Siamo grati a Drs. Diane Palmieri e Patricia Steeg di NSC per averci fornito le cellule MDA-MB231Br. Ringraziamo il Dr. Ralph Mason, Mr. Jason Reneau e la signora Ramona Lopes per il supporto tecnico e collegiale. Questo lavoro è stato sostenuto in parte dal IDEA DOD Awards W81XWH-08-1-0583 e W81XWH-12-1-0317. Esperimenti di risonanza magnetica sono stati eseguiti in Advanced Imaging Research Center, una struttura NIH BTRP # P41-RR02584, e l&#39;iniezione intracardiaca ecoguidata è stato eseguito con VisualSonics Vevo 770 sotto 1S10RR02564801.

Tutte le procedure di animali sono stati approvati dal Comitato Istituzionale cura degli animali ed uso del University of Texas Southwestern Medical Centro. 1. Preparazione delle cellule MDA-MB231/Br-GFP <ol><li> Recuperare e coltivare le cellule MDA-MB231/Br-GFP (gentilmente fornite da DRS. Palmieri e Steeg, NCI) in DMEM contenente 10% FBS, 1% glutamina e 1% di penicillina / streptomicina. </li><li> Osservare la condizione delle cellule e colore del mezzo, e sostituire medio ogni 2-3 giorni. </li><li> Trypsinize e raccogliere le cellule quando l&#39;80% di confluenza è raggiunto, come indicato dal passo 1.3.1-1.3.5: <ol><li> Rimuovere il vecchio mezzo completamente e aggiungere 5 ml di PBS (1x) per lavare le cellule delicatamente. Rimuovere la PBS dal piatto. </li><li> Aggiungere 1,5 ml di tripsina nel piatto; inclinare il piatto delicatamente per avere tutte le celle sono coperti da tripsina. Mettere il piatto di nuovo al incubatore cella e la tiene a 37 ° C per 1 min. </li><li> Prendete il piatto di incubatore e osservarele cellule sotto il microscopio ottico per assicurare le cellule si staccano dal piatto. </li><li> Aggiungere 3 ml mezzo per fermare l&#39;effetto della tripsina. Raccogliere la miscela di cellule in una provetta da centrifuga. Centrifugare la miscela a 2.000 rpm per 5 min. </li><li> Rimuovere accuratamente il mezzo e sospendere nuovamente le cellule in 5 mezzo privo di siero ml fino omogeneo. </li></ol></li><li> Contare numero appropriato di cellule e risospendere in mezzo DMEM privo di siero con una concentrazione finale di 1,75 x 10 5 cellule in 100 microlitri di volume. <ol><li> Prendere 50 microlitri miscela di cellule e aggiungere 50 microlitri Trypan Blue. Dopo la miscelazione, prendere 10 microlitri miscela e con attenzione aggiungere alla diapositiva conteggio delle cellule. Utilizzare campioni multipli per garantire il numero di cellule stima accurata. </li><li> Stick nella diapositiva conteggio delle cellule, un capo alla volta e il contatore delle cellule inizia a contare automaticamente. Prendere media di tutti i risultati del conteggio e calcolare il numero totale delle cellule. </li><li> Centrifugare nuovamente le cellule e risospendere inil volume appropriato di mezzo privo di siero per determinare la concentrazione finale di 1,75 x 10 5 cells/100 microlitri volume. </li></ol></li><li> Cellule posto sul ghiaccio prima di iniezione intracardiaca. </li></ol> 2. Ultrasound Imaging guidata iniezione intracardiaca <ol><li> Utilizzare topi nudi femminili (BALB / c nu / nu) tra 6-8 settimane. </li><li> Accedere al sistema di imaging. Inizializzare il trasduttore 704 (40 MHz). </li><li> Inizia un nuovo studio e inserire le informazioni. </li><li> Anestetizzare (3% isoflurane/100% O 2 in una camera di induzione) e mantenere gli animali con isoflurano (2%) in 100% O 2 (1 dm 3 / min) durante l&#39;intera procedura tramite un&#39;ogiva. L&#39;anestesia è confermata quando nessun ritiro riflesso si osserva con pizzico punta. </li><li> Impostare la temperatura del tavolo di imaging a 37 ° C. Nastro il topo anestetizzato alla tabella di imaging riscaldata in posizione supina. </li><li> Mantenere il gel ultrasuoni a 37 ° C prima di iminvecchiamento. Applicare il gel