Name: surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis
Uploaded: 2017-07-29T13:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

Gli autori ringraziano Jo Hill per l&#39;assistenza istopatologica, il Dr. John Gleysteen per l&#39;istruzione in tecnica chirurgica, Tessa Diebel per l&#39;assistenza video, tutti i membri dei laboratori Wong e Coussens per una visione e discussioni critiche e l&#39;OHSU Knight Cancer Institute per il sostegno finanziario. Gli autori riconoscono il supporto di T32GM071388-10 e T32CA106195-11 a CEG, il NCI / NIH, il Dipartimento della Difesa, il programma per la ricerca sul seno del seno, la Fondazione Susan G Komen, la Fondazione per la Ricerca sul Cancro del seno e una Fondazione per il Cancro - Lustgarten (SU2C-AACR-DT14-14) a LMC, un premio alla Fondazione per la Salute delle Donne di Salute a MHW e al Centro Brenden-Colson per la Salute Pancreatica a MHW e LMC.

Gli animali utilizzati nel protocollo seguente sono coperti dal comitato istituzionale per la cura e l&#39;uso degli animali (IACUC) dell&#39;Oregon Health &amp; Science, che è stato progettato per essere conforme alle normative vigenti in materia di benessere degli animali e della politica sanitaria pubblica (PHS). Manutenzione delle condizioni sterili: devono essere utilizzati strumenti sterilizzati e tra i topi devono essere puliti con garza sterile, sciacquati in PBS e sterilizzati con disinfettante al 70% di etanolo per almeno 15 minuti. È necessario indossare un cappello chirurgico, una maschera facciale, un abito e guanti per gli interventi di sopravvivenza. La preparazione pre-operativa dell&#39;animale per interventi di sopravvivenza è inclusa nel seguente protocollo. Fare riferimento alla tabella 1 per un elenco di reagenti e attrezzature. 1. Isolamento e preparazione di sospensioni di cellule singole da tumori primari del mammario <ol><li> Anestetizzare i topi transgenici MMTV-PyMT (FVB / n) femmina donatrice di 100 giorni e mantenere sotto cOntinuosa sedazione somministrando 2% di isoflurano attraverso una maschera di anestesia. Confermare che il mouse sia correttamente anestetizzato con un riflesso a pinza assente. </li><li> In un ambiente sterile, i respirazione di tumori mammari primari da topi femmina transgenici MMTV-PyMT (FVB / n) di 100 giorni, separando il tumore mammario dalla pelle sovrapposta e dai tessuti adiposi circostanti e / o dai linfonodi usando forbici sterili. Eutanizzare i topi anestetizzati per dislocazione cervicale. </li><li> Con forbici sterili o un bisturi, sminuzzare tumori primari manualmente in piccoli pezzi (~ 1,0 millimetri 3). Posizionare i tumori in 3,0 mg / mL collagenasi A e 4,0 U / mL DNasi I sciolti in DMEM. Utilizzare ~ 10 mL del suddetto mezzo di digestione per un tumore di diametro di 1,0 cm. <ol><li> Effettuare la digestione in una bottiglia sterile da 25 ml con una barra di stirata sterile a ~ 125 giri / min e 37 ° C per 40 min. </li></ol></li><li> Fermare la digestione aggiungendo il siero fetale fetale (FBS) ad una diluizione finale del 10% e collocare l&#39;intera miscela su wDove si mantiene. </li><li> Filtrare la sospensione tumorale digerita attraverso un filtro in nylon da 0,7 μm in un tubo conico da 50 ml e scartare qualsiasi tumore rimasto nel filtro. Centrifugare il surnatante a 300 xg a 4 ° C. </li><li> Resuspendere il pellet in 10 mL di DMEM per 1,0 cm di tumore e ri-filtrare attraverso un filtro in nylon da 0,7 μm. Conta concentrazione delle cellule, quindi centrifugare a 300 xg a 4 ° C. </li><li> Resuspendere il pellet nel 10% dimetilsolfossido con il 90% di FBS ad una concentrazione di 2 x 10 7 cellule vive / mL. Conservare le sospensioni a cellule singole di intero tumore primario a -80 ° C. </li></ol> 