Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

Quattro a sei settimane di età femminile C57BL / topi CJ (Harlan, Gerusalemme, Israele) sono stati utilizzati nell&#39;esperimento secondo l&#39;Associazione per la valutazione e l&#39;accreditamento delle specifiche Laboratory Animal Care. Tutte le procedure sperimentali sono state fatte secondo Istituzionale cura degli animali e del Comitato uso e il Dipartimento di Agricoltura regolamenti. 1. La raccolta del midollo spinale <ol><li> Euthanize il mouse utilizzando una camera di CO 2. Evitare dislocazione cervicale in quanto potrebbe causare danni alle radici gangliari a causa di forze di taglio. Da qui in poi, eseguire tutte le operazioni in condizioni sterili. </li><li> Bagnare animale verso il basso con il 70% di etanolo. Questo passaggio è importante per la sterilizzazione e per prevenire i capelli da trascinare attraverso gli organi interni. </li><li> Lasciare il mouse per asciugare da etanolo. Evitare qualsiasi contatto di etanolo con gli organi interni a causa di neurotossicità. </li><li> Dopo essiccamento del mouse, posizionate utilizzando perni in posizione prona(faccia in giù). Posizionare i perni su ogni degli arti posteriori e arti anteriori. </li><li> Utilizzando pinze, fare una incisione mediana che si estende cranio-caudale dal collo posteriore alla colonna lombare. Successivamente, sollevare lembi dermici bilateralmente con pinze ed esporre il tessuto sottocutaneo. </li><li> Palpare cranio del mouse fino al bivio craniocervicale. Utilizzando le forbici perpendicolari per l&#39;animale, tagliare i muscoli cervicali e della colonna vertebrale a livello della giunzione cranio-cervicale (il 7 ° vertebre cervicali), con pinze pesanti. Sezionare la colonna vertebrale caudale e accise della colonna vertebrale con una pinza pesanti fino a raggiungere il livello lombare (posteriori livello degli arti). </li><li> Eseguire un transezione caudale completa a livello della colonna lombare (la 5 ° vertebra lombare) con pinze pesanti. </li><li> Arrotolare il mouse sopra (faccia in su). Non vi è alcuna necessità di coprire l&#39;incisione dal DRG viene estratto dai livelli vertebrali inferiori e non cervicale. </li><li> Tagliare sulla linea mediana dal collo all&#39;addome, ritrarre ilpelle lateralmente e poi tagliato per aprire il peritoneo. </li><li> Rimuovere gli organi interni all&#39;interno del peritoneo (fegato, milza, pancreas, stomaco e intestino) in blocco. Quindi, rimuovere gli organi retroperitoneali (vale a dire, i reni e pancreas). Cranialmente, aprire la parete toracica. </li><li> Utilizzando pinze e una lama chirurgica, tagliare le nervature, lasciando 5 mm di costole bilateralmente distanza dalla colonna vertebrale. A questo punto, la colonna vertebrale è separata dal resto del corpo. </li><li> Lavare la colonna vertebrale, dal sangue freddo (4 ° C) fosfato fresco salina tamponata (PBS) due volte. </li></ol> 2. Isolare la gangli dorsali (DRG) <ol><li> Utilizzare uno stereomicroscopio con 4X ingrandimento. </li><li> Posizionare la colonna vertebrale su una piattaforma assorbente non non aderente (Telfa / nylon). Posizionare la faccia colonna vertebrale fino allo stesso orientamento cranio-caudale. </li><li> Rimuovere tutti i muscoli della colonna vertebrale e del tessuto connettivo. Utilizzare le costole come punto di riferimento per i nervi, come hannolasciare la colonna vertebrale. Costole anche proteggono i nervi dagli strumenti chirurgici. Il DRG si trova alle cervicali, toraciche e lombari siti delle vertebre. </li><li> Successivamente, utilizzando forbici tagliare il corpo vertebrale sulla linea mediana ed espongono il midollo spinale e le radici DRG delicatamente retrazione laterale del corpo vertebrale. </li><li> Nello spazio intercostale, seguire il nervo periferico medialmente lungo il laterale costola al DRG. </li><li> Identificare il DRG. Sembra che il tuorlo d&#39; &#39;uovo fritto&#39; sdraiato sul nervo. </li><li> Tagliare il distale nervi intercostali al DRG lasciando 2-3 mm di distale nervo gangli, da