sul torace del mouse. </li><li> Montare il trasduttore nel supporto. Abbassare il trasduttore fino a raggiungere la profondità delle immagini desiderata. Spostare lo stadio fino a quando il ventricolo sinistro si identifica con l&#39;aorta ascendente il punto di riferimento (Figura 1A). Bloccare la fase in cui una chiara visione del ventricolo sinistro viene visualizzato. </li><li> Disegnare 100 ml di miscela di cellule in una siringa da 1 ml con un ago G 22. Posizionare e fissare la siringa sulla siringa montaggio. </li><li> Spostare la siringa in avanti verso il petto del mouse e spostare con cura un lato all&#39;altro fino a che la punta dell&#39;ago sia nel campo dell&#39;imaging di vista (prima di entrare il mouse). </li><li> Regolare l&#39;altezza dell&#39;ago e l&#39;angolo di puntare verso il basso il ventricolo sinistro. </li><li> Penetrare l&#39;ago della siringa nello spazio intercostale tempestivamente, attraverso la pelle e muscoli strati nel ventricolo sinistro sotto la guida di imaging ad ultrasuoni. Un&#39;indicazione del successo di inserimento dell&#39;ago nel ventricolo di sinistracolo è il reflusso del sangue arterioso fresco (colore rosa in contrasto con sangue venoso rosso scuro) nella siringa </li><li> Iniettare la miscela di cellule lentamente (Figura 1B). </li><li> Al termine, togliere l&#39;ago, sollevare il trasduttore, pulire il gel per ultrasuoni con una garza umida e rimuovere il nastro. </li><li> Preparare una gabbia pulita con un tampone preriscaldato. </li><li> Mettere l&#39;animale sul pad e osservare l&#39;animale fino a completa guarigione. </li><li> Monitorare il comportamento dell&#39;animale ogni 24 ore per due giorni. </li><li> Condizioni generali e segni neurologici di complicanze nei topi sperimentali sono regolarmente monitorate. </li></ol> 3. RM Monitoraggio dello sviluppo del tumore intracranico <ol><li> Utilizzare un Tesla magnete 9.4 per monitorare lo sviluppo intracranica di metastasi cerebrali. </li><li> Avviare MRI due settimane dopo l&#39;impianto del tumore e ripetere una volta a settimana per un massimo di tre settimane. </li><li> Sedare gli animali con il 3% isoflurano e mantenerli sotto geneanestesia ral (1,5% isoflurano). </li><li> Monitorare e mantenere la temperatura corporea degli animali e costante respirazione tutto l&#39;esperimento. </li><li> Risoluzione multistrato alta (14 fette con spessore di 1 mm, senza gap) T 1 - e T 2-pesate coronali, che copre la regione del lobo frontale della fossa posteriore, sono acquisiti con i seguenti parametri: T immagini 1 ponderati: girare eco fetta multipla (SEMS), TR / TE = 400 msec msec/20, matrice: 256 x 256, FOV 20 x 20 mm, nella risoluzione aereo: 78 x 78 micron 2. T immagini 2-pesate: fast spin echo fetta multipla (FSEMS) sequenze, TR / TE = 2500 msec/48 msec, 8 treni eco, matrice: 256 x 256, FOV 20 x 20 mm, nella risoluzione aereo: 78 x 78 micron 28,9. Lesioni tumorali appaiono più luminoso rispetto ai normali tessuti cerebrali su T immagini 2-pesate. </li><li> Dimensioni del tumore è determinata su magliette immagini 2-ponderata delineando manualmente il enhanziamento parte della massa su ogni immagine utilizzando programmi MATLAB scritti da noi 8. Considerata la maggior parte del tumore diametri sono più piccoli rispetto allo spessore fetta (1 mm), la dimensione del tumore è presentato come nell&#39;attuale piano anziché il volume. </li></ol> 4. H &amp; E colorazione Confermando le metastasi <ol><li> Sacrifica i topi subito dopo la scansione l&#39;ultima MR, sezionare il cervello di tumore e incorporarlo in ottobre a medio e congelati in -80 ° C. </li><li> Sezione di una serie di 10 micron di spessore cervello coronale campioni con criostato. </li><li> Eseguire la colorazione H &amp; E sulle sezioni cerebrali. </li></ol>