2. Iniezione ortotopica del tumore mammario <ol><li> Parzialmente scongelare le sospensioni tumorali primarie congelate a 37 ° C fino a quando il pellet congelato può essere rilasciato dal tubo criogenico in 20 mL di DMEM e contare le cellule. Centrifugare a 300 xg a 4 ° C e risospendere le cellule in 1: 1 DMEM: preparazione solubilizzata di membrana basale a fattore di crescita ridottaEstratto dal sarcoma del mouse di Engelbreth-Holm-Swarm (EHS) ad una concentrazione di 1 x 10 7 cellule / ml (vedi tabella dei materiali ). </li><li> Mettere il lato ventrale femminile sinergico del ricevente anestetizzato e mantenere in sedazione continua somministrando 2% di isoflurano tramite una maschera di anestesia. </li><li> Sterilizzare il 4 ° sito di iniezione della ghiandola mammaria usando etanolo al 70% aerosolizzato e applicando Poly (vinilpirrolidone) -Iodina con un tampone sterile in cotone. </li><li> Iniettare 100 μL (1 x 10 6 cellule vive dalle sospensioni tumorali primarie congelate) in senso inverso verso l&#39;alto fino alla 4a ghiandola mammaria destra del 6- 10 settimane FVB / n femminile utilizzando una siringa da 29 g 0,3 ml di insulina. </li></ol> 3. Resezione chirurgica del tumore ortotopico del mammario <ol><li> 38-60 giorni dopo l&#39;iniezione delle cellule tumorali, eutanizzano i topi che non presentavano volumi tumorali ortotopici che vanno da 172 a 450 mm 3 in volume [lunghezza x (larghezza 2 </Sup&gt;) / 2] (circa il 75% dei topi iniettati). </li><li> Posizionare i topi anestetizzati visualizzando il lato ventrale appropriato del tumore fino alla sedazione continua somministrando 2% di isoflurano tramite una maschera di anestesia e confermare che il mouse è correttamente anestetizzato con un riflesso a pinza assente. Applicare unguento veterinario agli occhi per prevenire la secchezza durante la sedazione. </li><li> Spruzzare il 70% di etanolo per sterilizzare l&#39;area chirurgica che circonda il tumore primario, quindi applicare Poly (vinilpirrolidone) -Iodina con un tampone sterile di cotone. NOTA: La rimozione dei capelli presso il sito chirurgico ha provocato estrazioni cutanee e / o infezioni per topi del ~ 5% e fu pertanto escluso da questo passaggio (dati non mostrati). </li><li> Come mostrato in Figura 1C , effettuare un&#39;incisione iniziale della pelle usando forbici sbarrate medial-caudali al tumore. </li><li> Successivamente, fare un&#39;escissione superiore della pelle ( Figura 1C ) mediale al tumore. Prestate attenzione alla necessità di cauterizzare qualsiasi vascolarizzazioneIl tumore posto sulla pelle prima di estendere l&#39;incisione. </li><li> Continuare l&#39;incisione della pelle lateralmente (posteriormente al tumore), quindi fare un&#39;escisione superiore della pelle (laterale al tumore) e l&#39;escissione mediale (superiore al tumore) ( Figura 1C ). </li><li> Dopo che la pelle è stata tagliata circonferenzialmente intorno al tumore ( Figura 1C ), sollevare la pelle sovrapposta attaccata al tumore utilizzando le pinze, mentre sbucchiando il tumore lontano dalla muscolatura della parete addominale e mantenendo intatte le ghiandole mammarie. </li><li> Identificare (per via smussa) e cauterizzare grandi vasi che attraversano i 4 ° e 5 ° ghiandola mammaria. </li><li> Excise circa la metà dei 4 ° e 5 ° ghiandole mammarie nel sito cauterizzazione per liberare il tumore, pelle sovrastante, e segmenti delle ghiandole mammarie (Figura 1C). </li><li> In caso di sanguinamento, identificare attivamente il sanguinamentoImbarcazioni e immediatamente cauterizzarle. Se si perde più di 250 μL di sangue, escludere il topo da studi e eutanizzarlo. </li><li> Chiudere i siti di escissione con le clip della ferita utilizzando un applicatore a clip ferito ( Figura 1C ). NOTA: Rimuovere le clip della ferita 10 giorni dopo l&#39;intervento chirurgico con un rimontaggio della ferita. </li><li> Amministrare salina sterile calda subcutanea (4% del peso corporeo dell&#39;animale) e mantenere l&#39;animale caldo con una lampada di calore. Controllare gli animali ogni 5-10 minuti durante il recupero dall&#39;anestesia. NOTA: L&#39;amministrazione di bupivacaina o lidocaina ha aumentato la mortalità post-operatoria (dati non mostrati). I topi che mostravano segni di dolore (colpi, mancanza di spostamento, mancanza di sposo) sono stati eutanizzati 1 h dopo la chirurgia. <ol><li> Una volta che il mouse è completamente recuperato, restituirlo alla compagnia di altri topi. </li></ol></li></ol> 4. Isolamento e trattamento di sangue e polmone per la citometria e l&#39;istologia di flusso &lt;ol&gt; <li> All&#39;inizio degli studi, preparare i topi per diverse valutazioni istopatologiche, se lo si desidera. 90 minuti prima dell&#39;eutanasia, dare a ciascun topo un&#39;iniezione intraperitoneale di bromodeossiridina (BrdU, 50 μg / g peso del mouse) a una concentrazione di 6,25 μg / μL in 1x PBS. NOTA: Le scorte congelate di bromodeossiridina sciolte vengono utilizzate entro un mese dalla preparazione. </li><li> 10 minuti prima dell&#39;eutanasia, con il mouse anestetizzato, raccogliere il sangue retroorbitale (&gt; 500 μL) utilizzando tubi capillari eparinizzati e successivamente trasferirli in tubi rivestiti con EDTA di potassio tenuto in ghiaccio. </li><li> Per rimuovere polmoni e restante tessuto mammario, fare un&#39;incisione mediana con le forbici dal basso addome alla bocca per esporre la cavità toracica e peritoneali (Figura 2A), e staccare la pelle lateralmente per esporre anche i restanti right 4 ° e 5 ° mammaria ghiandole. </li><li> Accisa il residuo tessuto ghiandolare mammario e esamina iT per escludere la ricrescita tumorale primaria mediante la valutazione di un tessuto a base di paraffina (FFPE) fissato a forma di formalina a serie con la colorazione di ematossilina ed eosina (H &amp; E) ( Figura 1 supplementare ). </li><li> Per rimuovere i polmoni, aprire la parete addominale al diaframma con forbici, quindi eutanizzare l&#39;animale tagliando l&#39;aorta addominale per scaricare il sangue prima della perfusione dei polmoni ( Figura 2B -2C ). </li><li> Tagliare il diaframma lungo la gabbia toracica da un approccio addominale, assicurandosi di evitare il polmone e il cuore. Quindi esporre il torace tagliandone i lati laterali della gabbia toracica ( figura 2D ). </li><li> Usando un ago da 23 gauge su una siringa da 20 ml, perfezionare i polmoni con ~ 5,0 mL DPBS (~ 10 mL / min) attraverso il ventricolo destro del cuore fino a quando i polmoni diventano completamente bianchi ( Figura 2E -2F ). Immediatamente tagliare il cuore dalla vesse principaleCosì il sangue non ri-perfusa nel polmone. </li><li> Per i tessuti polmonari da fissare e lavorare per valutazioni istopatologiche, iniettare ~ 1,0 mL di formalina 10% a 4 ° C nel lato opposto alla trachea esposta fino ai polmoni usando un ago da 23 gauge ( Figura 2G - 2H ). Interrompere l&#39;iniezione quando i polmoni sono completamente espandibili e riempiti con fissativo ( Figura 2I ). </li><li> I lobi polmonari di accisa dalla trachea e l&#39;emersione fissano il tessuto polmonare in formalina neutra-tamponata per il successivo incorporamento di paraffina o il mezzo di congelamento OCT, secondo procedure istopatologiche standard. </li><li> Valutare quantitativamente l&#39;onere metastatico nei polmoni mediante la sequenza seriale del tessuto polmonare FFPE e valutare le sezioni del microtomo (100 μm, tredici sezioni) mediante la colorazione H &amp; E ( Figura 1B ). </li></ol>

