Diese Arbeit wurde vom National Institute of Neurological Diseases and Stroke (R01 NS048353 nach SLC), das National Cancer Institute (R01 CA122804 auf SLC, R01 CA134773 zu Kevin A. Roth und SLC und CA13148-35 zu KRZ) und dem Department of unterstützt Defense (X81XWH-09-1 bis 0086 auf SLC). Fonds zur Förderung der Betrieb der UAB Comprehensive Cancer Center Small Animal Imaging Gemeinsame Einrichtung wurden von einem NCI-Core-Unterstützung zu gewähren (; E. Partridge, PI P30 CA13148-35) zur Verfügung gestellt. Wir danken dem Alabama Neuroscience Blueprint Core Center (P30 NS57098) und der UAB Neuroscience Core Center (P30 NS47466) für ihre Unterstützung. Der Inhalt ist ausschließlich in der Verantwortung der Autoren und stellen nicht notwendigerweise die offizielle Meinung der National Institutes of Health oder das Department of Defense.

<ol>
	<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>

Die detaillierte orthotopen xenografting hier vorgestellte Methode ist eine, die wir entwickelt mit ST88-14 MPNST Zellen, eine Linie, die weit in Studien dieser NF1-assoziierten peripheren Nervenscheidentumoren verwendet wird. Allerdings ist diese Methode leicht anpassbar für präklinische Studien mit anderen MPNST Zelllinien. Zum Beispiel haben wir ebenfalls durchgeführt orthotopen xenografting mit STS-26T 10 Zellen, eine Zeile aus einem sporadisch auftretenden MPNST und T265-2c 11 Zellen, die aus einer NF1-assoziierten MPNST abgeleitet abgeleitet werden, mit Erfolg ähnlich, dass wir mit ST88 beobachtet -14 Zellen. Having said that, würden wir zur Kenntnis, dass die genauen Bedingungen beschreiben wir hier für orthotopen xenografting der ST88-14-Zellen nicht direkt für die Veredelung von anderen MPNST Linien angenommen werden. Stattdessen müssen wichtige Parameter der Methodik empirisch für jede Zelllinie bestimmt werden. Zu diesen Parametern gehören die Anzahl der MPNST Zellen zunächst veredelt, die Zeit für die Graft-Entwicklung und, in geringerem Maße erlaubt, die Lautstärke, in der die Tumorzellen injiziert. Um festzustellen, diese Parameter für eine neue Zeile, inhaltlicher Kontrolle übernehmen wir Transplantat Gruppen von Mäusen (5 Mäuse pro Gruppe) mit 3 bis 5 Oktober x 10 6 Tumorzellen, zwei Konzentrationen von Zellen für jede Größenordnung (dh 10 3 Zellen, 5 x 10 3 Zellen, 10 4 Zellen, 5 x 10 4 Zellen, etc.). Größere Zahlen von Tumorzellen (&gt; 10 6) muss in einem größeren Volumen injiziert werden, und wir haben festgestellt, dass bis zu 5 ml ohne Verlust von Zellen aus der veredelten Nerv injiziert werden kann. Wir haben auch festgestellt, dass nicht mehr als 5 x 10 6 Tumorzellen pro 5 ml Volumen injiziert werden kann, als dichter Suspensionen zunehmend anfällig zu scheren, die die Tumorzellen abtötet zu werden. Wir folgen dieser Mäuse, bis mindestens drei Tieren innerhalb jeder Gruppe tastbaren Tumoren, an welcher Stelle beenden wir diese Gruppe und prüfen die Nerven histologisch zu pfropfen Wachstum bestätigen müssen. Offensichtlich ist die erforderliche Zeit, um diesen Punkt erreichen unterscheiden sich je nach der Anzahl der Zellen injiziert, im allgemeinen, suchen wir eine Konzentration von Zellen, die maximal zulässige Tumorwachstum innerhalb von 30-60 Tagen zu erreichen. Mehr schnelle Wachstum machen es schwierig, eine effektive therapeutische Konzentrationen vor erreichen der Beendigung des Experiments und / oder verhindern, dass die Sammlung von ausreichenden Biolumineszenz-Bildgebung Datenpunkte im Laufe der Experimente. Eine zeitliche Verlauf mehr als 60 Tage macht das Einstellen experimentellen Parameter unhandlich und unnötig verlängert die Dauer der geplanten Experimente. Schließlich möchten wir auch zur Kenntnis, dass wir diesen orthotopen xenografting Methodik mit ST88-14-Zellen in NIH III Mäuse transplantiert, um die therapeutische Wirksamkeit von Tamoxifen 6 demonstrieren verwendet. Eine detaillierte Beschreibung der Methoden, die wir routinemäßig verwenden, um die Auswirkungen der Kandidat Therapeutika auf orthotopen Xenotransplantate beurteilen kann in diesem Manuskript zu finden. Es wurde auch gezeigt, dass Neurofibrom Zellen erfolgreich in peripheren Nerven 12 gepfropft werden, was darauf hindeutet, dass mit Änderungen, die in diesem Protokoll beschriebenen verwendet werden, um präklinischen Studien mit Kandidaten Therapeutika gegen Neurofibrome Regie durchzuführen.

