Supported by National Institutes of Health (NHLBI) R01 HL056888 (LFG), National Cancer Institute (NCI) RO1 CA134573NIH (LFG), P30 GM103488 (LFG), WV CTR-IDEA NIH 1U54 GM104942, the Alexander B. Osborn Hematopoietic Malignancy and Transplantation Program, and the WV Research Trust Fund. We are grateful for the support of Dr. Kathy Brundage and the West Virginia University Flow Cytometry Core Facility, supported by NIH S10-OD016165 and the Institutional Development Award (IDeA) from the NIH Institute of General Medical Sciences of the National Institutes of Health (CoBRE P30GM103488 and INBRE P20GM103434).

1. Advanced Vorbereitung <ol><li> Vorbereiten Dextran G10 Partikel. <ol><li> Bereiten G10 Aufschlämmung durch Zugabe von 50 ml 1x PBS zu 10 g G10 Teilchen. Mischen durch Umdrehen und lassen G10 aus phosphatgepufferter Salzlösung (PBS) bei 4 ° CO / N zu regeln. </li><li> Der Tag des G10-Säule Trennung absaugen PBS aus der ständigen G10-Partikel und 50 ml frischem PBS hinzufügen. Mischen durch Umdrehen. Zweimal wiederholen, Zugabe von 50 ml frischem PBS ständiger G10 Teilchen und lagern bei 4 ° C bis zur Verwendung. </li></ol></li><li> Kultivieren BMSC und OB. <ol><li> Halten beide BMSC oder OB bei 37 ° C in 6% CO 2 und 10 cm-Gewebekulturplatten gezüchtet, bis 90% Konfluenz erreicht wird. </li><li> Trypsinize BMSC oder OB-Zellen und spalten 1: 2 auf neue 10 cm-Platten. Die Zellen werden auf diese Normen herangezogen, bis für leukämische Co-Kultivierung benötigt wird. </li></ol></li></ol> 2. Aufbau und Pflege einer Co-Kultur <ol><li> In 5-20 x 10 6 Leukämiezellen in 10 ml tumorspezifischen Kulturmedien auf einem 80% -90% konfluent BMSC oder OB Platte. HINWEIS: Unser Labor hält Co-Kulturen bei 37 ° C in 5% O 2, um besser die Knochenmark-Mikroumgebung rekapitulieren, die von 1% bis 7% auf einen Bereich 16-18 gezeigt ist. Doch bei diesem Sauerstoffspannung Kokulturen Aufrechterhaltung ist nicht kritisch für die Festlegung der drei Leukämie Subpopulationen und liegt im Ermessen des Labors. </li><li> Jeden 4. Tag alle, aber 1 ml Medium (einschließlich leukämischen Zellen in Suspension) entfernen und mit 9 ml frisch Leukämiekulturmedien ersetzen. Beim Entfernen von 9 ml Medium von der Platte, sein nicht vorsichtig die BMSC oder OB haftende Schicht zu stören. <ol><li> Entfernen Sie das Medium durch Platte zur Seite und absaugen Medien in der Ecke der Platte zu kippen. Außerdem, wenn frisches Medium hinzufügen, achten Sie darauf, tropfenweise in die Ecke der Platte gegen die Seitenwand hinzuzufügen minimaler Unterbrechung des BMSC oder OB haftende Schicht zu gewährleisten. </li></ol></li><li> Nach dem 12. Tag der Co-Kultur, spülen Leukämiezellen von BMSC oder OB Schicht durch Pipettieren von Kulturmedien von Schale nach oben und unten leicht über der Schale etwa 5 bis 10 mal und dann in 15 ml konischen Röhrchen sammeln. Reseed auf neu 80% -90% konfluent BMSC oder OB Platte als 2,1 in Schritt beschrieben. HINWEIS: Die schonende Spülung der Co-Kultur wie in Schritt 2.3 wird S und PB leukämischen Zellen ohne Störung des BMSC oder OB-Monoschicht zu