<ol>
	<li>Schouten, L. 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=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., 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=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. F., 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=The+biology+of+brain+metastases-translation+to+new+therapies.">The biology of brain metastases-translation to new therapies.</a> Nat. Rev. Clin. Oncol. , (2011).</li><li>Lockman, P. R., 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=Heterogeneous+blood-tumor+barrier+permeability+determines+drug+efficacy+in+experimental+brain+metastases+of+breast+cancer.">Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer.</a> Clin. Cancer Res. 16, 5664-5678 (2010).</li><li>Yoneda, T., 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=A+bone-seeking+clone+exhibits+different+biological+properties+from+the+MDA-MB-231+parental+human+breast+cancer+cells+and+a+brain-seeking+clone+in+vivo+and+in.">A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in.</a> 16, 1486-1495 (2001).</li><li>Palmieri, D., 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=Her-2+overexpression+increases+the+metastatic+outgrowth+of+breast+cancer+cells+in+the+brain.">Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.</a> Cancer Res. 67, 4190-4198 (2007).</li><li>Percy, D. 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Radiol. 46, 718-725 (2011).</li><li>Zhou, 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=Longitudinal+MRI+evaluation+of+intracranial+development+and+vascular+characteristics+of+breast+cancer+brain+metastases+in+a+mouse+model.">Longitudinal MRI evaluation of intracranial development and vascular characteristics of breast cancer brain metastases in a mouse model.</a> PLoS One. 8, (2013).</li><li>Zhou, 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=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, (2009).</li><li>Zhang, R. 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Nessun conflitto di interessi dichiarati.

Nel presente studio, abbiamo dimostrato che l&#39;ecografia iniezione ventricolare sinistra di imaging guidata garantisce l&#39;accuratezza modo che è stata osservata ogni animale in questo studio sviluppato metastasi cerebrali e non la morte degli animali. La bellezza di iniezione immagine-guida è che il percorso di penetrazione dell&#39;ago della pelle e, infine, nel ventricolo sinistro può essere monitorato e regolato sotto la foto, che è distinta dalla procedura manuale che richiede severe punti di repere anatom...

Cervello metastasi è il tumore maligno intracranica più comune negli adulti. La prognosi è estremamente povero, con una sopravvivenza mediana di 4-6 mesi, anche con un trattamento aggressivo. Il cancro al seno è uno dei tre principali tumori primari con alta morbilità di metastasi cerebrali 1-3. Diverse linee di cancro al seno brain-tropico sono in grado di sviluppare metastasi cerebrali dopo intracardiaca o iniezione intracarotidea 4. Iniezione diretta di cellule tumorali nel ventricolo sinistro può ignorare il letto capillare polmonare e quindi aumentare l&#39;incidenza di formare metastasi cerebrali minimizzando metastasi viscerali. La linea MDA-MB231Br è una delle linee di cancro al seno più utilizzati umane per sviluppare metastasi cerebrali in modelli di roditori 5,6. Come molti altri studi 4,7, abbiamo eseguito una procedura manuale di iniezione intracardiaca nei nostri studi precedenti. Tuttavia, solo il 50% tasso di successo è stato ottenuto con il manualeiniezione e una frazione di topi morti per le procedure invasive ripetute se studi precedenti fallito. Qui, stiamo introducendo l&#39;uso di una procedura di imaging-guidata per garantire l&#39;iniezione di cellule di carcinoma mammario-cervello cerca nel ventricolo sinistro di topi atimici. Longitudinale alta risoluzione MRI è applicata a seguire lo sviluppo intracranica di metastasi cerebrali.

