נתמך על ידי subproject של NIH מענק P20 RR16440 ומענק גשר בין משרד אוניברסיטת וירג&#39;יניה המערבית של מחקר וחינוך בוגר כדי ויד ס. סיוע טכני בין לופז ל-סקינר במהלך השלבים המוקדמים של פיתוח הפרויקט היא הודתה בהכרת תודה. המחברים הם גם אסיר תודה על סיוע טכני OSC19 תאים ג&#39;מאיירס ומ יונס (המחלקה לכירורגיה ראש וצוואר, MD Anderson Cancer Center, ביוסטון, טקסס), פ &#39;טרנר ק סקרסט (מערב וירג&#39;יניה אוניברסיטת המחלקה פתולוגיה בנק רקמות) לעיבוד ונהלים היסטולוגית, ר &#39;Wysolmerski (אוניברסיטת וירג&#39;יניה המערבית, המחלקה לנוירוביולוגיה אנטומיה) על הקמת LifeAct-mCherry וג&#39; בר (אוניברסיטת צפון קרוליינה) עבור וקטור pLL7.0 lentiviral. השימוש במתקן אוניברסיטת וירג&#39;יניה המערבית הדמיה מיקרוסקופית (נתמך על ידי NIH מענק P20 RR16440 ואת באב מרי רנדולף Cancer Center) מעבדה שאינו ליניארי שלה מיקרוסקופיה אופטית (NLOM, נתמך על ידי פעולה משותפת בין וירג&#39;יניה המערבית המרכז האוניברסיטאי למדעי המוח ואת וירג&#39;יניה המערבית אוניברסיטת המחלקה לפיסיקה / מערב וירג&#39;יניה הננו יוזמת) היא גם הודתה בהכרת תודה. NLOM הוא נתמך בחלקו על ידי מענק NIH P30 RR031155 למרכז Neuroscience.

<ol>
	<li>Sano, D., Myers, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+models+of+head+and+neck+cancers.">Xenograft models of head and neck cancers.</a> Head Neck Oncol. 1, 32-32 (2009).</li><li>Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+cancer+in+the+head+and+neck+region.">Animal models of cancer in the head and neck region.</a> Clin Exp Otorhinolaryngol. 2, 55-60 (2009).</li><li>Myers, J. N., Holsinger, F. C., Jasser, S. A., Bekele, B. N., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+nude+mouse+model+of+oral+tongue+squamous+cell+carcinoma.">An orthotopic nude mouse model of oral tongue squamous cell carcinoma.</a> Clin Cancer Res. 8, 293-298 (2002).</li><li>Sano, D., Myers, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+of+squamous+cell+carcinoma+of+the+oral+tongue.">Metastasis of squamous cell carcinoma of the oral tongue.</a> Cancer Metastasis Rev. 26, 645-662 (2007).</li><li>Sano, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+combination+anti-endothelial+growth+factor+receptor+and+anti-vascular+endothelial+growth+factor+receptor+2+targeted+therapy+on+lymph+node+metastasis:+a+study+in+an+orthotopic+nude+mouse+model+of+squamous+cell+carcinoma+of+the+oral+tongue.">The effect of combination anti-endothelial growth factor receptor and anti-vascular endothelial growth factor receptor 2 targeted therapy on lymph node metastasis: a study in an orthotopic nude mouse model of squamous cell carcinoma of the oral tongue.</a> Arch Otolaryngol Head Neck Surg. 135, 411-420 (2009).</li><li>Kupferman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrkB+induces+EMT+and+has+a+key+role+in+invasion+of+head+and+neck+squamous+cell+carcinoma.">TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.</a> Oncogene. 29, 2047-2059 (2010).</li><li>Ammer, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saracatinib+Impairs+Head+and+Neck+Squamous+Cell+Carcinoma+Invasion+by+Disrupting+Invadopodia+Function.">Saracatinib Impairs Head and Neck Squamous Cell Carcinoma Invasion by Disrupting Invadopodia Function.</a> J Cancer Sci Ther. 1, 52-61 (2009).</li><li>Rosenthal, E. L., Kulbersh, B. D., King, T., Chaudhuri, T. R., Zinn, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorescent+labeled+anti-epidermal+growth+factor+receptor+antibody+to+image+head+and+neck+squamous+cell+carcinoma+xenografts.">Use of fluorescent labeled anti-epidermal growth factor receptor antibody to image head and neck squamous cell carcinoma xenografts.</a> Mol Cancer Ther. 6, 1230-1238 (2007).</li><li>Gleysteen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeled+anti-EGFR+antibody+for+identification+of+regional+and+distant+metastasis+in+a+preclinical+xenograft+model.">Fluorescent labeled anti-EGFR antibody for identification of regional and distant metastasis in a preclinical xenograft model.</a> Head Neck. 30, 782-789 (2008).</li><li>Bhirde, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+killing+of+cancer+cells+in+vivo+and+in+vitro+with+EGF-directed+carbon+nanotube-based+drug+delivery.">Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery.</a> ACS Nano. 3, 307-316 (2009).</li></ol>

