Work in Dr. Ogunwobi&#8217;s laboratory is supported by a Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.

הצהרת אתיקה: כל המחקרים בבעלי החיים אושרו על ידי הוועדה המוסדית הטיפול בבעלי חיים והשימוש (IACUC) של האנטר קולג &#39;של האוניברסיטה של ​​העיר ניו יורק. 1. נהלים טרום ניסוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עם ההגעה למתקן בעלי החיים, בתחילה בית 3 עכברי BALB / ג לכלוב עם מצעים העליונים ועץ שבב מסונן. ודא שיש לי העכברים גישה חופשית למזון, אוכל מכרסם פורינה, ומים. מצעים ומזון צריכים להיות מוחלפים פעמיים בשבוע בזמן שיש לנקות כלובי פעם בשבוע. </li><li style=";text-align:right;direction:rtl"> לגרום להרדמה כללית בעכברים באמצעות מכשיר אדים isoflurane יחד עם חמצן בקצב זרימה קבוע של 1 ליטר לדקה. המערכת היא עצמאית וכוללת מאדה דיוק. </li><li style=";text-align:right;direction:rtl"> בעלי החיים מיקום בקופסא פלסטיק שיש מאדה הדיוק וגז / מערכת הרדמה המחוברת אליו. בתחילה להגדיר את מאדה 2.5 עד בעלי החיים הוא חסרי הכרה, ובשלב זה להפחית את האידוי 2. </li><li style=";text-align:right;direction:rtl"> באותו זמן, לעבורקו שסתום, איטום אותו לתיבה ולפתוח אותו לחרטום. </li><li style=";text-align:right;direction:rtl"> הזז את בעלי החיים לאזור ההכנה, עם החרטום ממוקם כראוי ומחובר (להרדמת תחזוקה). </li><li style=";text-align:right;direction:rtl"> הכן 5 × 10 6 תאי HCC עכבר BNL 1ME A.7R.1 להשתלה לכל עכבר. BNL 1ME A.7R.1 צריך להיות גדל במדיום (10% חום מומת FBS, 1% פניצילין / סטרפטומיצין ו -2 מ&quot;מ L- גלוטמין) דוגרים בצלחת תרבית תאים על 37 מעלות צלזיוס בחממה humidified עם אוויר ו -5% CO 2. </li><li style=";text-align:right;direction:rtl"> עם מזרק שיש 20 מחט G, של PBS 100 μl השתל המכיל 5 x 10 6 תאים קרצינומה hepatocellular עכבר BNL 1ME A.7R.1 לאונה של הכבד של עכברים / ג BALB לייצר גידולים הראשוניים. לאחר השתלה, בית העכברים / ג BALB בכלוב עם אותן דרישות הבית כטרום השרשה. </li><li style=";text-align:right;direction:rtl"> בנקודת סיום הניסוי, עדות קלינית של קרצינומה hepatocellular היא נצפית כירידה משמעותית במצב גוףציון (בדרך כלל מזוהה לאחר 4 שבועות). בשלב זה, יש סיכוי שגידולים ראשוניים פיתחו בכבד ואילו פיקדונות גרורתי יצרו את הריאות. לאחר מכן, להתחיל התהליך של הבידוד והתפשטות תאים סרטניים במחזור. </li></ol> 2. אוסף של דם מלא למחזור בידוד תאים סרטניים הערה: כראוי מחוטאת אזור הניתוח ולוודא שהוא נטול עומס. החיטוי כל מכשירי הניתוח או לטבול בחומר חיטוי בהתאם להמלצת יצרן <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להקריב עכברים על ידי המתת חסד הומנית בנקודת סיום ניסוי (עדות קלינית של התפתחות גידולים). מתת חסד של עכברים צריכה לערב CO 2 מחנק. שים לב לעכברים כראוי כדי לאשר הרדמה מופץ בצורה נכונה. פחמן דו חמצני יש לשחרר באיטיות לתוך התא על פני תקופה של 5 - 10 דקות, גורמים לדום נשימה. עקוב ידי exsanguination וcervic intracardiacאל עקירה. 5 </li><li style=";text-align:right;direction:rtl"> שימוש במחט 25 G מצורפת מזרק 1 מיליליטר לאוסף של כל דם. Heparanize המזרק עם 0.01 מיליליטר הפרין. עור לנקב מייד עם המחט נחותה xyphisternum הזווית של 45 מעלות בכ. קידום מחט לאט לנקב את הלב, ואז להנמיך את הזווית של המחט. הולך ומתפשט המחט ומזרק במקום, ולצייר את 500 μl - 1,000 μl של כל דם מהלב. </li><li style=";text-align:right;direction:rtl"> הסר את המזרק ומחט ולהעביר את הדם לתוך צינור ללא pyrogen 1.5 מיליליטר שכותרתו מראש. צנטריפוגה כל הדם שנאסף לתוך 1.5 מיליליטר צינור ללא pyrogen ב1,167 XG במשך 5 דקות. הערה: צנטריפוגה שמפריד בין כל דגימת הדם לשלוש שכבות: השכבה התחתונה או המטוקריט המורכב מתאי דם אדומים, שכבת ביניים דקה של מעיל באפי, ושכבה עליונה פלזמה. </li><li style=";text-align:right;direction:rtl"> הסר