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.

Tutti gli esperimenti di topo sono stati fatti secondo protocolli approvati dalla cura degli animali e Istituzionale Usa Comitati (IACUC) di LSU School of Medicine. Tutti gli strumenti devono essere sterilizzati in autoclave prima della dissezione. 1. Preparazione di cellule primarie di tumore al seno del mouse <ol><li> Utilizzare un modello di topo che è stato geneticamente con il Tumor Virus mouse mammaria Polyoma Medio T-Antigen (topo MMTV-PyMT transgenico), che si sviluppano spontaneamente tumori come descritto in precedenza 6. </li><li> Sacrificare topi con isoflurano (99,9%) e spruzzare il 75% di etanolo per assicurare un ambiente sterile (Figura 1A). </li><li> Aprire la pelle dalla superficie dell&#39;orifizio uretrale al collo con le forbici. Pin la pelle verso il basso per consentire un facile accesso agli organi (Figura 1B). </li><li> Isolare il tumore al seno mammaria e lavare con freddo fosfato sterile salina tamponata (PBS) (pH 7,4) (Figura 1C). </li><li> Trasferire il tumoreuna piastra di coltura tissutale sterili 10 centimetri e tritare con una lama di rasoio sterile e forbici a circa 1 mm 3 pezzi. Fasi successive devono essere eseguite in una cappa a flusso laminare sterile. </li><li> Incubare il tessuto tumorale tritato con collagenasi disciolto nel siero mezzi DMEM ad una concentrazione finale di 1 mg / ml su un bilanciere per 2 ore a 37 ° C. Centrifugare la sospensione tessuto tumorale per 3 min a 200 x g. NOTA: L&#39;incubazione Collagenase è essenziale per la dissociazione enzimatica del tessuto. </li><li> Eliminare il supernatante e lavare il precipitato 3 volte con PBS (pH 7,4) per centrifugazione per 3 min a 200 x g. Dopo l&#39;ultimo lavaggio, decantare il surnatante. </li><li> Risospendere il tessuto con 2 ml di tripsina EDTA per 5 min a 37 ° C mentre oscillante. Centrifugare la sospensione per 3 min a 200 xg e ripetere i lavaggi. </li><li> Risospendere il pellet in tessuto normale terreno di coltura (DMEM, 10% FBS, penna 2X / strep) su un piatto 10 cm. Cambiamentoi media ogni giorno per i prossimi 3 giorni. Cellule coltura fino numero di cellulare desiderato si ottiene. NOTA: circa 1x10 7 cellule saranno isolati da questa procedura. Altri modelli tumorigenesi del mouse mammari, come MMTV-Neu, danno minore resa delle celle rispetto a MMTV-PyMT. Quando si utilizzano i modelli che producono un rendimento inferiore, è utile cominciare con una più grande cellule tumorali e cultura più lunghi. </li></ol> 2. polmone mouse estrazione di visualizzare Lung Metastasi <ol><li> Prima di procedere all&#39;estrazione del polmone, rimuovere il fegato e il diaframma. Rimuovere le costole senza disturbare i polmoni. </li><li> Con un bel paio di forbici, aprire la parte superiore della trachea che è più vicino alla vena giugulare. </li><li> Utilizzando un catetere, iniettare un agente chimico come 1 ml di Z-fix o inchiostro India attraverso la trachea per riempire i polmoni (Figura 2A). Incorpora la Z-fix Polmoni in paraffina. </li><li> De-macchia polmoni iniettati con inchiostro di china in soluzione di Fekete(100 ml 70% alcool, 10 ml di 10% formalina tamponata, e 5 ml di acido acetico glaciale) per 5 min. Definire noduli tumorali nei siti secondari con un ≥ 0,5 mm di diametro, determinare i noduli come metastasi. NOTA: a causa di un blocco bronchi e bronchioli dal tumore, noduli tumorali non possono assorbire il 15% inchiostro di china, e quindi normale tessuto polmonare sarà in nero, e noduli tumorali saranno di colore bianco 7. Questo permetterà la visualizzazione dei noduli tumorali individuali (Figura 2B). </li></ol> 3. Preparazione embrionali fibroblasti di topo (MEF) da embrioni di topo <ol><li> Sacrificio topi in gravidanza con isoflurano (99,9%) e spruzzare il 75% di etanolo per garantire un ambiente sterile. </li><li> Aprire la pelle dalla superficie dell&#39;orifizio uretrale al collo con le forbici e isolare gli embrioni dall&#39;utero. </li><li> Fare attenzione a non disturbare gli embrioni quando si apre la pelle. </li><li> Rimuovere le corna uterine e sciacquarli sotto ste freddorile PBS (pH 7,4). </li><li> Separare l&#39;embrione dalla sua placenta con molta attenzione e mettere gli embrioni in un piatto 10 centimetri contenente ghiaccio freddo PBS (pH 7,4). </li><li> Togliere la testa, fegato e intestino dall&#39;embrione e posizionare la parte restante in una piastra di coltura tissutale 12 pozzetto contenente PBS (pH 7,4). </li><li> Trasferire il corpo dell&#39;embrione ad un altro 12 pozzetti contenente tripsina EDTA e tagliato in piccoli pezzi (~ 1 mm 3) con una lama di rasoio sterile e forbici. Incubare il materiale tagliato con 5 ml di tripsina EDTA per 5 min a 37 ° C. </li><li> Inattivare tripsina aggiungendo 1 ml di siero fetale bovino (FBS) per gli embrioni tritate. Centrifugare la sospensione a 200 velocità xg per 10 minuti, rimuovere con attenzione il surnatante, risospendere il pellet con 1 ml di terreno DMEM caldo e trasferire tutti i contenuti per un piatto di 10 centimetri contenente 10 ml di DMEM (con il 20% FBS e 2X penicillina / streptomicina ). </li><li> Cambia il mezzo ogni giorno per due giorni. Coltivare le cellule fino cella desideratail numero è raggiunto. </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>

