עבודה זו נתמכה על ידי מכון קנדי ​​לבריאות מחקר שלב 1 המטפל-פרס מדען (AG) ושל גרנט תפעולי מהאגודה לחקר הסרטן (AG). המחברים מודים לד&quot;ר ג&#39;ון דיק לתמיכה בפרויקט זה שלו.

<ol>
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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

יש לנו תארנו מגוון של טכניקות כדי להקים תת עורי וxenografts HCC האנושי בעכברים עם מערכת חיסון חלשים intrahepatic שיכול להיות מיושמים על מגוון רחב של שאלות ומבחנים ניסיוניים. בעוד xenografts תת עורי הייתה בשימוש נרחב ללמוד היבטים שונים של הביולוגיה HCC, xenografts intrahepatic מתוארות בספרות רק לע...

קרצינומה hepatocellular (HCC) היא הסרטן החמישי הנפוץ ביותר בעולם והסיבה הכי בקצב המואץ למוות מסרטן בצפון אמריקה. גורם הסיכון הנפוץ ביותר עבור HCC הוא שחמת כבד, בתדירות הגבוהה ביותר מתרחש עקב דלקת כבד כרונית נגיפית, שימוש לרעה באלכוהול, מחלות אוטואימוניות, או הפרעות מטבוליות תורשתיות 1. למרות נטל מחלת הכבד שהוטל על ידי HCC על אוכלוסיות ברחבי העולם, הפתופיזיולוגיה של HCC מובנת יחסית גרוע בהשוואה לסוגי סרטן שכיח אחרים כגון מעי גס, שד, או סרטן הערמונית. לדוגמא, אירועים מולקולריים ותאיים ספציפיים נהיגה tumorigenesis להישאר להיות מוגדרים 2 באופן ברור. כמו רוב סוגי סרטן אפיתל מוצקים אחרים, גישות הגנומי חשפו את ההטרוגניות בסטיות קשורות עם HCC 3. מספר המחקרים גילו פעילות מסודרות של מגוון רחב של מסלולי איתות מעורבים בהתפשטות תאים, survival, בידול ואנגיוגנזה 4. בנוסף, תפקידם של תאי גזע סרטני בpathobiology HCC נשאר להתברר 5. עם הבנה מוגבלת של הפתופיזיולוגיה HCC, בארסנל של טיפולים יעילים לHCC גם נשאר מוגבל יחסית. חולים בשלב מוקדם, עם גידולים מוגבלים לכבד הם מועמדים לטיפול מרפא באמצעות אבלציה גידול או כריתה כירורגית, אם כי ההישנות היא שכיחה. עבור חולים עם מחלה מתקדמת יותר, כימותרפיה והקרנות הם בעלי יעילות מוגבלת ומשמשים בעיקר לבקרת מחלות בכוונה פליאטיבי 6. באיכות גבוהה במודלי ניסויי vivo של HCC האנושי ובכך מספקת פלטפורמה רבת ערך למחקר בסיסי נחוץ להפתופיזיולוגיה של HCC האנושי, כמו גם להערכה של גישות טיפוליות חדשניות. בהשוואה לשימוש בשורות תאים או מודלים עכבר מוגדרים מאוד, xenografts של פריגידולים אנושיים בעכברים עם מערכת חיסון חלשים מרי צמחו ככלים רבי ערך למחקרים מסוג זה שכן הם מסוגלים משחזרים מחלות של בני אדם עם נאמנות גבוהה גם בעת לכידת הטרוגניות שקיימת בתוך ובין חולים השונים 7,8. לשם כך, פיתחנו מגוון רחב של שיטות להקמת xenografts HCC האנושית בעכברים עם מערכת חיסון חלש. בעוד שרוב המחקרים שפורסמו מעורבים xenografts HCC לתאר את השימוש בשורות תאי HCC האנושי מבוססות היטב למטרה זו, אנו מתמקדים באופטימיזציה של מבחני שלנו כדי ליצור xenografts מדגימות HCC עיקריות מתקבלת מייד לאחר כריתה כירורגית של חולים. טכניקות xenografting שונות עשויות להידרש למחקר יישומים שונים. לדוגמא, xenografts תת עורי שנוצרה מתוך שברי גידול נוצרות במהירות, נמצאת במעקב בקלות, ועשוי להיות מתאים יותר לממשל מקומי של הרפוי רומן עם נוחניטור של תגובת גידול. xenografts intrahepatic עשויה להיות רלוונטית יותר למחקרים הנוגעים לתפקידו של המיקרו בכבד בביולוגיה HCC. Xenografts שנוצרה מהשעיות תא גידול נחוצות לזיהוי והאפיון של תת תא ייזום גידול או לניסויים הדורשים במניפולציות מבחנה של תאים סרטניים לפני xenotransplantation. וכך פיתחנו ומאומתים הפרוטוקולים הבאים להקים תת עורית או xenografts intrahepatic מהשעיות תא או שברי גידול נבעו מדגימות HCC האנושי ראשוניות.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dulbecco's Mod. Eagle Medium/Ham's F12 50/50 Mix x1(DMEM-F12)</td>
			<td>WISENT Bioproducts</td>
			<td>319-075-CL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase TypeIV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Stemcell Technologies</td>
