We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

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. הערה: שמונה עכברי BALB / ג שמשו בהליך ניסיוני זו. חמישה עכברים / ג BALB הושתלו עם 5 X 10 6 תאי קרצינומה hepatocellular BNL 1ME A.7R.1 עכבר כדי לייצר גידולים הראשוניים. שלושה עכברי BALB / ג ללא השתלות שמשו כקבוצת ביקורת. BNL 1ME A.7R.1 היא שורת תאי קרצינומה עכברית hepatocellular שנגזרה על ידי שינוי כימי משורת תאי hepatocyte שאינו סרטנית בעכברי BNL CL.2. BNL 1ME A.7R.1 התקבל מהסוג האמריקאי תרבות האוסף (ATCC). 1. טרום ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא עכברים שלכל ניתוחי ההישרדות בריאים. כדי להבטיח שעכברים בריאים, לבדוק סימנים של התייבשות על ידי האהלה של העור. אין להשתמש בעכברים עם כל ראיה של מחלה או דאיסטרס בניסויים אלה. </li><li style=";text-align:right;direction:rtl"> לשקול עכברים כדי לוודא שהם לא בתת משקל. ודא שהמשקל הוא לפחות 16-20 גרם. </li><li style=";text-align:right;direction:rtl"> הכן 5 × תאי HCC עכבר 10 6 BNL 1ME A.7R.1 להשתלה לעכבר: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תקשורת לשאוב מ- 10 מיליליטר 60% - 70% צלחת תרבית תאים ומחוברות של תאי HCC עכבר A.7R.1 BNL 1ME. לשטוף פעמיים עם 5 מיליליטר PBS. הוסף 2 מיליליטר של טריפסין לצלחת ולדגור על 37 מעלות צלזיוס חממת humidified עם האוויר ו 5% CO 2 למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> בהמשך לכך, פיפטה 10 μl trypsinized תאים לתוך hemocytometer ידני לספירת תאים תחת מיקרוסקופ בהגדלה של 10X. לספור את המספר הכולל של תאים הנמצאים בארבעה הריבועים הפינתיים הגדולים ולהכפיל את הגורם לדילול במספר הכולל של תאים. לאחר מכן, לחלק במספר הריבועים נספרו ולהכפיל ב -10 6 לריכוז התאים (תאים למ&quot;ל). </li><li style=";text-align:right;direction:rtl"> שימוש בחישוב, לאסוף 5 מיליון תאים לכל עכבר בסטרילי1.5 מיליליטר צינורות microcentrifuge. תווית 1.5 מיליליטר צינורות microcentrifuge. </li></ol></li><li style=";text-align:right;direction:rtl"> Resuspend 5 × 10 6 תאי BNL 1ME A.7R.1 עכבר HCC בשל נאגר מלוח פוספט 100 μl (PBS). לשמור על תאים אלה על קרח עד השתלה בניתוח. </li><li style=";text-align:right;direction:rtl"> לבצע פעולות הנדרשות להכנת עכברים לניתוח כגון אינדוקציה הרדמה ושיער גזיר מציוד וחומרי סטרילי כדי למנוע זיהום. </li><li style=";text-align:right;direction:rtl"> לגרום להרדמה כללית בעכברים על ידי שאיפה של isoflurane באמצעות מכשיר אדים isoflurane יחד עם חמצן בקצב זרימה מתמיד של ריכוז 1 ליטר לדקה (כ. 2.1%). הערה: מערכת היא עצמאית, וכוללת מאדה דיוק, אספקת חמצן ופחם מיכל שצמוד למעגל נשימה הדרוש להדחת גז / הרדמה. </li><li style=";text-align:right;direction:rtl"> ודא שהרדמה היא נאותה כדי למנוע את החיה מעובר כאב במהלך הניתוח. בצע הבוהן קמצוץ ולצפות בבעלי