entfernen. Dies ermöglicht nur Tumorzellen auf die nächste Co-Kulturplatte überführt werden. Diese 12-Tage-Zyklus kann so oft wie erforderlich auf Bedürfnisse der Nutzer wiederholt werden. </li></ol> 3. Vorbereitung G10 Bead Columns HINWEIS: Wenn sterile Downstream-Analyse oder die Kultivierung folgenden G10-Säule Trennung erforderlich ist, die folgenden Schritte steriler Technik zunutze machen sollten, und G10 Spalten sollte in einem sterilen biologischen Haube sein Setup durchgeführt. <ol><li> Pre-warm Zellkulturmedien auf 37 ° C in einem Wasserbad (~ 30 ml pro Säule). Mit einem 10-ml-Einwegspritze, zu entfernen und Kolben verwerfen. In Glaswolle Spritze. </li><li> Mit einer Pinzette auseinanderziehen Glaswolle in dünne lose Stränge. In mehreren Schichten aus leicht verpackt Glaswolle an der Spritze, bis 2/3 der Spritze mit Glaswolle gefüllt ist. HINWEIS: Die Glaswolle ist entscheidend Verunreinigungen der Leukämiezell Sammlung lose G10 Partikel zu verhindern. Stellen Sie sicher, Glaswolle gepackt genug, um die G-10-Partikel zu unterstützen, aber nicht zu dicht gepackten Medienstrom durch die Säule zu blockieren. </li><li> Bringen 1-Wege-Hahn auf die Spitze der Spritze in die geschlossene Position. </li><li> Clamp Spritze Spalte Ring stehen hoch genug, so eine 50 ml konischen Röhrchen (Sammelrohr) kann unter Absperrhahn platziert werden. Zeigen Sammelröhrchen unter Spritze Spalte. </li><li> Mit einem 10 ml-Pipette hinzufügen, tropfenweise, Partikel G10 in PBS auf die Säule oben aufdie Glaswolle. Fügen Sie weitere G10 Partikel bis zu einem ~ 2 ml Pellet aus G10-Teilchen bildet sich auf der Oberseite des Glaswolle (wie von Abstufungen auf die Spritze gemessen). </li><li> Gleichgewicht kommen die G-10-Säule, die mit vorgewärmten Medien. <ol><li> 2 ml vorgewärmten Medien Spalte. Offene Absperrventil langsam, so dass Medien fließt aus der Spalte tropft. </li><li> Wiederholen Sie Schritt 3.6.1 bis insgesamt 10 ml vorgewärmtes Medium wurden über die Säule lief. HINWEIS: Wenn G10 Partikel in der Strömung durch die Sammelrohr gesehen werden, entweder 1) mehr G10-Partikel in den halten ~ 2 ml Pellet dafür, dass keine zusätzlichen G10 Partikel aus der Kolonne entweichen oder 2) ersetzen Säule mit einem unbenutzten ein und wiederholen Schritte 3.4-3.6.1. </li><li> Nach den vorgewärmten Medien Abflüsse aus der Säule, den Wasserhahn schließen und Sammelrohr mit Fluss durch verwerfen. Hinzufügen neuer Sammelröhrchen unter Spalte. Spalte ist bereit, mit Medien + Zellgemisch geladen werden. HINWEIS: Spaltensofort und nicht erlaubt sollten zum Trocknen verwendet werden. </li></ol></li></ol> 4. Die Trennung 3 Subpopulationen in Co-Kultur <ol><li> Sammlung von Suspension (S) Tumor Subpopulation. <ol><li> Absaugen Medien von Co-Kulturplatte mit Pipette und sanft wieder gelten die gleichen Medien, um die Platte zu spülen und Medien, die Leukämiezellen in einem 15 ml konischen Röhrchen sammeln. Die Leukämiezellen gesammelt sind die S-Subpopulation. </li></ol></li><li> Sammlung von Phase Bright (PB) Tumor Subpopulation. <ol><li> 10 ml frisches Medium wieder auf