<ol>
	<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 Res. 8, 212 (2006).</li><li>Howlader, N. N. A., Krapcho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SEER Cancer Statistics Review 1975-2008. , (2011).</li><li>National Comprehensive Cancer Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Breast Cancer (Version 3.2014). , (2016).</li><li>Kataja, V., Castiglione, M., Group, E. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+recurrent+or+metastatic+breast+cancer:+ESMO+clinical+recommendations+for+diagnosis,+treatment+and+follow-up.">Locally recurrent or metastatic breast cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up.</a> Ann Oncol. 19 Suppl 2, 11-13 (2008).</li><li>Margolese, R. G., Hortobagyi, G. N., Buchholz, T. A., Kufe, D. W., Pollock, R. E., Weichselbaum, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Management of Metastatic Breast Cancer. Holland-Frei Cancer Medicine. , (2003).</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, 954-961 (1992).</li><li>Kouros-Mehr, H., 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=GATA-3+links+tumor+differentiation+and+dissemination+in+a+luminal+breast+cancer+model.">GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model.</a> Cancer Cell. 13, 141-152 (2008).</li><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+the+lung.">Genes that mediate breast cancer metastasis to the lung.</a> Nature. 436, 518-524 (2005).</li><li>Qian, B. Z., 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=FLT1+signalling+in+metastasis-associated+macrophages+activates+an+inflammatory+signature+that+promotes+breast+cancer+metastasis.">FLT1 signalling in metastasis-associated macrophages activates an inflammatory signature that promotes breast cancer metastasis.</a> J Exp Med. 212 (9), 1433-1448 (2015).</li><li>Verkooijen, H. M., 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=Patients'+refusal+of+surgery+strongly+impairs+cancer+survival.">Patients' refusal of surgery strongly impairs cancer survival.</a> Ann Surg. 242 (2), 276-280 (2005).</li><li>Peinado, H., 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=Pre-metastatic+niches:+organ-specific+homes+for+metastasis.">Pre-metastatic niches: organ-specific homes for metastasis.</a> Nat Rev Cancer. 17 (5), 302-317 (2017).</li></ol>

