Gli autori desiderano ringraziare Michele Allen del National Heart, Lung, and Blood Institute (NHLBI) dei National Institutes of Health (NIH) per la condivisione di generosamente la sua esperienza nei metodi di fenotipizzazione murine e per la sua assistenza tecnica in corso, così come per Timothy Hunt di NHLBI per aiutare a sviluppare il dispositivo di schermatura. Questo studio è sostenuto dalla Divisione di Intramural Research del National Institute of Nursing Research del NIH, e una parte del processo di validazione è supportata da una sovvenzione della Oncology Nursing Society Foundation.

Etica Dichiarazione: Questo studio è stato approvato dal National Institutes of Health (NIH) cura degli animali e del Comitato uso. Tutti i ricercatori che partecipano al trattamento degli animali e la misurazione dei risultati dello studio sono stati adeguatamente formati dall&#39;Ufficio NIH di cura degli animali e uso e il National Heart, Lung, and Blood Institute murino Fenotipizzazione core. Tutti gli aspetti della sperimentazione animale, gli alloggi, e le condizioni ambientali utilizzati in questo studio erano in conformità con la guida per la cura e l&#39;uso di animali da laboratorio 11. 1. L&#39;alloggio e animali da esperimento NOTA: Casa maschi C57BL / 6 topi (circa cinque settimane all&#39;arrivo) individualmente durante l&#39;esperimento e fornire ad libitum l&#39;accesso al cibo e all&#39;acqua. Tutte le gabbie sono tenuti al ciclo luce-buio 12:12 h con la fase di luce che inizia alle 6 del mattino e la fase di buio alle 6 del pomeriggio. <ol><li> Identificare i topi e assegnarli a singoli ventilare seriegabbie d mouse. Consentire 24 ore dopo la procedura di identificazione per il recupero. NOTA: il tatuaggio di coda è raccomandato come un mezzo di identificazione per eliminare la possibilità che un marchio auricolare potrebbe rimanere intrappolati in una ruota in esecuzione. Tatuaggio un numero sulla coda di ciascun topo, con il numero sulla coda corrispondente al numero scritto sulla gabbia del mouse. </li><li> Consentire topi si adatti alla loro gabbie per almeno tre giorni, manipolazione ogni mouse delicatamente per un periodo di tre minuti al giorno. </li></ol> 2. L&#39;esecuzione della rotella acclimatazione e Baseline <ol><li> Introdurre i topi alle singole gabbie VWRA, ciascuna dotata di una ruota in esecuzione collegato ad un contatore elettronico per la registrazione continua. NOTA: Tutti i contatori delle ruote connettersi a un computer tramite una sola interfaccia USB (vedere elenco dei materiali). Il software calcola il numero di giri delle ruote, la distanza percorsa e la velocità media di ciascun intervallo di tempo designato della duratio totale specificaton. Una volta che la registrazione si interrompe, i dati vengono automaticamente salvati sia come testo e fogli di calcolo. </li><li> Avviare la registrazione del VWRA attraverso l&#39;interfaccia software per computer. Impostare intervalli di registrazione a un&#39;ora e la durata di almeno cinque giorni. Continuare la registrazione VWRA per almeno cinque giorni. NOTA: Al termine della fase 2.2, tutti i topi dovrebbero raggiungere una quantità relativamente consistente di attività ruota gestione quotidiana. In caso contrario, quindi identificare ed escludere eventuali valori anomali. </li><li> Interrompere la registrazione VWRA tramite l&#39;interfaccia software, e restituire il mouse per loro gabbie standard descritti al punto 1.1 (gabbie senza correre ruote). </li><li> Randomize topi in nessuna delle due controllo sham-irradiati o gruppi irradiati. </li></ol> 3. L&#39;irradiazione Nota: eseguire i seguenti passaggi per tutti i mouse in entrambi i gruppi, una volta al giorno per tre giorni consecutivi. Trattare topi nello stesso ordine ogni giorno. <ol><li> Anestetizzare ogni topo mediante iniezione intraperitoneale diun ketamina (100 mg / kg) e xilazina (10 mg / kg) della miscela. </li><li> Confermare l&#39;anestesia con un pizzico punta e usare pomata sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Trasferire il mouse anestetizzato in un dispositivo di schermatura di piombo. Disporre il mouse nella schermatura in modo che solo la regione pelvica / addominale inferiore è esposta. NOTA: La schermatura è composta da due lead &quot;scatole&quot;, con uno spazio aperto stretto tra che permette l&#39;esposizione alle radiazioni ad una piccola regione di destinazione del mouse (vedi Figura 1). </li><li> Utilizzare nastro medico per fissare la base della coda del mouse nella posizione all&#39;interno della schermatura. NOTA: Passo 3.4 è facoltativo, ma in questo modo può contribuire a garantire che la posizione del mouse non cambia durante il passo successivo. </li><li> Trasportare il dispositivo di schermatura nel irradiatore, assicurando che la posizione degli animali all&#39;interno della schermatura viene mantenuta. </li><li> Se il mouse è nel gruppo irradiazione, consegnare 800 cGy a dose velocità di circa 110 cGy / min. Se il mouse è nel gruppo di controllo sham-irradiazione, lasciare il mouse nella irradiatore inattivo per il tempo equivalente. NOTA: le impostazioni ottimali irradiatore dipendono dal particolare dispositivo. Il rateo di dose di 110 cGy / min fornita da una fonte Cesio 137 è il rateo di dose centrale del irradiatore usato qui. Il tempo di esposizione è stata regolata a raggiungere la dose totale desiderata di 800 cGy. </li><li> Rimuovere il mouse dalla irradiatore e schermatura, e poi tornare alla sua gabbia originale, standard di cui al punto 1.1. </li><li> Monitorare costantemente il mouse fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. </li></ol> 4. Misura fatica indotta da radiazioni <ol><li> Il giorno dopo il completamento di tre giorni successivi di irradiazione, trasferire i topi ai loro gabbie individuali VWRA descritte nel punto 2.1. </li><li> Record VWRA, come descritto al punto 2.2, tranne qui impostando la registrazionedurata a più di 15 giorni. Al termine dei 15 giorni, interrompere manualmente VWRA registrazione tramite l&#39;interfaccia software. NOTA: I dati di ciascun periodo di registrazione vengono automaticamente salvati come fogli di calcolo, ognuno dei quali comprende le rotazione, la distanza e le misurazioni di velocità per tutti gli animali (colonne) e in tutti gli intervalli (righe) per tutta la durata della registrazione. Alla fine dell&#39;esperimento, ci sono due fogli generati dal software di registrazione: uno per il VWRA pre-irradiazione, e uno per il VWRA post-irradiazione. </li></ol>