Co-Kulturplatte. Spülen Sie kräftig durch hinzugefügte Medien Auf- und Abpipettieren etwa 5 mal haftLeukämieZellen zu entfernen, aber nicht hart genug haft BMSC / OB Komponente zu entfernen. </li><li> Saugen Sie mit Pipette und sammeln Medien in einem 15 ml konischen Röhrchen. Die gesammelten Zellen sind die PB Subpopulation. </li></ol></li><li> Sammlung von Phase-Dim (PD) Tumor Subpopulation. <ol><li> Rinse-Platte mit 1 ml PBS zu remove Stoffreste. Trypsinize Co-Kulturplatte mit 3 ml Trypsin und Ort in 37 ° C Inkubator für 5 min. </li><li> Entfernen Platte von Inkubator und sanft tippen Seiten der Platte haft BMSC / OB zu entfernen. </li><li> 1 ml fötales Rinderserum (FBS) und Pipette auf und ab 3-5 mal abgesehen große Zellaggregate zu brechen. </li><li> Sammeln Medien mit Zellen in einem 15 ml konischen Röhrchen. Diese Zellen sind das ungereinigte PD Subpopulation mit BMSC / OB als auch. </li></ol></li><li> Zentrifuge 3 Subpopulationen bei 400 g für 7 min isoliert. Saugen und Überstand verwerfen dann individuell resuspendieren Pellets in 1 ml vorgewärmten Medien. Zellen sind bereit, auf einer G10-Säule geladen werden. </li></ol> 5. Laden Co-Kulturzellen auf G10-Säule HINWEIS: Stellen Sie sicher, dass Hahn wird vor der Zugabe von Medien, die Zellen zu G10-Säule vollständig geschlossen. Außerdem muss jede Unterpopulation über einen separaten G10-Säule lief, so nicht zu introducE keine Vorspannung zwischen Populationen in Downstream-Analyse. <ol><li> Mit einem 1000 ul Pipette, 1 ml von jeder Zell-Subpopulation in vorgewärmten Medien zu einem separaten G10-Säule tropft. Stellen Sie sicher, dass die Medien die Zellen bleibt an der Spitze oder innerhalb G10 Pellet enthält. Erlauben Zellen für 20 min bei RT auf G10 Pellet inkubiert. HINWEIS: Absperrhahn geschlossen bleibt für die Dauer der Inkubation. </li></ol> 6. Sammeln Leukämiezellen von G10-Säule <ol><li> In 1-3 ml vorgewärmt Medien zu den G-10-Säule. Offene Absperrventil und erlauben Medien die Spalte tropfenweise langsam zu beenden. HINWEIS: Es ist wichtig, eine langsame Fließgeschwindigkeit aus der Säule oder der G10-Pellet, das BMSC / OB zu pflegen können aus der Säule zu waschen und die isolierten Leukämiezellen kontaminieren. </li><li> Weiter hinzuzufügen vorgewärmten Medien in kleinen Schritten (1-2 ml) auf G10-Säule, bis insgesamt 15 bis 20 ml hat durch die Säule laufen und gesammelt wurde. Absperrhahn schließen vaSammelröhrchen lve und Kappe. HINWEIS: Wenn ein G10 Partikel-Pellet am Boden des Sammelrohrs gesehen wird, sanft entfernen Medien aus dem Rohr verlassen G10 Partikel-Pellet ungestört und Übertragung auf neue Röhre. </li><li> Zentrifuge gesammelt Medien bei 400 × g für 7 min bei RT. Überstand entfernen und resuspendieren Zellpellet in Puffer geeignet für Folgeanwendungen. </li><li> Die Zellen sind nun eine reine Population von Leukämiezellen frei von BMSC oder OB Kontamination und sind bereit, bei der Benutzer Ermessen zu Downstream-Anwendungen angewendet werden. HINWEIS: Leukämiezelllebensfähigkeit sollten unverändert bleiben, wenn die Zellen durch G10 Säulen vorbei. </li></ol>

