עבודה זו נתמכה על ידי המכון הלאומי למחלות נוירולוגיות ושבץ (R01 NS048353 כדי SLC), המכון הלאומי לסרטן (R01 CA122804 כדי SLC, R01 CA134773 לקווין א רוט SLC ו CA13148-35 ל KRZ) ואת המחלקה ההגנה (X81XWH-09-1-0086 ל SLC). קרנות התומכות פעולה של מקיף במרכז לחקר הסרטן UAB במתקן הדמיה קטנים בעלי חיים משותפים סופקו על ידי Core NCI להעניק תמיכה (P30 CA13148-35; א פרטרידג&#39;, PI). אנו מודים Neuroscience אלבמה Blueprint Core Center (P30 NS57098) ואת Neuroscience UAB Core Center (P30 NS47466) על עזרתם. התוכן הוא באחריות הבלעדית של הכותבים ולא בהכרח מייצגים את הדעות הרשמיות של מכון הבריאות הלאומי או משרד ההגנה.

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	<li>Rosenberg, M. P., Bortner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+transgenic+and+knockout+animal+models+should+be+used+(for+drug+efficacy+studies+in+cancer).">Why transgenic and knockout animal models should be used (for drug efficacy studies in cancer).</a> Cancer Met. Rev. 17, 295-299 (1999).</li><li>Killion, J. J., Radinsky, R., 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=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Met. Rev. 17, 279-284 (1999).</li><li>Carroll, S. L., Ratner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+the+Schwann+cell+lineage+form+tumors+in+NF1?.">How does the Schwann cell lineage form tumors in NF1?.</a> Glia. 56, 1590-1605 (2008).</li><li>Woodruff, J. M., Kourea, H. P., Louis, D. N., Scheithauer, B. W., Kleihues, P., Cavenee, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology and Genetics of Tumours of the Nervous System. , 172-174 (2000).</li><li>DeClue, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+expression+in+neurofibromatosis+type+1-related+tumors+and+NF1+animal+models.">Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.</a> J. Clin. Invest. 105, 1233-1241 (2000).</li><li>Byer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+inhibits+malignant+peripheral+nerve+sheath+tumor+growth+via+an+estrogen+receptor-independent+mechanism.">Tamoxifen inhibits malignant peripheral nerve sheath tumor growth via an estrogen receptor-independent mechanism.</a> Neuro-Oncology. , (2010).</li><li>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=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Wu, J. C., Sundaresan, G., Iyer, M., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+imaging+of+firefly+luciferase+reporter+gene+expression+in+skeletal+muscles+of+living+mice.">Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.</a> Mol. Therapy. 4, 297-306 (2001).</li><li>Luker, G. D., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+real-time+bioluminescence+imaging+for+the+study+of+viral+pathogenesis.">Luciferase real-time bioluminescence imaging for the study of viral pathogenesis.</a> Methods Mol. Biol. 292, 285-296 (2005).</li><li>Dahlberg, W. K., Little, J. B., Fletcher, J. A., Suit, H. D., Okunieff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitivity+in+vitro+of+human+soft+tissue+sarcoma+cell+lines+and+skin+fibroblasts+derived+from+the+same+patients.">Radiosensitivity in vitro of human soft tissue sarcoma cell lines and skin fibroblasts derived from the same patients.</a> Int. J. Radiat. Biol. 63, 191-198 (1993).</li><li>Badache, A., DeVries, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrosarcoma-derived+Schwann+cells+overexpress+platelet-derived+growth+factor+(PDGF)+receptors+and+are+induced+to+proliferate+by+PDGF.">Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF.</a> BB. J. Cell. Physiol. 177, 334-342 (1998).</li><li>Muir, D., Neubauer, D., Lim, I. T., Yachnis, A. T., Wallace, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenic+properties+of+neurofibromin-deficient+neurofibroma+Schwann+cells.">Tumorigenic properties of neurofibromin-deficient neurofibroma Schwann cells.</a> Am. J. Pathol. 158, 501-513 (2001).</li></ol>

אין ניגודי אינטרסים הכריז.