<ol>
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N., 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+cancer-related+fatigue:+a+review+of+the+literature.">The biology of cancer-related fatigue: a review of the literature.</a> Support Care Cancer. 23 (8), 2461-2478 (2015).</li><li>Norden, D. 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=Tumor+growth+increases+neuroinflammation,+fatigue+and+depressive-like+behavior+prior+to+alterations+in+muscle+function.">Tumor growth increases neuroinflammation, fatigue and depressive-like behavior prior to alterations in muscle function.</a> Brain Behav Immun. 43, 76-85 (2015).</li><li>Ray, M. A., Trammell, R. A., Verhulst, S., Ran, S., Toth, L. 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L., Barry, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+responses+of+rats+to+acute+body+or+neck+restraint.">Stress responses of rats to acute body or neck restraint.</a> Physiol Behav. 5 (4), 443-448 (1970).</li><li>Iwakawa, 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=Different+radiation+susceptibility+among+five+strains+of+mice+detected+by+a+skin+reaction.">Different radiation susceptibility among five strains of mice detected by a skin reaction.</a> J Radiat Res. 44 (1), 7-13 (2003).</li><li>Travis, E. L., Peters, L. J., McNeill, J., Thames, H. D., Karolis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dose-rate+on+total+body+irradiation:+lethality+and+pathologic+findings.">Effect of dose-rate on total body irradiation: lethality and pathologic findings.</a> Radiother Oncol. 4 (4), 341-351 (1985).</li><li>Duran-Struuck, R., Dysko, 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=Principles+of+bone+marrow+transplantation+(BMT):+providing+optimal+veterinary+and+husbandry+care+to+irradiated+mice+in+BMT+studies.">Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies.</a> J Am Assoc Lab Anim Sci. 48 (1), 11-22 (2009).</li><li>Duran-Struuck, 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=Differential+susceptibility+of+C57BL/6NCr+and+B6.Cg-Ptprca+mice+to+commensal+bacteria+after+whole+body+irradiation+in+translational+bone+marrow+transplant+studies.">Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies.</a> J Transl Med. 6, 10 (2008).</li></ol>

Questa ricerca è stata sostenuta dalla Divisione di Intramural Research, Istituto Nazionale di ricerca infermieristica, National Institutes of Health.

Abbiamo descritto un protocollo mediante irradiazione periferico mirati per indurre una riduzione di VWRA in topi senza confondere morbilità o mortalità. È importante sottolineare che un semplice dispositivo di schermatura permette di irradiazione in questo protocollo per indirizzare una regione desiderata in modo coerente, imitando i trattamenti di radiazione ricevuta dai pazienti con cancro pelvico. A differenza dei modelli CRF esistenti che coinvolgono il cervello o irradiazione corporea totale <...