Gli autori non hanno alcuna dichiarazione di conflitto con i dati presentati.

Modifiche e risoluzione dei problemi: Quando il tumore sbarrato dissezione dalla parete addominale, il tumore può rimanere aderente alla parete addominale. Questo è stato osservato in &lt;5% di topi iniettati con tumore (dati non mostrati). Per i topi con tumori aderenti alla parete addominale, il topo deve essere eutanizzato in quanto la resezione è difficile senza ricrescita del tumore primario. Limitazioni del modello / tecnica:</st...

Le donne dell&#39;America settentrionale hanno un rischio di vita pari al ~ 12% per lo sviluppo del cancro al seno 2 ; La maggioranza di questi individui avrà i tumori primari rimossi tramite chirurgia e, a seconda del sottotipo del cancro, riceverà terapia mirata, endocrina, chemioterapia e / o radioterapia nell&#39;ambiente adiuvante 3 . Esempi includono, donne con diagnosi di cancro ormonale recettore-positivo che ricevono terapie anti-estrogeno e donne con tumori HER2-positivi che ricevono terapie con targeting HER2 con radiazioni / chemioterapia, mentre non sono disponibili terapie mirate per tumori negativi triplici 3 . Nonostante i progressi in radiazioni, chemioterapia, terapie personalizzate e ormonali che integrano la resezione chirurgica, la malattia si ripete nel 30-70% delle donne diagnosticate con la malattia di stadio II o III 4 , poiché le terapie sono in gran parte inefficaci nell&#39;eradicazione della malattia metastatica negli organi distanti, Polmone, osso, bPioggia e / o fegato 5 . Ciò è particolarmente significativo in quanto quando si verifica una malattia metastatica in assenza di ricrescita tumorale primaria, ciò implica che le cellule maligne disseminate erano già probabilmente presenti negli organi secondari al momento della chirurgia definitiva. Quindi terapie in grado di sradicare o lenta crescita dei tumori metastatici sono urgentemente necessari. Mentre i modelli de novo di cancro della carcinogenesi mammaria sono stati notevolmente informativi nei meccanismi rivelatori che regolano la progressione neoplastica 1 , i modelli esistenti hanno anche diverse limitazioni. Uno di questi è il fatto che i modelli de novo transgenici sviluppano tipicamente tumori primari in molteplici ghiandole mammarie, in cui il carico tumorale primario limita la durata degli studi. Mentre la fuga di cellule tumorali primarie e la semina metastatica probabilmente si verificano all&#39;inizio della progressione neoplastica in questi modelli, il sincero sviluppo di tumori metastatici si verifica in ritardo eN il modello del mouse e lo sfondo del ceppo, è spesso parzialmente penetrante 1 . Ciò limita ulteriormente l&#39;utilità dei modelli de novo per la scoperta di molecole che regolano la metastasi negli organi secondari e per la valutazione dell&#39;efficacia preclinica dei terapeutici nell&#39;ambiente adiuvante. Per eludere queste limitazioni, abbiamo sviluppato un modello de novo autochthonous di metastasi del carcinoma mammario ai polmoni. Le femmine transgeniche parentali ( ovvero MMTV-PyMT sullo sfondo del ceppo FVB / n per gli studi qui descritti) che portano tumori mammari di tarda fase de novo sono invecchiati a ~ 100 giorni 6 , a quel punto i loro tumori primari sono resettati chirurgicamente e dissociati enzimaticamente in Sospensioni singole cellule. Le sospensioni (1 x 10 6 cellule) sono a loro volta esplorate ortotropicamente in topi femmine singenici riceventi di 6-7 settimane, dove tumori mammari primari singoli si sviluppano in un periodo di 38-60 giorni ( 3), topi riceventi sono anestetizzati e tumori primari sono chirurgicamente resecati tale che la ricrescita del tumore al sito chirurgico è minimizzato, coerente con la chirurgia nelle donne (figura complementare 1). Sullo sfondo del ceppo FVB / n, i topi sviluppano foci metastatiche rilevabili istologicamente nei polmoni con 45% di penetranza per ~ 115 giorni dopo la chirurgia ( Figura 1B ). Con questa lunga latenza di crescita tumorale metastatica, il modello è posizionato in modo univoco per la consegna di terapie adiuvante e per chiarire e valutare la biologia sottostante che influenza la progressione metastatica dopo la rimozione chirurgica dei tumori primari.