את הפלזמה בקפידה על ידי pipetting ואיסוף אותו לתוך צינור 1.5 מיליליטר pyrogen ללא נפרד לפירטניסויים אה כגון זיהוי של חומצות גרעין תא ללא 7. </li><li style=";text-align:right;direction:rtl"> לבידוד של תאי גידול במחזור (CTCs), לאסוף את המעיל באפי לתוך צינור ללא pyrogen נפרד 1.5 מיליליטר בהכנה לתאי דם אדומים תמוגה (RBC). זה הכרחי כדי להסיר RBCs שייר בשכבת המעיל באפי. </li></ol> 3. מתכון לתאי דם אדומים (RBC) תמוגה מאגר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפוך פתרון 1 ליטר של חיץ תמוגה RBC. </li><li style=";text-align:right;direction:rtl"> הפוך 500 מיליליטר של אמוניום כלוריד ו -500 מיליליטר של טריס. אז לערבב כדי ליצור פתרון 1 ליטר של חיץ תמוגה RBC עם ריכוז סופי של 155 מ&quot;מ של אמוניום כלוריד ו -10 מ&quot;מ של טריס. </li><li style=";text-align:right;direction:rtl"> חיץ הפתרון לpH של 7.5. אחסן את מאגר תמוגה RBC ב2-8 מעלות צלזיוס, ולהביא לRT מייד לפני שימוש. </li></ol> 4. RBC תמוגה של מעיל באפי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 1 מיליליטר של חיץ תמוגה RBC לצינור 1.5 מיליליטר סטרילי ללא pyrogen מכיל מעיל באפי שנאסף. </li><li style=";text-align:right;direction:rtl"> מערבבים את התוכן של הצינור על ידיהיפוך מספר רב של פעמים. דגירה התוכן מעורב של הצינור במשך 5 דקות ב RT על הספסל. צנטריפוגה התוכן המעורב במשך 5 דקות ב1,167 x גרם. </li><li style=";text-align:right;direction:rtl"> לאחר צנטריפוגה, תתקבל גלולה לבנבן. בטל supernatant האדמדם לאט ובזהירות לאקונומיקה 10% ללא כל הפרעה לגלולה. אם גלולה היא לא לבנבן, מחדש לעשות צעדים 3.1-3.5. הגדלת זמן הדגירה במאגר תמוגה RBC עד 10 - 15 דקות יכולה להיות גם מועילה. </li><li style=";text-align:right;direction:rtl"> בבטחה להיפטר הפסולת הביולוגית פעם טיהור שפכים ביולוגיים באקונומיקת 10% תושלם לאחר 24 שעות. </li></ol> 5. ריבוי בתרבית תאים הערה: תאי גידול בדרך כלל גדלו במהירות תאי תאי דם לבנים שנמצאים בשפע בשילוב המקורי של תאי זרע לתוך הצלחת. בעקבות שינוי חוזר ונשנה של מדיום, והמעברים הבאים של CTCs, כל תאי הדם הלבנים יוסרו ואוכלוסייה טהורה יחסית של CTCs נשארה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחרהשלכת supernatant לתוך 10% אקונומיקה, לשטוף את הכדור לבנבן ב1 נאגר מלוח פוספט מיליליטר (PBS) לפחות פעם אחת, וגלול במדיום התרבות השלם לריבוי בתרבית תאים. </li><li style=";text-align:right;direction:rtl"> ברגע לשטוף הושלם, להוסיף 1 מיליליטר של מדיום מלא BNL 1ME תרבות A.7R.1 (חום 10% מומת FBS, 1% פניצילין / סטרפטומיצין ו2 מ&quot;מ L-גלוטמין) לגלולה וresuspend גלולה במדיום התרבות. </li><li style=";text-align:right;direction:rtl"> זרעי תערובת resuspended של תאים לתוך צלחת תרבית תאים. דגירה צלחת תרבית תאים על 37 מעלות צלזיוס בחממה humidified עם האוויר ו 5% CO 2. </li><li style=";text-align:right;direction:rtl"> לשנות את מדיום תרבית תאים בכל יומיים-שלושה. לאחר חמישה עד שבעה ימים, CTCs יהיה דבק בצלחת תרבית תאים והתחיל מתרבים. תאים אלה יישארו קיימא מעבר 25 קטעים ואחרי מחזורי הקפאה להפשרה חוזרים ונשנים 5. </li></ol> 6. אימות של Hepatocellular Carcinoma מחזור קו תאים סרטניים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בצע APolymerase תגובת שרשרת שיטה מבוססת (PCR), כדי להגביר קטע DNA ספציפי אחד של גן העכבר β-גלובין כדי לאשר שהרומן הוקם שורות תאי CTC הן תאי עכבר כמו הקו המקורי המושתל BNL 1ME A.7R.1 HCC. שיטה שתוארה במקור ומאומתים על ידי Steube et al. 5,8 </li><li style=";text-align:right;direction:rtl"> בצע immunostaining לאלמנט התגובה cAMP סמן hepatocyte הספציפי מחייב חלבון 3 כמו-3 (CREB3L3) באמצעות נוגדן ספציפי. זה יאשר שהרומן CTCs הוקם אכן hepatocytes כמו קו התא המקורי המושתל BNL 1ME A.7R.1. 5 </li></ol>