Affaticamento cancro-correlata (CRF) è una condizione dolorosa e costosa, che spesso colpisce i pazienti che ricevono trattamenti contro il cancro 1. La fatica non è né proporzionale alla recente attività né alleviato dal riposo, ed è associata ad una vasta gamma di disturbi legati allo stato d&#39;animo, la motivazione, l&#39;attenzione e la cognizione 2. Le cause biologiche di CRF sono sconosciute, anche se è stato dimostrato in molti casi correlati con livelli di infiammazione e citochine, in alcuni casi con livelli di emoglobina e la funzione dei vari sistemi ormonali (vedi Saligan et al. 3 per una revisione della biologica studi di CRF). Studi controllati su modelli animali sono necessari per capire il comportamento e la biologia associata a questa condizione complessa. Mentre tumore legati 4 o chemioterapia connessi 5, 6 grassoIgue è stato studiato in modelli di roditori, l&#39;eziologia della CRF può essere specifico trattamento. Per indagare CRF relative a radioterapia, il nostro gruppo ha recentemente sviluppato un modello murino di irradiazione stanchezza indotta 7. A differenza dei modelli CRF esistenti che coinvolgono il cervello o irradiazione corporea totale 8, 9, questo modello esplora come un cambiamento nel comportamento in posizione centrale-driven, come la fatica, può essere attivato da una procedura di irradiazione perifericamente mirati. La procedura descritta qui è stato progettato per modellare radioterapia somministrata a pazienti con tumore pelvico, utilizzando schermatura di piombo per indirizzare la regione addominale inferiore / pelvica con irradiazione. Tuttavia, modificando la schermatura di piombo o la sua posizione rispetto agli animali da esperimento, questa procedura potrebbe essere adattato per modellare irradiazione di altre parti del corpo. Volontario attività ruota corsa (VWRA) viene utilizzato per misurare la fatica behavio simileR; perché è un comportamento volontario e normale 10, dovrebbe consentire l&#39;uso concomitante di altri test comportamentali e biologici. Abbiamo scoperto che l&#39;irradiazione periferica è sufficiente a ridurre VWRA nei topi senza causare morbilità palese 7. Esperimenti futuri con questo modello possono aiutare a rivelare effetti dell&#39;irradiazione periferico sul segnale biologico immunitario e altri, così come i cambiamenti a valle del sistema nervoso centrale che può produrre deficit associati CRF.

<table border="0" cellpadding="0" cellspacing="0" width="1098">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">C57BL/6 Mice</td>
			<td style="width:135px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:591px;">Strain code 027 (http://www.criver.com/products-services/basic-research/find-a-model/c57bl-6n-mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ketamine HCl</td>
			<td style="width:135px;">Putney</td>
			<td style="width:119px;"></td>
			<td style="width:591px;">100 mg/ml stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Xylazine HCl</td>
			<td style="width:135px;">Lloyd Laboratories</td>
			<td></td>
			<td style="width:591px;">100 mg/ml stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rodent Tattoo System</td>
			<td style="width:135px;">AIMS</td>
			<td style="width:119px;">ATS-3</td>
			<td>http://animalid.com/lab-animal-identification-systems/ats-3-general-rodent-tattoo-system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Lead Shielding Apparatus</td>
			<td style="width:135px;">(custom made)</td>
			<td style="width:119px;"></td>
			<td style="width:591px;">One-inch thick lead shielding arranged as two boxes with a one-inch thick gap between them for targeted irradiation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Plexiglass shielding container</td>
			<td style="width:135px;">(custom made)</td>
			<td style="width:119px;"></td>
			<td style="width:591px;">Plexiglass container filled with styrofoam. Styrofoam cutouts hold the lead shielding in place.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GammaCell 40 Exactor</td>
			<td style="width:135px;">Best Theratronics</td>
			<td style="width:119px;"></td>
			<td>http://www.theratronics.ca/product_gamma40.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">RAD Disk Ultra</td>
			<td style="width:135px;">Best Theratronics</td>
			<td style="width:119px;"></td>
			<td>http://www.theratronics.ca/product_rad.html</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouse Single Activity Wheel Chamber</td>
			<td style="width:135px;">Lafayette Instrument Company</td>
			<td style="width:119px;">#80820</td>
			<td>http://www.lafayetteneuroscience.com/product_detail.asp?itemid=980</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Activity Wheel Counter for Computer Monitoring</td>
			<td style="width:135px;">Lafayette Instrument Company</td>
			<td style="width:119px;">#86061</td>
			<td>http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1052</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Modular Cable for Wheel Counters</td>
			<td style="width:135px;">Lafayette Instrument Company</td>
			<td style="width:119px;">#86051-7</td>
			<td>http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1046</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">USB Computer Interface for Activity Wheel Counters</td>
			<td style="width:135px;">Lafayette Instrument Company</td>
			<td style="width:119px;">#86056A</td>
			<td>http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1047</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Activity Wheel Monitor Software</td>
			<td style="width:135px;">Lafayette Instrument Company</td>
			<td style="width:119px;">#86065</td>
			<td>http://www.lafayetteneuroscience.com/product_detail.asp?itemid=1053</td>
		</tr>
	</tbody>
</table>