חיים לאי-responתאוות כדי לאשר הרדמה. </li><li style=";text-align:right;direction:rtl"> באותו זמן, לעבור את קו השסתום, חותם לתיבה ופתוח לחרטום. </li><li style=";text-align:right;direction:rtl"> ברגע שהעכבר הוא מורדם, למרוח כמות קטנה של משחת עיניים לקרניות כאמצעי מניעה מייבוש והנזק. </li><li style=";text-align:right;direction:rtl"> בהמשך לכך, להעביר את בעלי החיים לאזור ההכנה, עם החרטום ממוקם כראוי ומחובר (להרדמת תחזוקה). הכן את האתר של החתך הניתוחי לניתוח על ידי הסרת שיער באזור הכנה זו. </li><li style=";text-align:right;direction:rtl"> ודא שהאזור של ניתוח הוא במיקום ללא כל הפעילות אחרת. </li><li style=";text-align:right;direction:rtl"> ודא שמשטח העבודה יציב ומחוטא כראוי, לא הוכתם ולפני הניתוח ללא עומס. מניחים פיסת פשתן או מגש כירורגית על זה סטרילי. </li><li style=";text-align:right;direction:rtl"> ודא שהשדה האופרטיבי הוא זמין רק אנשי ביצוע הניתוח. ודא שחדר הניתוח הוא ניקה ושטף עם חומר ניקוי מתאים וריסס עם disinfe מתאיםctant. </li><li style=";text-align:right;direction:rtl"> לוודא שהוא גם ממוקם במרחק של דלתות וחלונות כזרמי אוויר יכול להתפשט אבק מוביל לזיהום של האתר. </li><li style=";text-align:right;direction:rtl"> לבצע את ניתוח ההישרדות עם מכשירים סטריליים. כך או לספוג מכשירים בחומר חיטוי בהתאם להמלצתו של היצרן או החיטוי. השתמש בחומרים מתכלים בסיסיים סטרילי כי הם מוכנים לשימוש. </li><li style=";text-align:right;direction:rtl"> מכשירים נפרדים לפי פונקציה כדי למנוע זיהום של אזורים סטריליים. </li><li style=";text-align:right;direction:rtl"> לגלח כ 1 אינץ &#39;סביב האתר של חתך (חתך קו האמצע) כדי לגשת לכבד של בעלי החיים. להסיר את השיער מבעלי החיים מהמקום של ניתוח. </li><li style=";text-align:right;direction:rtl"> לאחר גילוח, לשפשף את האזור עם סבון חיטוי (לשפשף בטאדין כירורגית) ולשטוף את חומר החיטוי העודף משם עם פתרון PBS סטרילי. בצע את זה כמה פעמים, בתהליך שנמשך כ לשפשף כל 5 דקות. הסר והושלך כפפות משומשות. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, לשנות לביגוד מגן כגון surgicשמלת אל, ראש לשפשף, ופניו מסכה. בשלב הבא, לטהר את הידיים עם פתרון לשפשף נכון לפני שתרכיב את כפפות מנתחים. הערה: זה הוא צעד הכרחי כדי למנוע שפיכה ומזהם את האתר כירורגית עם מזהמים פוטנציאליים. </li></ol> 2. ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לספק מקור חום חיצוני לחום לאורך כל התקופה של הרדמה. בעלי החיים עלולים להיתקל כמה היפותרמיה בגלל הרדמה גורמת להפרעות בויסות החום; לכן, לשמור על טמפרטורת הגוף. </li><li style=";text-align:right;direction:rtl"> הנח את העכבר על גבה לאורך כל הניתוח. </li><li style=";text-align:right;direction:rtl"> תקן את הגפיים קדמי, גפיים אחוריות והזנב של העכבר על שולחן הניתוחים עם חתיכות של קלטות כירורגית. </li><li