<ol>
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The authors have no competing financial interests.

Minimaler Resterkrankung (MRD), das mit Rückfall der Krankheit beiträgt wie vor eine große klinische Herausforderung bei der Behandlung von aggressiven refraktärer ALL, sowie eine Vielzahl von anderen hämatologischen Malignitäten zu sein. Das Knochenmark-Mikroumgebung ist die häufigste Lokalisation eines Rückfalls in ALLEN 3,8. Als solche Modelle, die das Knochenmark-Mikroumgebung sind wichtige Werkzeuge zu testen Hypothesen während der Chemotherapie Exposition gegenüber leukämischen Überleben von...

The overall goal of the method described is to provide an efficient, cost-effective in vitro approach that supports investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure. It is well documented that surviving residual tumor cells that persist after treatment contribute to relapse of disease that is often more aggressive than that at diagnosis and is often less effectively treated1-8. Models that include leukemic cells in isolation, such as those limited to culture of cells in media alone, for testing of therapeutic approaches do not factor in these critical signals, or the heterogeneity of disease that occurs in response to availability of niche derived cues in which tumor cell subpopulations with very specific interactions with niche cells derive enhanced protection. Standard 2D co-culture models that co-culture bone marrow derived stromal cells and leukemic cells have somewhat addressed the contribution of the marrow niche and have shown that interaction with bone marrow microenvironment cells increases their resistance to chemotherapy and alters their growth characteristics9-14. These models however often fail to recapitulate long term survival of tumor cells and do not accurately inform the outcomes associated with the most resistant leukemic cell populations that contribute to MRD. In vivo models remain critical and define the "gold standard" for investigation of innovative therapies prior to clinical trials but they are often challenged by the time and cost required to test hypotheses related to resistant tumors and relapse of disease. As such, development of more informative 2D models would be of benefit for pilot investigations to better inform the design of subsequent murine based pre-clinical design. 
The 2D in vitro model presented in this report lacks the complexity of the true in vivo microenvironment, but provides a cost effective and reproducible means to interrogate tumor interactions with the microenvironment that lends itself specifically to enrichment of the chemoresistant subpopulation of tumor cells. This distinction is valuable as evaluation of the entire population of tumor cells may mask the phenotype of a minor group of therapy resistant tumor cells that comprise the most important target. An additional advantage is the scalability of the model to fit the analysis of interest. Bulk cultures can be established for those analyses requiring significant recovery of tumor cells, while small scale co-cultures in multi-well plates can be utilized for PCR based analysis or microscopy based evaluations. 
Based on this need we developed an in vitro model to address the heterogeneity of disease that is characteristic of B-lineage acute lymphoblastic leukemia (ALL). We demonstrate that ALL cells, which share many characteristics in common with their healthy counterparts, localize to distinct compartments of BMSC or OB co-culture. Three populations of tumor cells are generated that have distinct phenotypes that are valuable for investigation of therapeutic response. Specifically, we demonstrate that (ALL) cells recovered from the "phase dim" (PD) population of co-culture are consistently refractory to therapy with survival that approximates tumor cells that have not been exposed to cytotoxic agents. These ALL cells, from either established cell lines or primary patient samples, migrate beneath adherent stromal cells or osteoblast layers but can be captured following trypsinization of cultures and separation of cell types by utilization of gel type 10 cross-linked dextran (G10) particle columns15. 
Here we present a setup of a 2D co-culture that can be employed to model interactions between bone marrow microenvironment stromal cells (BMSC/OB) and leukemic cells. Of particular importance is the observation that leukemic cells form three spatial subpopulations relative to the stromal cell monolayer and that the PD population represents a chemotherapy resistant tumor population due to its interaction with the BMSC or OB. Furthermore, we demonstrate how to effectively isolate the leukemic cell populations by G10 columns. Of note, we have found that isolation of these subpopulations allows for downstream analysis of the most resistant PD population to determine potential modes of resistance that are conferred to these cells due to their interaction with the bone marrow microenvironment stromal cells or osteoblasts. Techniques that we have utilized downstream of this co-culture and isolation model include flow cytometric evaluation, proteomic analysis and targeted protein expression evaluation as well as more recently developed laser ablation electrospray ionization (LAESI) and Seahorse analysis to evaluate metabolic profiles. Through use of this model in combination with the techniques above we have found that the PD population of leukemic cells has a chemotherapy resistant phenotype that is unique when compared to leukemic cells cultured in media alone or those recovered from the other subpopulations in the same co-culture. As such, this model lends itself to more rigorous evaluation to test strategies targeting the most chemotherapy resistant leukemic cells which derive their resistant phenotype through interaction with the bone marrow microenvironment.