a mouse model of fatigue induced by peripheral irradiation

affaticamento cancro-correlata (CRF) è una condizione dolorosa e costosa, che spesso colpisce i pazienti che ricevono trattamenti contro il cancro, tra cui la radioterapia. Qui si descrive un metodo che utilizza irraggiamento periferico mirati per indurre comportamento a fatica, come nei topi. Con adeguata schermatura, l&#39;irraggiamento si rivolge la regione inferiore addominale / pelvica del mouse, risparmiando il cervello, nel tentativo di modellare il trattamento di radiazione ricevuta dagli individui con tumori pelvici. Forniamo una dose di irradiazione che è sufficiente per indurre comportamento a fatica, come nei topi, misurati con l&#39;attività ruota corsa volontaria (VWRA), senza provocare evidenti morbilità. Dal momento che ruota in esecuzione è un normale, comportamento volontario nei topi, il suo uso dovrebbe avere scarso effetto di confusione su altri test comportamentali o misure biologiche. Quindi, ruota in esecuzione può essere usato come misura di esito realizzabile nella comprensione dei correlati comportamentali e biologiche di fatica. CRF è una condizione complessa con frequenti comorbidità, e probabilmente ha cause legate sia al cancro e dei suoi vari trattamenti. I metodi descritti in questo documento sono utili per studiare variazioni indotte da radiazioni che contribuiscono allo sviluppo di CRF e, più in generale, per esplorare le reti biologiche che possono spiegare lo sviluppo e la persistenza di un comportamento perifericamente-triggered ma centralmente guidato come affaticamento .

Tre gruppi di topi sono stati eseguiti con il protocollo descritto sopra. Ci sono stati un totale di 16 sham e 20 irradiate (2.400 cGy, 3 x 800 / giorno cGy) topi. Dopo tre giorni consecutivi di irradiazione, il gruppo irradiato ha mostrato significativamente ridotto rispetto al VWRA farsa (misti misure ripetute ANOVA: effetto principale del trattamento di irradiazione, F 1,13 = 19,233, p &lt;0.001). L&#39;effetto è significativo per i primi sette giorni dopo l&#39;irraggiame...

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A Mouse Model of Fatigue Induced by Peripheral Irradiation

Descriviamo un metodo che utilizza irraggiamento periferico mirati per indurre comportamento a fatica, come nei topi. La dose di irradiazione non letale selezionati porta ad una riduzione di una settimana in attività di ruote in esecuzione volontaria.

Un modello murino di fatica indotta da irradiazione Peripheral

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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JoVE Journal

Cancer Research

7.8K Views.  National Institutes of Health.  Descriviamo un metodo che utilizza irraggiamento periferico mirati per indurre comportamento a fatica, come nei topi. La dose di irradiazione non letale selezionati porta ad una riduzione di una settimana in attività di ruote in esecuzione volontaria. 

Video: A Mouse Model of Fatigue Induced by Peripheral Irradiation

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Fluorescente ortotopico modello murino di cancro al pancreas

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

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Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Trasduzione-trapianto modello murino di neoplasia mieloproliferativa

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Isolamento delle cellule tumorali circolanti in un modello di topo ortotopico del tumore del colon-retto

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Un modello di mouse geneticamente modificato del cancro colorettale sporadico

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

Attecchimento del tumore in un modello murino di xenotrapianto di umano linfoma a cellule mantellari

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Sviluppo di un modello preclinico umano di osteoclastogenesi dai monociti del sangue periferico co-coltivati con linee cellulari di cancro al seno

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

PET e MRI guidate irradiazione di un modello del ratto di Glioblastoma utilizzando un Micro-irradiatore

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Valutare il ruolo della funzione mitocondriale in affaticamento legato al cancro

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Endpoint importanti e marcatori proliferativi per valutare piccole lesioni intestinali e adattamento utilizzando un modello murino di mucosite indotta dalla chemioterapia

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Un modello di topo ortotopico oncogenico indotto dall'epatocito del cancro epatocellulare in aumento nell'impostazione dell'infiammazione epatica e della fibrosi

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...