<table><tbody><tr><td>Isofluorane</td><td>Piramal Healthcare</td><td>N/A</td><td>Prescription order</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>11088793001</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>12634010</td><td></td></tr><tr><td>25 mL Pyrex bottle</td><td>Sigma-Aldrich</td><td>CLS139525</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Bio&amp;nbsp;</td><td>S11150</td><td></td></tr><tr><td>0.7 &amp;micro;m nylon strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>50 mL conical tube&amp;nbsp;</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</td><td>BD</td><td>354230</td><td>Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma</td></tr><tr><td>Poly(vinylpyrrolidone)&amp;ndash;Iodine complex</td><td>Sigma-Aldrich</td><td>PVP1</td><td></td></tr><tr><td>29 gauge 0.3 mL insulin syringe</td><td>BD</td><td>324702</td><td></td></tr><tr><td>Small Vessel Cauterizer Kit</td><td>FST</td><td>18000-00</td><td></td></tr><tr><td>Wound clips</td><td>Texas Scientific</td><td>205016</td><td></td></tr><tr><td>AutoClip wound clip applier&amp;nbsp;</td><td>BD</td><td>427630</td><td></td></tr><tr><td>AutoClip wound clip remover&amp;nbsp;</td><td>BD</td><td>427637</td><td></td></tr><tr><td>Bromodeoxyuridine</td><td>Roche</td><td>10280879</td><td></td></tr><tr><td>Heparinized capillary tubes&amp;nbsp;</td><td>Fisher</td><td>22362566</td><td></td></tr><tr><td>Microtainer tubes with dipotassium EDTA&amp;nbsp;</td><td>BD</td><td>365974</td><td></td></tr><tr><td>20 mL syringe&amp;nbsp;</td><td>BD</td><td>309661</td><td></td></tr><tr><td>DPBS</td><td>Thermo-Fisher</td><td>14190-250</td><td></td></tr><tr><td>OCT-freezing medium&amp;nbsp;</td><td>VWR</td><td>25608930</td><td></td></tr><tr><td>Formalin, Buffered, 10% (Phosphate Buffer)</td><td>Fisher</td><td>SF100-4</td><td></td></tr><tr><td>23g needle</td><td>Fisher</td><td>14-826-6B</td><td></td></tr><tr><td>FVB/n mouse</td><td>Jackson Laboratories</td><td>001800</td><td></td></tr></tbody></table>

surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Più del 75% dei topi riceventi che ricevono 1 x 10 6 cellule da tumori mammari primari derivati ​​da topi MMTV-PyMT, sviluppano singoli adenocarcinomi mammari che vanno da 172 a 450 mm 3 entro 38-60 giorni (dati non mostrati). I topi ammessi alla randomizzazione sono quindi iscritti in gruppi di studio dopo la resezione chirurgica dei tumori primari come mostrato ( Figura 1C ). La ricrescita tumorale primaria è stata identificata ...

Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Mancano modelli pre-clinici che valutano la terapia adiuvante che mirano alla metastasi del cancro al seno. Per risolvere questo problema, abbiamo sviluppato un modello murino di metastasi adenocarcinomi mammarie polmonari de novo , in cui le terapie somministrate nell&#39;ambiente adiuvante (resezione chirurgica postale dei tumori primari) possono essere valutate per efficacia nell&#39;effetto delle metastasi polmonari precedentemente seminate.

Procedure chirurgiche e metodologia per un modello preclinico murino di<em&gt; De Novo</em&gt; Metastasi del cancro del mammario

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

surgical-procedures-methodology-for-preclinical-murine-model-de-novo

Research

JoVE Journal

Cancer Research

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

13.8K Views.  Oregon Health & Science University.  Mancano modelli pre-clinici che valutano la terapia adiuvante che mirano alla metastasi del cancro al seno. Per risolvere questo problema, abbiamo sviluppato un modello murino di metastasi adenocarcinomi mammarie polmonari de novo , in cui le terapie somministrate nell'ambiente adiuvante (resezione chirurgica postale dei tumori primari) possono essere valutate per efficacia nell'effetto delle metastasi polmonari precedentemente seminate. 

Video: Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Un modello di iniezione vena porta del fegato per studiare metastasi del cancro al seno

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Ricerca

Ricerca sul cancro

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

Orthotopic iniezione delle cellule di cancro al seno in topi cuscinetto grasso mammario

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Modello Murine dei tumori metastatici del fegato nell'impostazione della lesione alla riperfusione dell'ischemia

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Trapianto ortotopico di tumori al seno come modelli preclinici per il cancro al seno

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Modellazione delle metastasi cerebrali attraverso l'iniezione intracraniciale e la risonanza magnetica

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Avvio del carcinoma metastatico della mammella dalla Targeting del duttale con Adenovirus-Cre: A Novel modello di topo transgenico di cancro al seno

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Medicina

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Un preclinici Modello murino di metastasi epatiche

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

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.

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 ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Un modello murino ortotopico di spontanea Breast Cancer metastasi

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

Generazione di un modello di cancro al seno metastatico murino ortotopico ed eseguendo murino mastectomia radicale

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Procedure chirurgiche e metodologia per un modello preclinico murino di

Riepilogo

Esplora Altri Video

Capitoli in questo video