Name: primary tumor and mef cell isolation to study lung metastasis
Uploaded: 2015-05-20T13:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

We would like to thank NIH, LACaTS and Ochsner clinic foundation for the financial support.

Alle Maus-Experimente wurden in Übereinstimmung mit Protokollen von der Institutional Animal Care und Verwenden Committees (IACUC) von LSU School of Medicine genehmigt wurde. Alle Instrumente müssen vor der Dissektion autoklaviert werden. 1. Herstellung von primären Zellen aus Mausbrusttumoren <ol><li> Eine Maus-Modell, das genetisch mit dem Maus-Brusttumorvirus Polyoma Middle-T-Antigen (MMTV-PyMT transgenen Maus), das spontan entwickeln Tumore wie zuvor beschrieben 6 konstruiert wurde. </li><li> Opfern Mäuse mit Isofluran (99,9%) und 75% Ethanol zu sprühen, um eine sterile Umgebung (1A) zu gewährleisten. </li><li> Öffnen die Haut vor dem Bereich der Harnröhrenöffnung, um den Hals mit einer Schere. Pin der Haut nach unten, um einfachen Zugang zu Organen (1B) zu ermöglichen. </li><li> Isolieren Sie die Brustbrusttumor und mit kaltem sterile phosphatgepufferte Kochsalzlösung (PBS) (pH 7,4) (Abbildung 1c). </li><li> Übertragen Sie den Tumoreine sterile 10 cm Gewebekulturplatte und hacken sie mit einer sterilen Rasierklinge und Schere auf ca. 1 mm 3 Stück. Die nachfolgenden Schritte sollten in einer sterilen Sterilbank durchgeführt werden. </li><li> Inkubiere die gehackte Tumorgewebe mit Collagenase in serumfreiem DMEM-Medium mit einer Endkonzentration von 1 mg / ml gelöst auf einer Wippe für 2 h bei 37 ° C. Zentrifugieren Sie das Tumorgewebe Suspension 3 min bei 200 x g. HINWEIS: Die Collagenase-Inkubation ist für enzymatische Dissoziation des Gewebes. </li><li> Überstand verwerfen und wäscht den Niederschlag 3-mal mit PBS (pH 7,4) durch Zentrifugation für 3 min bei 200 x g. Nach dem letzten Waschen, Dekantieren des Überstandes. </li><li> Resuspendieren des Gewebes mit 2 ml Trypsin-EDTA für 5 min bei 37 ° C, während Taumel. Zentrifugieren Sie die Suspension 3 min bei 200 xg und wiederholen Sie die Wäschen. </li><li> Resuspendieren Gewebepellet in normalem Wachstumsmedium (DMEM, 10% FBS, 2X Pen / Strep) auf eine 10 cm Platte. Veränderungdie Medien jeden Tag für die nächsten 3 Tage. Kulturzellen, bis die gewünschte Anzahl von Zellen erhalten wird. HINWEIS: Etwa 1x10 7 Zellen werden aus diesem Verfahren isoliert werden. Andere Maus-Mammatumorgenese Modellen wie MMTV-Neu ergeben niedrigere Ausbeute an Zellen im Vergleich mit MMTV-PyMT. Bei der Verwendung von Modellen, die einen niedrigeren Ertrag erzeugen, ist es vorteilhaft, mit einem größeren Tumor und Kulturzellen länger zu beginnen. </li></ol> 2. Maus Lung Extraction auf Lungenmetastasen visualisieren <ol><li> Vor der Lunge Extraktion, entfernen Sie die Leber und die Membran. Verdrängen die Rippen ohne die Lungen zu stören. </li><li> Mit einer feinen Schere, öffnen Sie den oberen Teil der Luftröhre, die näher an der Halsschlagader ist. </li><li> Verwendung eines Katheters, injiziert ein Mittel wie beispielsweise 1 ml Z-fix oder Tusche durch die Luftröhre in die Lunge (2A) zu füllen. Betten Sie die Z-fix Lungs in Paraffin. </li><li> De-Fleck die Lungen mit Tusche in Fekete-Lösung eingespritzt(100 ml 70% -igem Alkohol, 10 ml 10% igem gepuffertem Formalin und 5 ml Eisessig) für 5 min. Definieren Tumorknoten an sekundären Standorten mit ≥ 0,5 mm im Durchmesser, bestimmen die Knötchen als Metastasierung. HINWEIS: Aufgrund der Bronchien und Bronchiolen Blockade durch den Tumor, können Tumorknoten nicht absorbieren 15% Tusche und damit normalem Lungengewebe wird in schwarz, und die Tumorknoten wird in weißer Farbe 7 sein. Dies wird die Visualisierung individueller Tumorknoten (2B) zu ermöglichen. </li></ol> 3. Vorbereitung embryonalen Maus-Fibroblasten (MEFs) aus Maus Embryonen <ol><li> Opfern trächtige Mäuse mit Isofluran (99,9%) und 75% Ethanol zu sprühen, um eine sterile Umgebung sichergestellt. </li><li> Öffnen die Haut vor dem Bereich der Harnröhrenöffnung, um den Hals mit einer Schere und Isolierung der Embryonen aus dem Uterus. </li><li> Achten Sie darauf, um die Embryonen stören beim Öffnen der Haut. </li><li> Entfernen Sie die Gebärmutterhörner und spülen Sie sie in kaltem sterile PBS (pH 7,4). </li><li> Trennen Sie den Embryo von seiner Plazenta sehr sorgfältig und Ort Embryonen in einer 10 cm Platte mit eiskaltem PBS (pH 7,4). </li><li> Entfernen Sie den Kopf, Leber und Darm aus dem Embryo und legen Sie den restlichen Teil in einer 12-Well-Gewebekulturplatte enthält PBS (pH 7,4). </li><li> Übertragen Sie die Embryo Körper in einen anderen 12 Well-Platte mit Trypsin EDTA und in kleine Stücke (ca. 1 mm 3) mit einer sterilen Rasierklinge und Schere. Inkubieren Häckselgut mit 5 ml Trypsin-EDTA für 5 min bei 37 ° C. </li><li> Inaktivierung von Trypsin durch Zugabe von 1 ml fötales Rinderserum (FBS), um die geschnittenen Embryonen. Zentrifugieren Sie die Suspension bei 200 xg Geschwindigkeit für 10 Minuten, entfernen Sie vorsichtig den Überstand, das Pellet mit 1 ml warmem DMEM-Medium, und all der Inhalt in einer 10 cm Schale mit 10 ml DMEM (mit 20% FBS und 2X Penicillin / Streptomycin ). </li><li> Ändern Sie das Medium jeden Tag für zwei Tage. Kultur die Zellen, bis die gewünschte ZelleZahl erreicht ist. </li></ol>