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			<td height="21" style="height:21px;width:413px;">DMEM</td>
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			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
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			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
			<td style="width:147px;"> </td>
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			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
			<td style="width:136px;">Biorad</td>
			<td style="width:87px;">TC10</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:147px;"> </td>
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			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
			<td></td>
			<td style="width:147px;"> </td>
		</tr>
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			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
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			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Sakura Finetek USA</td>
			<td style="width:87px;">4583</td>
			<td style="width:147px;"> </td>
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ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

Metastasi cerebrali Cancro al seno, che si verificano nel 30% dei pazienti con carcinoma mammario in stadio IV, è associata ad alta mortalità. La sopravvivenza mediana è di soli 6 mesi. È fondamentale disporre di adeguati modelli animali per imitare la diffusione emodinamico delle cellule metastatiche nello scenario clinico. Qui, stiamo introducendo l&#39;uso di piccolo ecografia animale per guidare un&#39;iniezione accurata delle cellule tumorali mammarie tropicale cervello nel ventricolo sinistro di topi nudi atimici. MRI longitudinale viene utilizzato per valutare iniziazione intracranica e la crescita delle metastasi cerebrali. Iniezione intracardiaca ecoguidata garantisce non solo un&#39;iniezione accurata e pertanto un più alto tasso di successo, ma anche diminuito significativamente il tasso di mortalità, rispetto al nostro precedente procedura manuale. In vivo alta risoluzione MRI permette la visualizzazione di lesioni iperintense multifocali, piccolo come 310 micron di diametro sulle immagini T 2-pesate a tre settimane dopo l&#39;iniezione. Follow-up MRI rivela la crescita del tumore intracranico e aumento del numero di metastasi che distribuiscono in tutto il cervello.

Con l&#39;alta risoluzione spaziale della RM (78 micron di risoluzione aereo), lesioni iperintense possono essere identificati piccolo come 310 micron di diametro (Figura 2). Dal momento che le metastasi in questo studio sono molto piccoli e lo sviluppo di necrosi ed edema è minima, la lesione iperintensa su magliette immagini 2-pesate davvero rappresentato la massa tumorale. MRI studi longitudinali permettono la valutazione vivo non invasiva della cresc...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Un seno cancro al cervello metastasi modello del mouse viene stabilito con ecografia guidata iniezione intracardiaca delle cellule MDA-MB231/Br-GFP. Sviluppo delle metastasi intracraniche multifocali è stato monitorato longitudinalmente utilizzando ad alta risoluzione di 9.4 T MRI.

Ultrasound Imaging guidata intracardiaca iniezione di sviluppare un modello murino di Breast Cancer metastasi cerebrali Seguito da longitudinale MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.7K Views.  University of Texas Southwestern Medical Center.  Un seno cancro al cervello metastasi modello del mouse viene stabilito con ecografia guidata iniezione intracardiaca delle cellule MDA-MB231/Br-GFP. Sviluppo delle metastasi intracraniche multifocali è stato monitorato longitudinalmente utilizzando ad alta risoluzione di 9.4 T MRI. 

Video: Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Ricerca

Medicina

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI-guidato Interruzione della barriera emato-encefalica transcranica con ultrasuoni focalizzati in un modello murino

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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

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

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

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

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

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Acuta cerebrale Trauma nei topi Seguito By longitudinale a due fotoni di imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

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, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Iniezione ortotopico di cellule di carcinoma mammario nel mammaria Fat Pad di Mice per studiare la crescita del tumore.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans Biofilm sviluppo sulla medicalmente rilevanti Foreign Bodies in un mouse sottocutaneo modello seguito da Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.