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The authors declare that they have no competing financial interests.

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis. 
A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy3. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are important in cancer metastasis and have potential for multiple clinical applications, such as for diagnosis3,9,10. This is supported by data from several clinical studies that have showed that CTCs have a strong correlation with the progression of the disease in various cancer types such as pancreatic, prostate and breast, among others3,11,12. Despite the fact that CTCs may be very useful for cancer diagnosis, isolating and characterizing CTCs are challenging because very few are present among billions of blood cells3,7.
A significance of isolating CTCs using our technique is that it provides a reproducible model which can be optimized for potential utility in a clinical setting. This may ultimately lead to the possibility of using this method to replace current biopsy procedures that are more invasive and associated with side effects3,13. Modifications of this method, such as peripheral intravenous blood collection, would be expected to utilize it in a clinical setting. Of course, it is acknowledged that isolating CTCs from humans is a much more challenging endeavor due to general low yield4,14. But, in many cases such as in pancreatic cancer, histo-pathologic or cytological validation using tissue biopsies is fruitless in spite of multiple invasive attempts4. Isolating CTCs has been described as &#8220;liquid biopsy&#8221; or &#8220;live biopsy&#8221; of a tumor which enables noninvasive cancer diagnosis and real-time monitoring of therapeutic response3. This methodology models the isolation of CTCs in a mouse model. Thus far, only one FDA- cleared assay, such as Veridex CellSearch System, has been approved to detect CTCs. In one study, this technology identified CTCs in only 11 of 26 patients. This system is associated with some inefficiency, limited purity and high cost4. Moreover, it lacks the important feature of being able to collect viable CTCs whether they are EpCAM-expressing or not for extensive analyses using multiple in vitro and in vivo methods.
An important feature of the described method is that it preserves the viability of the isolated CTCs3. Therefore, using this method allows for the subsequent extensive downstream analysis of both the molecular and functional characteristics of CTCs using a variety of techniques. 
In conclusion, a methodology for isolating and propagating CTCs that are viable and can be characterized molecularly and functionally is presented. There is potential that this method can be optimized for clinical applications.