<ol>
	<li>Minn, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Natur. 436 (7050), 518-524 (2005).</li><li>Poste, G., 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=The+pathogenesis+of+cancer+metastasis.">The pathogenesis of cancer metastasis.</a> Natur. 283 (5743), 139-146 (1980).</li><li>Wan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Metastasis+Moving+New+Biological+Insights+into+the+Clinic.">Tumor Metastasis Moving New Biological Insights into the Clinic.</a> Nat Med. 19 (11), 1450-1464 (2013).</li><li>Bergers, G., Benjamin, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenesis+and+the+angiogenic+switch.">Tumorigenesis and the angiogenic switch.</a> Nat Rev Cancer. 3 (6), 401-410 (2003).</li><li>Frese, K. K., Tuveson, 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=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7 (9), 654-658 (2007).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12 (3), 954-961 (1992).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Identification+of+Experimental+Pulmonary+Metastases.">Accurate Identification of Experimental Pulmonary Metastases.</a> J Natl Cancer Inst. 36 (4), 641-645 (1966).</li><li>Boczkowski, D., Nair, S. K., Nam, J. H., Lyerly, H. K., Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tumor+immunity+and+cytotoxic+T+lymphocyte+responses+using+dendritic+cells+transfected+with+messenger+RNA+amplified+from+tumor+cells.">Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells.</a> Cancer Res. 60 (4), 1028-1034 (2000).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat Rev Cancer. 7 (9), 659-672 (2007).</li><li>Watts, A. M., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+Tumor+Foci+in+an+Experimental+Murine+Tumor+Model+Using+Computer-Assisted+Video+Imaging.">Quantitation of Tumor Foci in an Experimental Murine Tumor Model Using Computer-Assisted Video Imaging.</a> Anal Bioche. 256 (2), 217-219 (1998).</li><li>Rosenberg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+Established+Pulmonary+Metastases+and+Subcutaneous+Tumor+Mediated+by+the+Systemic+Administration+of+High-Dose+Recombinant+Interleukin+2.">Regression of Established Pulmonary Metastases and Subcutaneous Tumor Mediated by the Systemic Administration of High-Dose Recombinant Interleukin 2.</a> J Exp Me. 161 (5), 1169-1188 (1985).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Re. 8 (4), 212 (2006).</li><li>Veer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+predicts+clinical+outcome+of+breast+cancer.">Gene expression profiling predicts clinical outcome of breast cancer.</a> Nature. 415 (6871), 530-536 (2002).</li></ol>

Die Autoren haben keine Angaben.