			<td>7923</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>354234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 % Buffered Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9 % Saline Solution (NaCl), sterile</td>
			<td>House Brand</td>
			<td>1011-L8001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine surgical scrub</td>
			<td>Purdue Pharma</td>
			<td>NPN 00158313</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LORIS 10% PVP-I Solution</td>
			<td>LERNA Pharma Inc.</td>
			<td>109-09</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Buprenorphine (Temegesic) NR 0.3 mg/ml</td>
			<td>Reckitt Benckiser</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isoflurane USP, 99.9 %, inhalation anesthetic</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>M60302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tear-Gel</td>
			<td>Novartis Pharmaceuticals</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Frozen section compound</td>
			<td>VWR</td>
			<td>95057-838</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryomold, Tissue -Tek</td>
			<td>Sakura Finetek</td>
			<td>4566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Precision Glide Needle 18G 1 &frac12;</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Precision Glide Needle 27G &frac12;</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>305109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Insulin syringe, 3/10 cc U-100, 29G&frac12;</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>309301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical blade No.10</td>
			<td>Feather Safety Razor Co.</td>
			<td>08-916-5A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>#5-0 Soft silk surgical suture, 3/8" taper point needle</td>
			<td>Syneture</td>
			<td>VS-880</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transpore surgical tape</td>
			<td>3M Health care</td>
			<td>1577-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cotton applicator</td>
			<td>Medpro</td>
			<td>018-425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgicel, oxidized regenerated cellulose</td>
			<td>Ethicon</td>
			<td>1951</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell strainer 100 &mu;m nylon</td>
			<td>Becton-Dickinson Biosciences</td>
			<td>352360</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnification lighting with mobile base</td>
			<td>Benson medical Industries Inc.</td>
			<td>model: RLM-CLT-120V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petridish sterile 100x20 mm</td>
			<td>Sarstedt</td>
			<td>821474</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue forcep, 1x2 teeth, 4-1/2"</td>
			<td>Almedic</td>
			<td>A10-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Adson dressing forcep 4-3/4"</td>
			<td>Almedic</td>
			<td>A10-220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eye dressing forcep, serrated, straight, 4"</td>
			<td>Almedic</td>
			<td>A19-560</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hartman Hemostatic Forceps, curved, 3-1/2"</td>
			<td>Almedic</td>
			<td>A12-142</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iris scissor, curved, 4-1/4"</td>
			<td>Almedic</td>
			<td>A8-690</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iris scissor, straight, 4-1/2"</td>
			<td>Almedic</td>
			<td>A8-684</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olsen-Hegan needle driver, 5-1/2"</td>
			<td>Almedic</td>
			<td>A17-228</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

generation of subcutaneous and intrahepatic human hepatocellular carcinoma xenografts in immunodeficient mice