<table border="0" cellpadding="0" cellspacing="0" width="734">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:224px;">G10 sephadex beads</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">G10120</td>
			<td style="width:243px;">Referred to in manuscript as gel type 10 cross-linked dextran particles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">10 ml sterile syringe</td>
			<td style="width:149px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Glass wool</td>
			<td style="width:149px;">Pyrex</td>
			<td style="width:119px;">3950</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">1-way stopcocks</td>
			<td style="width:149px;">World Precision Instruments, Inc.</td>
			<td style="width:119px;">14054-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">50 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021039</td>
			<td>Used as collection tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">15 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021037</td>
			<td>Used for cell collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Fetal Bovine Serum</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">F6178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">0.05% Trypsin&#160;</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">100X20mm Cell Culture Dishes</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:119px;">664160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Culture media</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Osteoblast culture media&#160;</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-27001</td>
			<td>For human osteoblast media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">RPMI 1640 media</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">15-040</td>
			<td>For tumor media prepation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Cell lines</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Adherent Cells</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Human Osteoblasts</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-12720</td>
			<td style="width:243px;">Human osteoblast were cultured according to the supplier&#8217;s recommendations.&#160;</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:224px;">Human Bone Morrow Stromal Cells</td>
			<td style="width:149px;">WVU Biospecimen Core</td>
			<td style="width:119px;"></td>
			<td style="width:243px;">De-identified primary human leukemia and bone marrow stromal cells (BMSC) were provided by the Mary Babb Randolph Cancer Center (MBRCC) Biospecimen Processing Core and the West Virginia University Department of Pathology Tissue Bank. BMSC cultures were established as previously described (*)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Leukemic Cells:</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">REH</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:119px;">ATCC-CRL-8286</td>
			<td style="width:243px;">REH cells were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">SD-1</td>
			<td style="width:149px;">DSMZ</td>
			<td style="width:119px;">ACC 366</td>
			<td style="width:243px;">SD-1 were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="48">
			<td colspan="4" height="48" style="height:48px;width:735px;">(*) Gibson LF, Fortney J, Landreth KS, Piktel D, Ericson SG, Lynch JP. Disruption of bone marrow stromal cell function by etoposide. Biol Blood Marrow Transplant J Am Soc Blood Marrow Transplant. 1997 Aug;3(3):122&#8211;32.&#160;</td>
		</tr>
	</tbody>
</table>

modeling chemotherapy resistant leukemia in vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Erfolgreichen Aufbau und die Kultur dieser Co-Kultur-Modell wird bei der Errichtung von drei Subpopulationen von Leukämiezellen gegenüber dem haft BMSC oder OB-Monoschicht führen. Abbildung 1 zeigt, wie alle in einem BMSC Monoschicht ausgesät Zellen zunächst als nur eine einzige Population von suspendierten erscheinen Leukämiezellen. Im Laufe von 4 Tagen mit dem BMSC Leukämiezellen interagieren 3 Raum Subpopulationen von Leukämiezellen zu bilden (gesperrt (S), Ph...

Watch this Scientific Journal Video about Modeling Chemotherapy Resistant Leukemia In Vitro at JoVE.com

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

Modeling Chemotherapie beständig Leukämie<em&gt; In Vitro</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

modeling-chemotherapy-resistant-leukemia-in-vitro

Research

JoVE Journal

Medicine

Modeling Chemotherapy Resistant Leukemia In Vitro

9.0K Aufrufe.  West Virginia University School of Medicine. The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation. 

Serie: Modeling Chemotherapy Resistant Leukemia In Vitro

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodale Bildgebung von Angiogenese in einem Nude Rattenmodell der Brustkrebs Knochenmetastasen mittels Magnetresonanztomographie, volumetrische Computertomographie und Ultraschall

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Forschung

Medizin

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotypische Dreidimensionale<em&gt; In Vitro</em&gt; Modellierung von Stromal-Epithelial Interaktionen beim Eierstockkrebs Initiation und Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Modeling Hirnblutung in Mice: Injektion von Eigenblut oder bakterielle Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

Quantitative Multispektrale Fluoreszenz-Analyse Nach Gewebetransplantation zur Visualisierung von Zell Origins, Typen und Wechselwirkungen

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Präklinische Evaluierung von Tyrosinkinase-Inhibitoren für die Behandlung von akuter Leukämie

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modellierung Spontane metastasiertem Nierenzellkarzinom (mRCC) in Mäusen nach Nephrektomie

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapie-induzierte vaskuläre Toxizität - Echtzeit-<em&gt; In-vivo-</em&gt; Imaging von Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Modellierung Schlaganfall bei Mäusen: Permanent Koagulation der distalen A. cerebri media

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

Von einem 2DE-Gel-Spot die Proteinfunktion: Lektion gelernt von HS1 in chronischer lymphatischer Leukämie

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Modellierung Muskeldystrophie 1 in C2C12 Myoblastenzellen

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...