Dieses Protokoll ist zur Isolierung von primären Zellen aus einer neuen Maus-Tumor. Lysate aus diesen Verfahren hergestellt werden, sind nützlich für die Protein-, DNA- und RNA-Analyse. Krebszellen aus dem primären Brusttumor metastasieren häufig zum Gehirn, Lunge und Knochen 1. Eingeschlossen ist eine Technik, die zur Detektion von Lungenmetastasen zu extrahieren. Anfärben der Lunge mit einem Tintenfleck wird zur Visualisierung von Tumorknoten zu ermöglichen, wie in 2B zu sehen. Zur Festsetzung der Lunge in Z-fix-Lösung wird uns erlauben, Teile der Lunge zu machen, und Fleck mit Antikörpern, die angemessen, metastasiertem Mechanismen zu studieren sind. Ebenfalls enthalten ist ein Protokoll für die Isolierung von embryonalen Maus-Fibroblasten. Dies ist nützlich für den Vergleich der Genexpression von der primären Tumorzellen mit der embryonalen Fibroblasten in der gleichen Tierart. Bei der Herstellung von Primärelementen und MEFs, ist der wichtigste Schritt die Hacken des Materials. Ohne die richtige Aufteilung, low Zellzahl erreicht wird. Wenn eine niedrige Zellzahl erreicht ist, kann noch Protokoll folgen, aber die Zellen benötigen, um für eine längere Zeitdauer kultiviert werden .. In der Lunge Extraktion, ist die richtige Färbung und Entfärbung wesentlich, damit Tumorknotens Visualisierung zu ermöglichen. Um RNA / Protein aus der Lunge zu extrahieren, wird es gut, Lunge, die ungefärbten und unbefestigten sind zu verwenden. Jedoch kann RNA aus fixierten Geweben sowie 8 extrahiert werden. Da der metastasierten Stadium von Brustkrebs stellt die größte Bedrohung für die betroffene Person 2, ist es wichtig, dass alle Komponenten des Brusttumorgenese zu visualisieren. Eine große Herausforderung für das Verständnis Mechanismen von Brustkrebs Progression ist die Verfügbarkeit von Modellen, die das Fortschreiten der Erkrankung 9 rekapitulieren. Aufklärung der molekularen Mechanismen von Brusttumorentstehung und Metastasierung sind durch Verwendung von Tiermodellen, wie Mäuse 9, 10 erzielt. Die gebräuchlichsten Methoden zur Induzierung mouse Brusttumoren gentechnisch den Mäusen mit prädisponierenden Mutationen von Onkogenen oder Tumorsuppressoren, die Einführung Karzinogene und Einspritzen sehr invasiv Krebszellen 10-12. Erhalten Tumoren können dann weiter für die Genexpression analysiert werden. Für Brustkrebs-Forschung, ist Genexpressions wichtig, da sie das klinische Ergebnis von Patienten 13 prognostiziert. Einige Einschränkungen gibt es mit der Maus-Modell zu studieren Mammatumorgenese. Obwohl Mäuse teilen molekularen und physiologischen Ähnlichkeiten mit dem Menschen, sie nicht immer zu rekapitulieren alle Aspekte des menschlichen Krankheiten 5. Dies sollte bei der Verwendung bestimmter gentechnisch veränderten Mäusen in Betracht gezogen werden. Insgesamt Mausmodelle bieten einen realistischeren Weg, um Maus-Mammatumorgenese über andere anwesende Modelle wie Gewebekultur und isolierten menschlichen Tumoren zu analysieren. Zusammengenommen werden, ermöglicht dieses Protokoll für die weitere Untersuchung der verschiedenen Aspektevon Brusttumorgenese.

Metastasis is the final step in the progression of breast cancer 1. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs 2. The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system 3. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes 4. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse models in cancer research are the molecular and physiological similarities they share with humans 5. 
Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse&#8217;s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body 5.
The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.