style=";text-align:right;direction:rtl"> לעשות חתך קו ישר באופן מיידי נחותים xyphisternum. </li><li style=";text-align:right;direction:rtl"> השתמש במפשק כירורגי שמירה עצמית סטרילי לחשוף את הכבד. </li><li style=";text-align:right;direction:rtl"> עם מזרק שמחט 20G, לאט ובזהירות להזריק של PBS 100 μl המכיל 5 × 10 6 BNL 1ME A.7R.1תאי העכבר HCC לאונה הימנית של הכבד. </li><li style=";text-align:right;direction:rtl"> לאחר הסרת המחט, תחול מקלון צמר גפן סטרילי דקות לפחות 1 לעצור כל דימום פוטנציאלי עקב גבוה כלי דם של הכבד. </li><li style=";text-align:right;direction:rtl"> סגור את הרקמה התת עורית עם תפר נספג ואחרי סגירת עור באמצעות קליפים כירורגית. בהמשך לכך, לנקות את הפצע שוב עם לשפשף בבטאדין. הערה: הפעולה זו תבטיח את סגירת פצע הניתוח. </li><li style=";text-align:right;direction:rtl"> תת עורי לתת למלח רגיל 100 μl. </li></ol> 3. לאחר ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מייד לאחר ניתוח, להעביר את העכבר לכלוב נקי עם כרטיס זיהוי שלאחר ניתוח להודיע ​​מטפלים של מצב העכבר. לספק מזון ומים. הערה: מניסיוננו, עכבר שזה עתה עבר ניתוח יעלה בגורלם הטוב ביותר אם שוכנו לבד עם אוכל ומים רטובים לתקופה שלאחר הניתוח 24 שעות הראשונית. </li><li style=";text-align:right;direction:rtl"> שמור חמים לבעלי החיים עם מנורת חום שיוצר חום לא מצוין 38 מעלות צלזיוס. Use מדחום למדידת הטמפרטורה עד העכבר התאושש לחלוטין. </li><li style=";text-align:right;direction:rtl"> בית החיה בכלוב רצוי עם מצעי נייר כדי להבטיח נוחות. </li><li style=";text-align:right;direction:rtl"> לאחר התאוששות ראשונית, לפקח על בעלי החיים לסימנים של אי נוחות, דימום, כאב ומצוקה. תן עצירות (0.05 -0.1 מ&quot;ג / קילוגרם) כדי להקל על כאב. באופן הדוק לפקח על הפעילות, ההתנהגות ומראה, כמו גם מים וצריכת מזון לכ 2 שעות. אם אמבולטורי, להעביר את בעלי החיים בחזרה לאזור לדיור רגיל רגיל של עכברים וימשיך לעקוב במרווחים של 6-12 שעות שללאחר ניתוח 24 שעות. לבצע בדיקת משקל אם תרצה בכך. </li><li style=";text-align:right;direction:rtl"> עם זאת, אם בעל החיים הוא שיש בעיות מחלים, לנקוט בפעולות טיפול תומך מתאימות. אם עכברים אינם מסוגלים לשחזר, הומני להרדים עכברים באמצעות CO 2 מחנק ונקע בצוואר הרחם. פעל לפי הנחיות מוסדיות מאושרות להמתת חסד. </li><li style=";text-align:right;direction:rtl"> לאחר ניתוח, לבדוק ראיות קליניות של HCC שהיא בדרך כלל observabהפחתה משמעותית כle בציון מצב גוף. הערה: בשלב זה, גידול ראשוני סביר שפותח בכבד ובפיקדונות גרורתי שסביר להניח שפותחו בריאות. </li><li style=";text-align:right;direction:rtl"> בשלב זה, להקריב את כל העכברים (ניסיון ושליטה) מעורבים בניסוי על ידי המתת חסד הומנית. להכפיף את העכברים למחנק פחמן דו חמצני: לשחרר באיטיות פחמן דו חמצני לתוך התא על פני תקופה של 5 -10 דקות. זה יגרום דום נשימה. </li></ol>