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<td>Foxchase outbred SCID mice</td>
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<td>Charles River </td>
<td>#236</td>
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<td>NIH III mice</td>
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<td>Taconic </td>
<td>#NIHBNX</td>
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<td>NOD-SCID&gamma; mice</td>
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<td>Jackson Laboratories </td>
<td>#005557</td>
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<td>Cell Stripper</td>
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<td>Fisher Scientific</td>
<td>25-056-cl</td>
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<td>Vetbond surgical glue</td>
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<td>Hamilton syringe</td>
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<td>Hamilton syringe needles</td>
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<td>Hamilton Company</td>
<td>#7803-05</td>
<td>33 Ga., 0.5 inch, PT4 (custom made)</td>
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<td>Ear tags</td>
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<td>National Band and Ear Tag Co.</td>
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<td>Ophthalmic ointment</td>
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orthotopic xenografting of human luciferase-tagged malignant peripheral nerve sheath tumor cells for in vivo testing of candidate therapeutic agents

Obwohl in vitro-Bildschirme sind für die erste Identifizierung von Kandidaten-Therapeutika, eine strenge Bewertung der Droge die Fähigkeit zur Hemmung des Tumorwachstums in einem geeigneten Tiermodell durchgeführt werden muss unerlässlich. Die Art des Tieres, das am besten für diesen Zweck ist ein Thema intensiv diskutiert. Einige Hinweise darauf, dass präklinischen Studien untersucht Wirkung von Medikamenten auf Tumoren, die in transgenen Mäusen mehr Vorhersagekraft der klinischen Ergebnisse 1 sind und so Kandidaten Therapeutika sind oft in dieser Modelle getestet. Leider sind transgene Modelle nicht verfügbar für viele Tumorarten. Weitere transgene Modelle haben oft andere Einschränkungen wie Bedenken, wie gut die Maus-Tumor-Modellen seiner menschlichen Gegenstück, unvollständiger Penetranz des Tumor-Phänotyp und eine Unfähigkeit, vorauszusagen, wann Tumoren entwickeln. Daher verwenden viele Forscher Xenotransplantatmodellen (humane Tumorzellen in immundefizienten Mäusen gepfropft) für die präklinische Studien gegebenenfalls transgenen Tumormodellen nicht verfügbar sind. Auch wenn transgene Modelle zur Verfügung stehen, sind sie oft mit Xenotransplantatmodellen Partnerschaft wie diese erleichtern die schnelle Bestimmung der therapeutischen Bereiche. Darüber hinaus ermöglicht diese Partnerschaft ein Vergleich der Wirksamkeit des Mittels in transgenen Tumoren und echte menschliche Tumorzellen. Historisch gesehen hat xenografting oft durch die Injektion von Tumorzellen subkutan (ektopische Xenotransplantate) durchgeführt. Diese Technik ist eine schnelle und reproduzierbare, relativ kostengünstig und ermöglicht eine kontinuierliche Quantifizierung des Tumorwachstums während der therapeutischen Periode 2. Allerdings ist die subkutane Raum nicht den normalen Mikroumgebung für die meisten Tumoren und so Ergebnisse mit ektopischen xenografting erhalten können aufgrund von Faktoren wie das Fehlen von Organ-spezifische Expression von Wirtsgewebe und Tumor-Gene in die Irre führen. Es wurde deshalb dringend empfohlen, dass ektopische Pfropfen Studien ersetzt oder durch Studien, in denen menschliche Tumorzellen in ihrem Gewebe Ursprungsbezeichnung (orthotopen xenografting) 2 aufgepfropft sind, ergänzt werden. Leider ist die Umsetzung dieser Empfehlung häufig durch die Tatsache, dass orthotopen xenografting Methoden noch nicht für viele Tumorarten entwickelt vereitelt. Bösartige peripheren Nervenscheidentumoren (MPNSTs) sind hoch aggressive Sarkome, die sporadisch oder in Verbindung treten mit Neurofibromatose Typ 1 3 und am häufigsten in den Ischiasnerv 4 entstehen. Hier beschreiben wir eine technisch einfache Methode, bei der Glühwürmchen-Luciferase-markierten menschlichen MPNST Zellen orthopically in den Ischiasnerv von immundefizienten Mäusen sind xenotransplantiert. Unser Ansatz zur Bewertung des Erfolgs der Transplantation Verfahren in einzelnen Tieren und nachfolgenden nicht voreingenommen Randomisierung in Lerngruppen wird ebenfalls diskutiert.