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 18691. Since then, CTCs have been shown to be important in tumor metastasis and disease progression2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis2-5. Notably, there is positive correlation between tumor size and CTC number3,4. 
An understanding of CTC biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment2. Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood6. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.
Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.
We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis5. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology. 
There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy4. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

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	<tbody>
		<tr>
			<td>Heparin Sodium Salt 1G</td>
			<td>VWR</td>
			<td>89508-852</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX Tube Micro 1.5mL Clear NS</td>
			<td>VWR</td>
			<td>89511-254</td>
			<td>1.5mL&#160; pyrogen-free eppendorf Tubes</td>
		</tr>
		<tr>
			<td>Needle Sterile Disp BD 25GX1IN</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
		<tr>
			<td>Slp Tip SRNG 1ml 200 each per pack</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>1mL syringe tip</td>
		</tr>
		<tr>
			<td>Syringe 1ml leur lok Pk 100</td>
			<td>VWR</td>
			<td>BD309628</td>
			<td>1 mL syringe</td>
		</tr>
		<tr>
			<td>VWR Forceps Tissue 6</td>
			<td>VWR</td>
			<td>82027-446</td>
			<td>Forceps</td>
		</tr>
		<tr>
			<td>Cyromold intrm 15X15X5MM PK 100</td>
			<td>VWR</td>
			<td>25608-924</td>
			<td>Cyromold</td>
		</tr>
		<tr>
			<td>Cryo-oct compund 4oz</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Oct compound</td>
		</tr>
		<tr>
			<td>VWR Slide sprfrst 25X75MM PK72</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td>Slides</td>
		</tr>
		<tr>
			<td>VWR Cover Glass #2 22X5oMM OZ</td>
			<td>VWR</td>
			<td>48-382-128</td>
			<td>Cover Glass</td>
		</tr>
		<tr>
			<td>VWR Slide Box True North Fm Pu</td>
			<td>VWR</td>
			<td>89140-278</td>
			<td>Slide Box</td>
		</tr>
		<tr>
			<td>Super HT PAP Pen</td>
			<td>VWR</td>
			<td>89427-058</td>
			<td>PAP pen</td>
		</tr>
		<tr>
			<td>Water RNASe and DNAse free 2L</td>
			<td>VWR</td>
			<td>101454-204</td>
			<td>Nuclease Free Water</td>
		</tr>
		<tr>
			<td>Buffer Tris Ultra Pure Grade 500G</td>
			<td>VWR</td>
			<td>97061-796</td>
			<td>Tris Buffer</td>
		</tr>
		<tr>
			<td>Ammonium Chloride ACS Grade 2&#160; 5KG</td>
			<td>VWR</td>
			<td>97062-048</td>
			<td>Ammonium Chloride Buffer</td>
		</tr>
		<tr>
			<td>Falcon Tissue Culture Dish 60 x 15mm Style polystyrene</td>
			<td>VWR</td>
			<td align="right">353002</td>
			<td>Tissue Culture Dish</td>
		</tr>
		<tr>
			<td>Clorox&#174; Germicidal Bleach, Regular</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Clorox Bleach</td>
		</tr>
		<tr>
			<td>PBS, 1X (Phosphate-Buffered Saline) without calcium &#38; magnesium (500mL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>21-040-CV</td>
			<td>PBS, 1X</td>
		</tr>
		<tr>
			<td>DMEM, 1X&#160; with 4.5 g/L glucose &#38; L-glutamine without sodium pyruvate</td>
			<td>Thermo Fischer Scientific</td>
			<td>10-017-CV</td>
			<td>DMEM 1X media for BNL 1ME A.7R.1 cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Seru8m</td>
			<td>Thermo Fischer Scientific</td>
			<td>35-010-CV</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution, 100X</td>
			<td>Thermo Fischer Scientific</td>
			<td>30-002-Cl</td>
			<td>Penicillin Streptomycin</td>
		</tr>
		<tr>
			<td>Sorvall Biofuge pico</td>
			<td>Thermo Fischer Scientific</td>
			<td align="right">75002411</td>
			<td>13000rpm Centrifuge</td>
		</tr>
	</tbody>
</table>

isolation and propagation of circulating tumor cells from a mouse cancer model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