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Questo protocollo è per isolare cellule primarie di un tumore del mouse fresca. I lisati preparati da questo metodo sono utili per le proteine, DNA e RNA analisi. Le cellule tumorali del tumore mammario primario spesso metastatizzano al cervello, polmoni e ossa 1. Incluso è una tecnica per estrarre il polmone per il rilevamento di metastasi. Colorazione polmone con una macchia di inchiostro permetterà la visualizzazione dei noduli tumorali, come mostrato nella Figura 2B. Fissaggio dei polmoni in soluzione Z-fix ci permetterà di fare sezioni di polmoni, e macchia con anticorpi che sono appropriati per studiare i meccanismi metastatici. Inoltre è incluso un protocollo per l&#39;isolamento di fibroblasti embrionali di topo. Ciò è utile per confrontare l&#39;espressione genica delle cellule tumorali primarie con l&#39;fibroblasti embrionali nello stesso tipo di animale. Nella preparazione di pile e MEF, la fase più critica è la macinazione del materiale. Senza ripartizione corretta, losarà raggiunto il numero di cellulare w. Se si raggiunge un conteggio di cellule basso, protocollo può ancora essere seguito, ma le cellule dovrà essere coltivate per un periodo di tempo più lungo .. Nell&#39;estrazione polmone, corretta colorazione e decolorazione è essenziale per consentire la visualizzazione del tumore nodulo. Per estrarre l&#39;RNA / proteine ​​dai polmoni, sarà bene utilizzare polmoni che sono senza macchia e non fissato. Tuttavia, l&#39;RNA può essere estratto da tessuti fissati nonché 8. Dal momento che la fase metastatica del tumore al seno costituisce la più grande minaccia per l&#39;individuo colpito 2, è importante visualizzare tutti i componenti della tumorigenesi seno. Una sfida importante per la comprensione dei meccanismi di progressione del cancro al seno è la disponibilità di modelli che ricapitolano la progressione della malattia 9. Il chiarimento dei meccanismi molecolari della tumorigenesi della mammella e delle metastasi vengono raggiunti utilizzando modelli animali, come i topi 9, 10. I modi più comuni per indurre mouse tumori mammari sono geneticamente ingegnere topi con mutazioni predisponenti di oncogeni o soppressori tumorali, introducendo sostanze cancerogene, e l&#39;iniezione di cellule tumorali altamente invasivo 10-12. I tumori ottenuti possono poi essere ulteriormente analizzati per l&#39;espressione genica. Per la ricerca sul cancro al seno, l&#39;espressione genica è importante in quanto predice il decorso clinico del paziente 13. Esistono alcune limitazioni con il modello murino di studiare tumorigenesi mammaria. Anche se i topi condividono similitudini molecolari e fisiologici con gli esseri umani, non sempre ricapitolano tutti gli aspetti delle malattie umane 5. Questo dovrebbe essere preso in considerazione quando si utilizzano alcuni topi geneticamente ingegnerizzati. Nel complesso, modelli murini forniscono un modo più realistico per analizzare il mouse mammaria tumorigenesi rispetto ad altri modelli presenti quali la cultura dei tessuti e tumori umani isolati. Nel loro insieme, questo protocollo permette di ulteriori indagini di diversi aspettidella tumorigenesi seno.