מודלים העכבר orthotopic הוכיחו שימושי לחקר היבטים רבים של סרטן הראש והצוואר 1,2. יש לנו שילוב מערכת orthotopic מבוססת היטב של הסקוטי 3 עם הדמיה מיקרוסקופית שני הפוטונים של תאים mCherry שכותרתו כמערכת ללמוד את האירועים הראשונים של הראש והצוואר הפלישה גידול התא. בהליך זה, יש לציין כי תאים יכול לדלוף מאתר ההזרקה הגידול, בעיקר בעכברים שישה שבועות או פחות בשל גודל הלשון מספקת. אנו משתמשים עכברים מבוגרים, כדי למנוע בעיה זו. גודל הלשון גדול עם עכברים מבוגרים גם מסייע למניעת קרע של עורק לשונית דימום יתר מן הלשון. לקחת הגידול הוא משפר באופן דרמטי כאשר הלשון מתנפחת באתר ההזרקה הראשוני. שוככת זה נפיחות בתוך 1-2 שעות כמו הנוזל המוזרק נספג. הגידול מופיע מאליו 1-2 שבועות זריקה הודעה עם מספר הנייד הצביע על מוזרק כמו בליטה לבנה קטנה על פני הלשון. כמו כן, אנו מציינים כי על המטרה 40X שימוש במחקרים שלנו שהוצגו כאן, אנחנו לא יכולים להבדיל תאים סרטניים בודדים אך מזדהים IGS כאשכולות תא פולשנית כי לשחזר את מצב טיפוסי של הפלישה HNSCC. עד כה במודל זה נעשה שימוש נרחב לבדיקת התפקיד של מולקולות ספציפיות, כמו גם מספר תרופות אנטי הגידול על הצמיחה Scot פלישה, עם יעילות נמדדת גרורות הצומת לימפה בצוואר הרחם לנטר באמצעות IHC או bioluminescent שיטות 4-7. גידולים שנוצרו לידי ביטוי במערכת זו קרוב לפני השטח לשון (איור 7), המאפשר את היישום של מיקרוסקופיה שני הפוטונים גידולים התמונה כולה באתרו כמו רב הסלולר אשכולות פולשנית. ההליך יכול גם להיות מנוצל כדי להמחיש הפלישה הגידול עם רזולוציה הסלולר. מיקרוסקופיה דו פוטון נוצל בעבר ללמוד טיפולים ניסיוניים עבור סרטן הראש והצוואר ב 8,9 ו orthotopic xenograft 8,10 מודלים. עם זאת, ישנם שני הבדלים משמעותיים בין דוחות אלה פרוטוקול שלנו. ראשית, מחקרים אלה להשתמש בתוויות תאיים למקד / לזהות תאים סרטניים הראש והצוואר, אולי רק כדי להגביל את גילוי תאים סרטניים עם גישה נאותה במחזור הדם. הפלישה השנייה של תאים סרטניים קרוב ל האתר העיקרי משחזר סביר פעילות גרורתי מוקדם לא הוערך כפרמטר ניסיונית. פרוטוקול שלנו מספק את היכולת לכמת באופן ישיר הפלישה תאים סרטניים בכל נקודה במהלך התקדמות הגידול בלשונות העכבר. בעוד השיטה המתוארת כאן מתאר את ההליך באמצעות לשונות גזור, אנחנו נמצאים כרגע בתהליך של הסתגלות בשיטה זו כדי הפלישה הגידול התמונה עכברים חיים לשימוש בשילוב עם פליטת אור בו זמנית לפקח על הפלישה מוקדם, מעורבות קשרי לימפה מקומיות ו גרורות רחוקות אותו בעל חיים. שינויים בפרוטוקול עבור in vivo הדמיה דורשים עיצוב הבמה המתאימה מיצוב ושמירה על עכברים במהלך הדמיה, כמו גם מערכת מעשית שצריך להשקות את חלל הפה של בעכברים מורדמים במהלך ההליך הדמיה. אופטימיזציה פעם, עיבודים אלה יספקו את היכולת ללמוד את התפקיד של הפרו פולשנית מולקולות פוטנציאליות נגד פולשני לבדיקת תרכובות על הפלישה המקומית ומעורבות גרורות מרוחקות יותר אצל בעלי חיים על פני תקופות זמן ממושך.