השיטה מפורט xenografting orthotopic המוצג כאן הוא אחד שפיתחנו באמצעות ST88-14 תאים MPNST, קו שנמצא בשימוש נרחב במחקרים אלה NF1 הקשורים גידולים מעטפת העצב ההיקפיים. עם זאת, מתודולוגיה זו ניתנת להתאמה בקלות מחקרים פרה עם שורות תאים אחרים MPNST. למשל, יש לנו גם ביצע orthotopic xenografting עם STS-26T 10 תאים, קו נגזר MPNST לסירוגין המתרחשים T265 ו-2c 11 תאים, אשר נגזרות MPNST NF1 הקשורים, עם הצלחה דומה לזו שראינו עם ST88 -14 תאים. אחרי שאמרתי את זה, היינו מציינים כי התנאים המדויקים אנו מתארים כאן orthotopic xenografting של ST88-14 תאים לא ניתן לאמץ ישירות עבור השתלת קווי MPNST אחרים. במקום זאת, הפרמטרים העיקריים של המתודולוגיה צריכה להיקבע באופן אמפירי לכל שורה התא. פרמטרים אלה כוללים את מספר התאים MPNST מורכבים בתחילה, את הזמן המותר עבור פיתוח שוחד, ובמידה פחותה, נפח שבו תאים את הגידול מוזרקים. כדי לבסס את הפרמטרים הללו עבור קו חדש, אנחנו קבוצות שתל של עכברים (5 עכברים בכל קבוצה) עם 03-05 אוקטובר x 10 6 תאים סרטניים, בדיקת שני ריכוזי של תאים עבור כל הזמנה של גודל (כלומר, 10 3 תאים, 5 x 10 3 תאים, 4 תאים 10, 5 x 10 4 תאים וכו &#39;). מספרים גדולים של תאים סרטניים (&gt; 10 6) חייב להיות מוזרק בנפח גדול יותר, מצאנו כי עד 5 מ&quot;ל ניתן להזריק ללא אובדן תאים העצב מורכבים. מצאנו גם כי לא יותר מ 5 x 10 6 תאים סרטניים לכל נפח 5 מ&quot;ל ניתן להזריק כמו המתלים נוטה להיות צפוף יותר ויותר גזירה אשר הורג את התאים הסרטניים. אנו בצע את העכברים עד לפחות שלוש חיות בתוך כל קבוצה יש גידולים מוחשית, ובנקודה זו אנו לסיים כי הקבוצה ולבחון את העצב בהיסטולוגיה לאשר צמיחה השתל. כמובן, הזמן הנדרש כדי להגיע לנקודה זו יהיה שונה, תלוי במספר התאים מוזרקים, באופן כללי, אנו מחפשים ריכוז של תאים יגיע הגידול המרבי המותר צמיחה בתוך 30-60 ימים. צמיחה מהירה יותר יכול לעשות את זה קשה להשיג ריכוזי טיפולית יעילה לפני תום הניסוי ו / או למנוע את איסוף datapoints הדמיה מספקת פליטת אור במהלך הניסויים. כמובן זמן רב יותר מאשר 60 ימים עושה התאמת הפרמטרים ניסיוני מסורבל ושלא לצורך מאריכה את משך הניסוי המתוכנן. לבסוף, היינו גם לציין כי השתמשנו במתודולוגיה זו xenografting orthotopic עם ST88-14 תאים מורכבים לעכברים NIH השלישי כדי להדגים את היעילות הטיפולית של טמוקסיפן 6. תיאור מפורט של שיטות אנו משתמשים באופן שגרתי כדי להעריך את ההשפעות של סוכני מועמד טיפולית על xenografts orthotopic ניתן למצוא את כתב היד הזה. כמו כן הוכח כי תאי neurofibroma יכול להיות מורכבים בהצלחה העצבים ההיקפית 12, דבר המצביע על כך, עם שינויים, הנהלים המפורטים בפרוטוקול זה יכול לשמש כדי לבצע ניסויים פרה עם סוכנים מועמד טיפולית מכוונת נגד neurofibromas.

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orthotopic xenografting of human luciferase-tagged malignant peripheral nerve sheath tumor cells for in vivo testing of candidate therapeutic agents