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.

Lo stadio progressione tumorale in cui sacrificare l&#39;animale dipende dalla particolare studio e orientamenti IACUC. Nella Figura 1A, un 100 giorni vecchio C57BL / 6 del mouse con il Tumor Virus mouse mammaria Polyoma Medio T-antigene (MMTV-PyMT) è stato sacrificato a tre mesi. La pelle è stata aperta dalla zona dell&#39;orifizio uretrale al collo con le forbici e la pelle era immobilizzato per consentire un facile accesso agli organi come mostrato nella Figura 1B. Questo ha permesso un facile accesso al tumore ottenuta nella Figura 1C. Le cellule primarie ottenute dai tumori sono visualizzate nella Figura 1D. Dal momento che molti geni mediano seno metastasi del cancro al polmone 1, abbiamo estratto i polmoni (Figura 2A) per studiare il processo metastatico. Il polmone è stato gonfiato con Z-fix e colorati con inchiostro di china (Figura 2B) per visualizzare i noduli tumorali. Figura 3 mostra topo FIBR embrionali<html> oblasts isolato dal FVB embrioni di topo sforzo. <img alt="Figura 1" src="/files/ftp_upload/52609/52609fig1.jpg" /> Figura 1. Preparazione di cellule primarie di tumori al seno. (A) A 100 giorni vecchio mouse femmina con la Polyoma Medio T-Antigen (PyMT) è stato sacrificato. (B) La pelle è stato immobilizzato per consentire un facile accesso al tumore mammario. (C) Il tumore al seno è stato tagliato dall&#39;animale . (D) Le cellule primarie isolate sono state coltivate in Dulbecco modificato (DMEM) supplementato con 10% di siero fetale bovino e antibiotici (barra della scala: 100 micron). Le immagini mostrano le cellule a giorno 3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> caricare / 52609 / 52609fig2.jpg &quot;/&gt; Figura 2. Estrazione del polmone del mouse per visualizzare metastasi polmonari. (A) Il polmone è stato gonfiato con Z-fix e (B), macchiate di inchiostro di china per visualizzare noduli tumorali (barra della scala: 500 micron). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52609/52609fig3.jpg" /> . Figura 3. Preparazione embrionali fibroblasti di topo (MEF) da embrioni di topo MEF cellule sono state estratte da un embrione di topo FVB (barra della scala: 100 micron). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</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.

Tumore primario e isolamento delle cellule MEF per studiare Lung Metastasi

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

15.8K Views.  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.

Video: 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.

Analisi di reclutamento delle cellule di tumore del polmone

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

Ricerca

Ricerca sul cancro

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.

Metastasi di analisi sperimentale

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

Medicina

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.

Considerazioni pratiche nello studio di colonizzazione del polmone metastatico nell'Osteosarcoma usando l'analisi di metastasi polmonare

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.

L'istituzione di un'analisi di colonizzazione del polmone per la visualizzazione di cellule del tumore nei tessuti polmonari di circolazione

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.

Modello del polmone acellulare e cellulari per lo studio di metastasi del tumore

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.

Generazione ed espansione delle linee tumorali primarie e maligne del mesotelioma pleurico

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

Esame della disseminazione delle cellule tumorali da metastasi polmonari

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.

Autore in primo piano: Decodifica della semina da metastasi a metastasi utilizzando una nuova tecnica in vivo per monitorare la diffusione del cancro al seno 

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.

Monitoraggio della disseminazione delle cellule tumorali dalle metastasi polmonari utilizzando la fotoconversione

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.

Tumore primario e isolamento delle cellule MEF per studiare Lung Metastasi

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Capitoli in questo video

Analisi di reclutamento delle cellule di tumore del polmone

Metastasi di analisi sperimentale

Considerazioni pratiche nello studio di colonizzazione del polmone metastatico nell'Osteosarcoma usando l'analisi di metastasi polmonare

L'istituzione di un'analisi di colonizzazione del polmone per la visualizzazione di cellule del tumore nei tessuti polmonari di circolazione

Modello del polmone acellulare e cellulari per lo studio di metastasi del tumore

Generazione ed espansione delle linee tumorali primarie e maligne del mesotelioma pleurico

Monitoraggio della disseminazione delle cellule tumorali dalle metastasi polmonari utilizzando la fotoconversione

Monitoraggio della disseminazione delle cellule tumorali dalle metastasi polmonari utilizzando la fotoconversione

Monitoraggio della disseminazione delle cellule tumorali dalle metastasi polmonari utilizzando la fotoconversione

Isolamento e caratterizzazione di neutrofili con proprietà antitumorali