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The authors declare that they have no competing financial interests.

במאמר זה תיאור מעמיק של השיטה הוא נתון שלאחרונה דווח על ידי Ogunwobi ועמיתים של ההקמה המוצלחת של מודל עכבר syngeneic orthotopic של גרורות HCC (איור 2) 3. שיעור המיצוי של גידולים להליך זה הוא בדרך כלל גבוה. יש לנו שנצפה קודם לכן שיעור מיצוי גידול של 100% 3. עם זאת, השיעו...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table border="0">
	<tbody>
		<tr>
			<td>Micro Dissecting Tweezer&#160;</td>
			<td>Roboz</td>
			<td>(RS-5040)</td>
			<td>Tip .20 x .12mm</td>
		</tr>
		<tr>
			<td>Graefe Micro Dissecting Forceps, serrated curved tip</td>
			<td>Roboz</td>
			<td>(RS-5111)</td>
			<td>1 X 2 teeth, curved tip width 0.6mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6501)</td>
			<td>Blunt 3 X 3 prongs, depth 4mm, spread 25mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6503)</td>
			<td>Blunt, 3 X 3 prongs, depth 4mm, spread 19mm</td>
		</tr>
		<tr>
			<td>Jameson Caliper (Measures tumors)</td>
			<td>Roboz</td>
			<td>(RS-6466)</td>
			<td>80mm/3 inch scale, chrome plated&#160;</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td>
			<td>Roboz</td>
			<td>(RS-5852)</td>
			<td>23mm blades, length 4 inches, flat shanks and large rings</td>
		</tr>
		<tr>
			<td>Scalpel with blades, for delicate dissecting procedures</td>
			<td>Roboz</td>
			<td>(RS-9861-36)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle&#160;</td>
			<td>Roboz</td>
			<td>(RS-9884)</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Littauer Stitch Sissors</td>
			<td>Roboz</td>
			<td>(RS-7074)</td>
			<td>Length 4.5 inches</td>
		</tr>
		<tr>
			<td>Brown needle holder, for easy suture tying</td>
			<td>Roboz</td>
			<td>(RS-7960)</td>
			<td>Convex jaw, fine serrations</td>
		</tr>
		<tr>
			<td>Reflex 7MM wound clips with reflex 7 clip applier</td>
			<td>Roboz</td>
			<td>(RS-9262)</td>
			<td>safe, secure alternative method of wound closure</td>
		</tr>
		<tr>
			<td>Instrument tray and lid</td>
			<td>Roboz</td>
			<td>RT-1350S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-clipper with detachable blade</td>
			<td>Roboz</td>
			<td>(RS-5903)</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td>
			<td>Roboz</td>
			<td>Ds-400, Ds-401, DS-501</td>
			<td>For fast decontamination of micro-dissecting instruments.&#160;&#160; Instruments decontaminate within 15 seconds.&#160;</td>
		</tr>
		<tr>
			<td>Microinjection needles</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
	</tbody>
</table>

a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

הכבדים של עכברים / ג BALB הושתלו עם 5 × 10 6 תאי HCC עכבר BNL 1ME A.7R.1. ראיות קליניות של פיתוח HCC הייתה נצפות 63 ימים שלאחר ניתוח. כתוצאה מכך, עכברים מורדמים אנושי. נתיחה לאחר המוות בוצעה בעכברים מורדמים. הריאות והכבד היו כריתה ובדיקת ברוטו זהירה בוצעה לזהות גידולים מקרוסקופי...

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

ההתפשטות הגרורה של הסרטן היא הסיבה העיקרית למוות הקשורים לסרטן. אנו מספקים תיאור מעמיק של מתודולוגיה ניתוח ההישרדות שלנו להקמת מערכת &quot;חולה-כמו&quot; orthotopic מודל עכבר syngeneic לחקר המנגנונים של גרורות בגידולים מוצקים איבר.

&quot;מטופל כמו&quot; Orthotopic syngeneic עכבר הדגם של Hepatocellular Carcinoma גרורה

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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Research

JoVE Journal

Medicine

12.2K Views.  Hunter College of the City University of New York.  ההתפשטות הגרורה של הסרטן היא הסיבה העיקרית למוות הקשורים לסרטן. אנו מספקים תיאור מעמיק של מתודולוגיה ניתוח ההישרדות שלנו להקמת מערכת "חולה-כמו" orthotopic מודל עכבר syngeneic לחקר המנגנונים של גרורות בגידולים מוצקים איבר. 

Video: A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

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 ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

מודל עכבר orthotopic של קרצינומה של בלוטת התריס אנפלסטית

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

דור של תת עורי וintrahepatic אדם xenografts קרצינומה hepatocellular בעכברים עם מערכת חיסון חלשים

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

מודל Murine orthotopic של אדם סרטן ערמונית גרורה

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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 ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

<em&gt; באוב</emAssay&gt; אפרוח chorioallantoic ממברנה (CAM) כמודל xenograft יעיל של Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

ניטור נייד Orthotopic השרשה והיקפי חיסון במודל השני-45 syngeneic עכברוש מזותליומה

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

"מטופל כמו" Orthotopic syngeneic עכבר הדגם של Hepatocellular Carcinoma גרורה

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