המתודולוגיה שתוארה כאן הוכיחה כי יכול להיות מבודד CTCs. עכברים מורדמים אנושיים בנקודת סיום ניסוי. מחנק התהליך מעורב פחמן דו חמצני, exsanguination פנים-לב, ונקע בצוואר הרחם. סכמטי של שלבים העיקריים של ההליך הם באיור 2. דגימות דם כל שנאספו על ידי exsanguination פנים-לב עובדו באמצעות הפרוטוקול שתואר לעיל. לאחר ש, צנטריפוגה שימשה כדי לקבוע את שכבת מעיל באפי שהיה נתון לתמוגה RBC. זה היה לאחר מכן על ידי צנטריפוגה צעד אחר לאסוף גלולה לבנבן שנשטפה ב- PBS, וresuspended במדיום תרבית תאים מלא לריבוי בתרבית תאים. בעיה שעלולה לבוא היא שהגלולה שהושגה לא יכולה להיות ברורה בצבע. בעיה זו ניתן לפתור בקלות על ידי שינוי זמן הדגירה במאגר תמוגה RBC על ידי הגדלתו עד 10 - 15 דקות, או אולי על ידי ביצוע פעמים נוספות זה.נציג תמונות של CTCs שמופצות בתרבות משלושה עכברים שונים מוצגים באיור 1, בהשוואה לתאי A.7R.1 BNL 1ME. התפשטות ומורפולוגיה מהירות יעזרו לזהות אם תאים שנזרעו הם CTCs. עם זאת, טכניקות אימות כגון PCR וimmunostaining המתוארים בצעדים 5.1 - נדרשות 5.2 כדי לאשר את הקמת קו CTC קיימא. <img alt="איור 1" src="/files/ftp_upload/52861/52861fig1.jpg" /> איור 1. ריבוי תאים סרטניים במחזור (CTCs) בתרבית תאים ממודל העכבר orthotopic syngeneic של Hepatocellular Carcinoma גרורה. תאים היו מופצים בתרבות מנות תא x 60 מ&quot;מ 15 מ&quot;מ במדיום תרבות BNL 1ME A.7R.1. תאים סרטניים מתרבים דבקו צלחת ומוצגים משלושה עכברים נפרדים. תאים סרטניים שנותרו קיימא דרך מעברים רציפים. תמונה בניגוד שלב של CTCOL1MEA081211, CTCOL1MEACTC082911 וCTCOLUF2419 נלקחו ב40X הגדלה (עדשה אובייקטיבית) בהשוואה לתאי BNL 1ME A.7R.1 (הגדלת 40X). <a href="https://www.jove.com/files/ftp_upload/52861/52861fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 1" src="/files/ftp_upload/52861/52861fig2.jpg" /> איור 2. סכמטי של ההליך של בידוד והפצת מחזור תאים סרטניים ממערכת מודל העכברי של הסרטן. כאשר ראיות קליניות של קרצינומה hepatocellular נצפו כהפחתה משמעותית בציון מצב גוף, כל הדם נאסף מעכברים. לאחר כמה centrifugations, תמוגה RBC, וכמה צעדים לשטוף, CTCs היה זרע לתוך תרבות מנות תא להתפשטות. התפשטות תאי גידול נצפתה לאחר כמה ימים. <a href="https://www.jove.com/files/ftp_upload/52861/52861fig2large.jpg" target="_blank">אנא לחץ עליו</a>מחדש כדי לצפות בגרסה גדולה יותר של דמות זו.

Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

בידוד וריבוי תאים סרטניים במחזור מסרטן מודל עכבר

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

isolation-propagation-circulating-tumor-cells-from-mouse-cancer

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11.9K Views.  Hunter College of The City University of New York. Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Video: Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

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

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

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

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

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

בתחום ההדמיה vivo של פליטת אור Dynamics היפוקסיה גידול של גרורות סרטן השד המוח במודל של עכברים

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

שילוב הקרנות של מודל העכבר Orthotopic המוח גידול

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

הקרנה למכפילים מלנומה תוך שימוש במודל גידול כבני מקום דג זברה

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

חי הדמיה של תגובות סמים בmicroenvironment הגידול במודלים עכבריים של סרטן השד

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

תרבות מטופל נגזר סלולרי ובידוד של CD133<sup&gt; +</sup&gt; תאי גזע סרטני משוערים מלנומה

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

חניכה של סרטן השד גרורתי על ידי מיקוד של האפיתל ductal עם Adenovirus-Cre: דגם רומן מהונדס עכבר של סרטן השד

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

עיבוד של semiautomated מחזור תא גידול מבחני (CTC) ליישומים קליניים ומחקר הקליני

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

מודל גידול מאוס של מתח כירורגי כדי לחקור את המנגנונים של דיכוי מערכת חיסוני לאחר ניתוח והערכת immunotherapies Perioperative רומן

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

בידוד של תאי גזע סרטני מדגימות אדם סרטן הערמונית

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...