<table><tbody><tr><td>DMEM</td><td>HyClone</td><td>SH30243.01</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life&amp;nbsp; technologies</td><td>25300-062</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Collagenase Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Pen Strep</td><td>Life&amp;nbsp; technologies</td><td>15140-122</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gemini Bio-Products</td><td>100-106</td><td>Store at &amp;ndash; 80 &amp;deg;C</td></tr><tr><td>Z-fix</td><td>ANATECH</td><td>#174</td><td>Store at RT</td></tr><tr><td>India Ink</td><td>Yasutomo</td><td></td><td>Store at RT</td></tr><tr><td>AERRANE (Isoflurane)</td><td>Baxter</td><td>NDC 10019-773-60</td><td>Store at RT</td></tr><tr><td>Ethanol</td><td>EMD</td><td>AX0441-3</td><td>Store at RT</td></tr></tbody></table>

primary tumor and mef cell isolation to study lung metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

Die Tumorprogression Stadium, in dem das Tier zu opfern, hängt von der jeweiligen Studie und IACUC Richtlinien. In 1A einer 100 Tage alte C57BL / 6-Maus mit der Maus-Mammatumorvirus Polyoma Middle-T-Antigen (MMTV-PyMT) wurde nach drei Monaten geopfert. Die Haut wurde von dem Bereich der Harnröhrenöffnung, um den Hals mit einer Schere geöffnet und die Haut wurde festgenagelt, um einfachen Zugang zu Organen, wie in 1B gezeigt, zu ermöglichen. Dies erlaubt einen einfachen Zugang zum Tumor, der in 1C erhalten. Die primären Zellen aus den Tumoren erhaltenen Ergebnisse sind in Figur 1D visualisiert. Seit vielen Genen vermitteln Brustkrebsmetastasen in der Lunge 1 extrahierten wir die Lunge (2A), um die metastatischen Prozess zu untersuchen. Die Lunge wurde mit Z-fix aufgepumpt und mit Tusche (2B), um die Tumorknötchen visualisieren gefärbt. Figur 3 zeigt embryonalen Maus fibr<html> den Oblast von FVB-Stamm Maus-Embryonen isoliert. <img alt="Abbildung 1" src="/files/ftp_upload/52609/52609fig1.jpg" /> Abbildung 1. Herstellung von primären Zellen von Brusttumoren. (A) Ein 100 Tage alte weibliche Maus mit dem Polyoma Middle T-Antigen (PyMT) wurde geopfert. (B) Die Haut wurde festgenagelt, um einen einfachen Zugang zu den Mamma-Tumor zu ermöglichen. (C) Das Brusttumor wurde von der Tier geschnitten . (D) Die isolierten primären Zellen wurden in Dulbeccos modifiziertem Eagle Medium (DMEM) mit 10% fötalem Rinderserum und Antibiotika ergänzt kultiviert (Maßstab: 100 &amp; mgr; M). Die Bilder zeigen die Zellen am Tag 3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> laden / 52.609 / 52609fig2.jpg &quot;/&gt; Abbildung 2. Maus-Lunge-Extraktion, um Lungenmetastasen zu visualisieren. (Maßstab: 500 &amp; mgr; M) (A) Die Lunge wurde mit Z-fix und (B) mit Tusche gefärbt, um Tumorknoten visualisieren aufgeblasen. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/52609/52609fig3.jpg" /> . Abbildung 3. Vorbereitung embryonalen Maus-Fibroblasten (MEFs) aus Maus Embryonen MEF-Zellen wurden aus einer FVB Mausembryo (Maßstab: 100 &amp; mgr; M) extrahiert. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Primärtumor und MEF Zellisolation zu Lungenmetastasen Studieren

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

primary-tumor-and-mef-cell-isolation-to-study-lung-metastasis

Nanyang Technological University

Research

JoVE Journal

Medicine

15.8K Aufrufe.  Louisiana State University Health Sciences Center. The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Serie: Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Lung Tumor Zelle Rekrutierung Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Forschung

Krebsforschung

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Experimentelle Metastase Assay

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medizin

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

Praktische Überlegungen an einem Studium von metastasierendem Lungenkrebs Besiedlung in Osteosarkom mit pulmonalen Metastasen-Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Die Einrichtung eines Lunge Kolonisation Assays für zirkulierenden Tumor Zelle Visualisierung im Lungengewebe

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Azelluläre und zellulären Lunge Modell Tumor Metastasen zu studieren

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

Erzeugung und Erweiterung primärer, maligner pleuraler Mesotheliom-Tumorlinien

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

Untersuchung der Ausbreitung von Tumorzellen aus Lungenmetastasen

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Autor im Rampenlicht: Entschlüsselung der Metastasen-zu-Metastasen-Seeding mit einer neuen In-vivo-Technik zur Verfolgung der Ausbreitung von Brustkrebs 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Verfolgung der Ausbreitung von Tumorzellen aus Lungenmetastasen mittels Photokonversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Primärtumor und MEF Zellisolation zu Lungenmetastasen Studieren

Zusammenfassung

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