In vivo מודלים ניסיוניים של קרצינומה hepatocellular (HCC) כי לשחזר את המחלה האנושית מספקים פלטפורמה רבת ערך למחקר להפתופיזיולוגיה מחלה ולהערכה קליני של טיפולים חדשניים. אנו מציגים מגוון רחב של שיטות ליצירה תת עורית או xenografts HCC האנושי בעכברים עם מערכת חיסון חלשים orthotopic שיכול להיות מנוצל במגוון רחב של יישומי מחקר. עם דגש על השימוש ברקמת גידול ראשונית מחולים שעברו כריתה כירורגית כנקודת התחלה, אנו מתארים את ההכנה של השעיות תא או שברי גידול לxenografting. אנו מתארים טכניקות ספציפיות לxenograft רקמות אלה תת עורי), או ii) intrahepatically, או על ידי השתלה ישירה של תאים סרטניים או שברים בכבד, או בעקיפין על ידי הזרקה של תאים לתוך טחול העכבר. אנו גם מתארים את השימוש של כריתה החלקית של כבד עכבר הילידים בזמן xenografting כאסטרטגיה ללגרום למצב של התחדשות כבד פעילה בעכבר הנמען שעשוי להקל על קליטתם intrahepatic של תאים סרטניים אנושיים ראשוניים. התוצאות הצפויות של טכניקות אלה באים לידי ביטוי. הפרוטוקולים שתוארו אומתו באמצעות דגימות אנושיות ראשוני HCC וxenografts, אשר בדרך כלל לבצע פחות וחסונה יותר משורות תאי HCC האנושי המבוססות היטב כי נמצא בשימוש נרחב ומצוטטות לעתים קרובות בספרות. בהשוואה לשורות תאים, אנו דנים גורמים אשר עשוי לתרום לסיכוי הנמוך יחסית של engraftment HCC העיקרי במודלי xenotransplantation ולהגיב על בעיות טכניות שעלולות להשפיע על קינטיקה של צמיחת xenograft. אנחנו גם מציעים שיטות שיש ליישם כדי להבטיח שxenografts השיגה במדויק דומה רקמות HCC הורה.

איור 3 מדגים את המראה האופייני של xenograft HCC האנושי תת עורי ואת מראה histopathological המקביל של הגידול. ההתפתחות והצמיחה של xenografts תת עורית ניתן לפקח בקלות על ידי בחינה יומית של עכברי נמען. מרווח הזמן בין xenografting ופיתוח של גידול עשוי להשתנות במידה רבה בהתאם לסוג של רקמה (ש...

Watch this Scientific Journal Video about Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice at JoVE.com

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

xenografts גידול האנושי בעכברים עם מערכת חיסון חלשים הם כלים רבי ערך כדי ללמוד ביולוגיה בסרטן. פרוטוקולים ספציפיים כדי ליצור xenografts תת עורי וintrahepatic מתאי אנושיים hepatocellular קרצינומה או שברי גידול מתוארים. התחדשות כבד הנגרמת על ידי כריתה חלקית בעכברי נמען מוצגת כאסטרטגיה כדי להקל על קליטתם intrahepatic.

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  ...

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Medicine

35.8K Views.  University Health Network.  xenografts גידול האנושי בעכברים עם מערכת חיסון חלשים הם כלים רבי ערך כדי ללמוד ביולוגיה בסרטן. פרוטוקולים ספציפיים כדי ליצור xenografts תת עורי וintrahepatic מתאי אנושיים hepatocellular קרצינומה או שברי גידול מתוארים. התחדשות כבד הנגרמת על ידי כריתה חלקית בעכבר...

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

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

השתלה של תאים סטרומה החלב ותאי קרצינומה של עכברים C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

הדור ניסויית של קרצינומה-Associated Fibroblasts (CAFs) מ Fibroblasts החלב אנוש

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

אינדוקציה של קרצינומה של תאי מעבר שלפוחית ​​השתן פולשנית בMUC1 עכברים הטרנסגניים חיסוניים אנושיים שלמים: מודל לפיתוח חיסוני

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

יצירה אנטומית מדויק והשחזור תוך גולגולת xenografts של גידולים במוח אדם

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

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 ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

TH17 דגם דלקת של לוע תחתון פטרת בעכברים immunodeficient

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

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.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&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 ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

תת עורית אנגיוטנסין II עירוי באמצעות אוסמוטי משאבות גורם אבי העורקים מפרצת בעכברים

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

חסך בתאי עכבר מן xenografts גידול האנושי משפר משמעותי ניתוח במורד זרם של תאי יעד

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

ביטוי של ציטוקינים אקסוגניים ב- Xenografts שמקורם בחולה באמצעות הזרקה באמצעות קו תאים סטרומל ציטוקינים

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...