אמנם מסכי במבחנה חיוניים לזיהוי ראשוני של סוכני מועמד טיפולית, הערכה קפדנית של היכולת של התרופה לעכב את צמיחת הגידול חייב להתבצע במודל חיה מתאים. סוג של מודל החיה, כי הוא הטוב ביותר למטרה זו היא נושא לדיון אינטנסיבי. הראיות עולה כי חלק בניסויים פרה בחינת השפעות התרופה על גידולים שמקורם העכברים הטרנסגניים הם חזוי יותר של התוצאה הקלינית 1 ולכן סוכני מועמד טיפולית נבחנים לעתים קרובות במודלים אלו. למרבה הצער, מודלים מהונדס אינם זמינים עבור סוגי גידולים רבים. בנוסף, מודלים מהונדס לעתים קרובות יש מגבלות אחרות, כגון חששות באשר לאופן היטב את הגידול מודלים העכבר מקבילו האנושי שלה, penetrance חלקית של הפנוטיפ הגידול וחוסר יכולת לחזות מתי יהיה לפתח גידולים. כתוצאה מכך, חוקרים רבים משתמשים במודלים xenograft (תאים סרטניים אנושיים מורכבים לעכברים immunodeficient) עבור ניסויים פרה אם מתאים מודלים גידול מהונדס אינם זמינים. גם אם מודלים מהונדס זמינים, הם לעתים קרובות שותפות עם מודלים xenograft כפי שהאחרון להקל נחישות מהירה של טווחי טיפולית. יתר על כן, שותפות זו מאפשרת השוואה של היעילות של הסוכן בגידולים מהונדס ואמיתי תאים סרטניים אנושיים. מבחינה היסטורית, xenografting פעמים רבות שבוצעו על ידי הזרקת תאים סרטניים תת עורי (xenografts חוץ רחמי). טכניקה זו היא המהירה לשחזור, זול יחסית ומאפשר quantitation רציפה של הגידול בתקופה הטיפולי 2. עם זאת, את המרחב התת עורית לא microenvironment נורמלי ביותר שאתות ולכן התוצאות שהושגו עם xenografting חוץ רחמי יכול להיות מטעה בשל גורמים כגון היעדרות של הביטוי איבר ספציפי של רקמת המארח גנים סרטניים. יש ובכך היה מומלץ מאוד מחקרים השתלת חוץ רחמי יוחלף או כהשלמה מחקרים בהם תאים סרטניים אנושיים הם מורכבים לרקמת מוצאם (xenografting orthotopic) 2. לרוע המזל, יישום המלצה זו סוכלה לעתים קרובות על ידי העובדה מתודולוגיות xenografting orthotopic טרם פותחו עבור סוגי גידולים רבים. ממאירה היקפי נדן עצב גידולים (MPNSTs) הם סרקומות אגרסיבי המתרחשות לסירוגין או בשיתוף עם נוירופיברומטוזיס סוג 1 ו - 3 בדרך כלל להתעורר עצב השת 4. כאן אנו מתארים שיטה פשוטה מבחינה טכנית שבה גחלילית בלוציפראז-tagged תאים MPNST האדם xenografted orthopically לתוך עצב השת של עכברים immunodeficient. הגישה שלנו כדי להעריך את ההצלחה של ההליך השתלת בחיות הפרט אקראיות בלתי משוחד הבאים לקבוצות המחקר נדון גם.

Watch this Scientific Journal Video about Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents at JoVE.c

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

שיטה אמינה עבור השתלת בלוציפראז-מתויג האדם ממאיר עצב היקפי נדן תאים סרטניים לתוך עצב השת של עכברים immunodeficient מתואר. השימוש הדמיה פליטת אור להפגין הקמת התקין של שתלי וקריטריונים גידול של הפרדה אקראית של בעלי חיים לקבוצות המחקר נדונות גם.

Orthotopic Xenografting של האדם בלוציפראז-Tagged ממאירה היקפיים נדן בתאי העצב גידול של In vivo בדיקה של סוכני המועמד טיפולית

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

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Medicine

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

18.5K Views.  University of Alabama at Birmingham - UAB. שיטה אמינה עבור השתלת בלוציפראז-מתויג האדם ממאיר עצב היקפי נדן תאים סרטניים לתוך עצב השת של עכברים immunodeficient מתואר. השימוש הדמיה פליטת אור להפגין הקמת התקין של שתלי וקריטריונים גידול של הפרדה אקראית של בעלי חיים לקבוצות המחקר נדונות גם.

Video: Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

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 ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

מסירה של סוכני טיפולית באמצעות Intracerebroventricular (ICV) ו תוך ורידי (IV) הזרקה של עכברים

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

מיים מודלים של הדמיה חיה של תאים 4D גידול: אינטראקציות microenvironment המשפיעים ממאירה התקדמות

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

בF-MRI 19 vivo למעקב סלולרי

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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  ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

לומד סרטן לבלב גזע מאפייני תא לפיתוח טיפול חדש אסטרטגיות

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

דור של Microtumors שימוש 3D אדם Biogel תרבות מערכת תאי Glioblastoma נגזרות חולות להרכבת פרופיל Kinomic ובדיקת תגובה לתרופה

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

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 ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

Dosators פנויה עבור ריאתי הֲפָחָה ממוקד של תרופות בעלי חיים קטנים

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

במבחנה and In Vivo זיהוי של Mitophagy ב תאים אנושיים, C. Elegansועכברים

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...