הגידול. 

Video: A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Murine ניסיוני דגם של התפתחות גידולים מקורית גרור הצפק<em&gt; דרך</em&gt; Orthotopic הרכבה עם קרצינומה של תאי השחלות

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

במודל עכבר של עייפות הנגרם על ידי הקרנה ההיקפית

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

בידוד של תאים סרטניים במחזור מודל עכבר אורתופטי של סרטן המעי הגס

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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

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 stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

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 ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

כרומטין יליד Immunoprecipitation באמצעות המוח מאתר הגידול Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

דגם Syngeneic Orthotopic זוהרים, פלורסנט מאתר של סירוס עמידים ו אנדרוגן תלויי סרטן הערמונית

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

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

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with 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 ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

תלת מימדי הרתי תרבות מערכת לייצר Murine המושרה תא גזע Pluripotent-הגידול הנגזר אנטיגן-מהגרים ספציפיים הרתי

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

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

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 colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

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 ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

מודל גידול Murine Orthotopic שלפוחית ​​השתן סרטן מערכת איתור

מודל גידול Murine Orthotopic שלפוחית השתן סרטן מערכת איתור