multi-photon imaging of tumor cell invasion in an orthotopic mouse model of oral squamous cell carcinoma

לוקו אזורי הפלישה של סרטן הראש והצוואר קשורה לסיכון גרורתי מציב אתגר קשה בעיצוב ויישום אסטרטגיות ניהול המטופל. מודלים העכבר orthotopic של סרטן הפה פותחו כדי להקל על מחקר של גורמים הפלישה להשפיע ולשמש מודל להערכת מערכת אנטי הגידול הרפוי. במערכות אלה, ויזואליזציה של תאים סרטניים המופץ בתוך רקמות חלל הפה יש בדרך כלל נערכו על ידי היסטולוגיה או קונבנציונלי או עם שיטות vivo bioluminescent. החיסרון העיקרי של שיטות אלה הוא חוסר היכולת לחזות במדויק את הטמון ולכמת תא בתחילת הפלישה הגידול הנובע האתר הראשי בשלושה ממדים. כאן אנו מתארים פרוטוקול המשלבת מודל הוקמה עבור קרצינומה של תאים קשקשיים של הלשון (סקוטי) עם הדמיה שני הפוטונים כדי לאפשר רב וקטורי להדמיה של התפשטות הגידול לשוני. OSC-19 ראש הגידול בצוואר קו התא תוכנן ביציבות לבטא את LifeAct F-אקטין פפטיד מחייב התמזגו לחלבון mCherry ניאון (LifeAct-mCherry). Fox1 נו / נו עכברים הוזרקו תאים אלה בצורה אמינה גידולים המאפשרים הלשון להיות דמיינו ידי יישום לשעבר vivo של מיקרוסקופיה שני הפוטונים. טכניקה זו מאפשרת ויזואליזציה orthotopic של מסת הגידול והמקומית פולשים לתאים לשונות נכרת ללא הפרעה של microenvironment הגידול האזורי. בנוסף, מערכת זו מאפשרת כימות של הפלישה תאים סרטניים על ידי חישוב מרחקים שפלשו להעביר תאים ממקום הגידול הראשוני. בסך הכל הליך זה מהווה מערכת מודל משופר לניתוח הגורמים שתורמים הפלישה Scot וטיפולים טיפולית המותאמת למנוע פלישה מקומית התפשטות גרורות רחוקות. שיטה זו גם יש את הפוטנציאל להיות משולב בסופו של דבר עם שיטות הדמיה אחרות בסביבה vivo.

Watch this Scientific Journal Video about Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma at JoVE.com

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

סקירה מקיפה של טכניקות מעורב ביצירת מודל עכברי של סרטן הפה ניטור כמותי של הפלישה הגידול בתוך הלשון באמצעות מיקרוסקופ רב פוטון של תאים שכותרתו מוצג. מערכת זו יכולה לשמש פלטפורמה שימושי להערכת יעילות התרופה מולקולרית של תרכובות אנטי פולשנית.

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 ...

multi-photon-imaging-tumor-cell-invasion-an-orthotopic-mouse-model

Research

JoVE Journal

Medicine

19.0K Views.  Mary Babb Randolph Cancer Center, West Virginia University. סקירה מקיפה של טכניקות מעורב ביצירת מודל עכברי של סרטן הפה ניטור כמותי של הפלישה הגידול בתוך הלשון באמצעות מיקרוסקופ רב פוטון של תאים שכותרתו מוצג. מערכת זו יכולה לשמש פלטפורמה שימושי להערכת יעילות התרופה מולקולרית של תרכובות אנטי פולשנית.

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

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

דגם orthotopic של סרטן שלפוחית ​​השתן Murine

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

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 ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

מידול והדמיה 3-Dimensional Cell קיבוציים Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

שלפוחית ​​השתן Orthotopic גידול דגם ואת הערכת טיפול סרנה השלפוחית

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

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 ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma עכבר דגם orthotopic שמירה על אילוצי המוח של רקמת ריאה, פיסיים ומתאים לשני פוטונים במיקרוסקופ Intravital

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

פגיעה דגם אלקלי לשרוף של הקרנית neovascularization בעכבר

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

RNAscope עבור<em&gt; באתרו</em&gt; איתור של נגיף הפפילומה אנושי תעתיק פעיל בראש וצוואר קשקש קרצינומה של תאים

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

הדמיית תהודה מגנטית משופרת ניגודיות דינמית של דגם orthotopic סרטן לבלב עכבר

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

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 ...