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

Ein Verfahren zur sicheren Pfropfen Luciferase-markierten menschlichen malignen peripheren Nervenscheide Tumorzellen in den Ischiasnerv von immundefizienten Mäusen beschrieben. Der Einsatz von Biolumineszenz-Bildgebung, um die ordnungsgemäße Errichtung von Tumor Transplantate und Kriterien für die zufällige Trennung der Tiere in Arbeitsgruppen zeigen, werden ebenfalls diskutiert.

Orthotopen Xenografting of Human Luciferase-Tagged malignen peripheren Nervenscheide Tumorzellen für In vivo Prüfung von Kandidaten Therapeutika

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

orthotopic-xenografting-human-luciferase-tagged-malignant-peripheral

Research

JoVE Journal

Medicine

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

18.5K Aufrufe.  University of Alabama at Birmingham - UAB. Ein Verfahren zur sicheren Pfropfen Luciferase-markierten menschlichen malignen peripheren Nervenscheide Tumorzellen in den Ischiasnerv von immundefizienten Mäusen beschrieben. Der Einsatz von Biolumineszenz-Bildgebung, um die ordnungsgemäße Errichtung von Tumor Transplantate und Kriterien für die zufällige Trennung der Tiere in Arbeitsgruppen zeigen, werden ebenfalls diskutiert.

Serie: 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-Photonen-Imaging der Tumorzellinvasion in einem orthotopen Mausmodell des oralen Plattenepithelkarzinoms

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

Forschung

Medizin

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.

Abgabe therapeutischer Wirkstoffe durch intracerebroventrikuläre (ICV) und intravenöse (IV) Injektion in Mäuse

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.

MAME Modelle für die 4D-Bildgebung lebender Zellen von Tumor: Mikroumgebung Wechselwirkungen, die Einfluss auf maligne Progression

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.

In-vivo-19 F-MRT für die Zellverfolgung

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.

Ein orthotopen Mausmodell der menschlichen Prostatakrebs-Metastasen

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.

Studieren Bauchspeicheldrüsenkrebs Stammzelleigenschaften für die Entwicklung neuer Behandlungsstrategien

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

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE Gene Silencing: Screening eines Heptamer-Typ Kleiner Leitfaden RNA-Bibliothek für potentielle Krebs Therapeutika

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

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.

Die Erzeugung von Microtumors Mit 3D Human Biogel Kultursystem und Patienten stammGlioblastomZellen für Kinomic Profilieren 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 ...

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.

Depletion von Maus-Zellen aus menschlichem Tumorxenografte deutlich verbessert Downstream-Analyse von Zielzellen

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.

Einweg-Dosierer für Lungen Insufflation von therapeutischen Wirkstoffen zu Kleintieren

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