utilizzare per la retrazione. Questa &quot;coda&quot; sarà rimosso dopo la raccolta. </li><li> Afferrare il nervo con pinze. Stringendo il corpo DRG stesso causerà danni alle cellule neurali e deve essere evitato. </li><li> Avvicinatevi al DRG prossimalmente lungo il suo attaccamento al midollo spinale, attraverso il suo anteriore e radici posteriori. </li><li> Applicare delicata retrazione al DRG tirando la efferenti nervo (nervo intercostale moncone) lateralmente, e tagliare l&#39;anteriore e radici posteriori vicino al DRG. </li><li> Mantenere il DRG in una Petri piastra da 35 mm riempita fresco, ghiaccio DMEM freddo supplementato con 10% di siero fetale di vitello (FCS), 1% di penicillina-streptomicina, aminoacidi non essenziali 1% e 1% di sodio piruvato. </li></ol> 3. L&#39;impianto nella capsula di Petri <ol><li> Eseguire i prossimi passi sul ghiaccio con punte pre-raffreddato pipette per evitare ECM solidificazione. </li><li> Sotto uno stereomicroscopio a 2-4x ingrandimento, mettere da 35 mm di vetro di Petri fondo piatto su una griglia di carta. IMPORTANTE: Lavorare sul ghiaccio al fine di mantenere l&#39;ECM allo stato liquido. Creare un punto diametro di 1 mm (circa 20 ml) di fattore di crescita impoverito ECM al centro della griglia. </li><li> Sotto visualizzazione diretta, posizionare il DRG al centro dello spot ECM vicino al fondo del piatto. Nota: Le cellule tumorali utilizzati in questo protocollo sono murini cellule tumorali pancreatiche (KPC) ha istituito ilhed da campioni tumorali appena isolate da topi KPC come descritto altrove 19. </li><li> Celle di raccolta 40.000 tumorali provenienti da culture confluenti, lavarli una volta con PBS, e li risospendere in 40 ml ECM sul ghiaccio. </li><li> Sotto la visualizzazione diretta della griglia utilizzando un micron misura stereomicroscopio 500 in ogni direzione dalla DRG. A questo punto seme lentamente 10.000 cellule / ml 10 ECM. Utilizzare una punta preraffreddato 2-10 ml per iniettare le cellule vicino al fondo piatto, evitando la loro diffusione nella matrice o distacco della matrice. (Figura 1). </li><li> Lasciare il piatto all&#39;interno di una cappa a flusso laminare per 10-15 min a solidificare. Evitare ECM essiccazione. </li><li> Aggiungere lentamente DMEM, preparata come precedentemente accennato, contro la parete laterale della piastra. Aggiungere abbastanza medio per coprire l&#39;ECM (circa 2 ml). </li><li> Mettere il piatto di nuovo per l&#39;incubatore a 37 ° C. </li><li> Sostituire il mezzo con terreno fresco il giorno seguente. </li><li> Sostituire la media ogni 2 giorni. &lt;/ Li&gt; </ol> 4. Acquisizione dati, Time-lapse di videomicroscopia <ol><li> Il giorno 7 dopo l&#39;impianto, prendere il piatto per la microscopia lasso di tempo. Si noti che alcune cellule potrebbero richiedere l&#39;acquisizione in precedenza a causa della migrazione più veloce. </li><li> Durante l&#39;imaging dal vivo, la capsula in un ambiente chiuso ad una temperatura media di 37 ± 0,1 ° C e 5% CO 2. </li><li> Registrare le celle utilizzando un accoppiamento di carica fotocamera dispositivo posto sul microscopio. </li><li> Prendere immagini digitalizzate ogni 10 minuti per un massimo di 72 ore a seguire locomozione cella da tre a sei celle diverse. </li><li> Sostituire media dopo 24 ore. </li><li> Eseguire acquisizione e analisi dei dati semplice utilizzando il software microscopio. </li></ol> Analisi 5. I dati <ol><li> Per applicazioni più avanzate (ad esempio, il monitoraggio delle cellule in 2D o 3D) utilizzare software specializzati (come Imaris 4D). Nota: Questo software determina le coordinate XY di una cella aqualsiasi dato tempo, insieme con la distanza dall&#39;origine, e la velocità di movimento. </li><li> Calcolare la velocità media misurando il percorso fra posizioni successive in intervalli di 10 min. Calcolare la migrazione Index Forward (FMI), che è la distanza della parte anteriore cella dal neuriti e descrive il movimento unidirezionale delle celle verso il DRG. </li></ol>

<ol>
	<li>Carter, R. L., Foster, C. S., Dinsdale, E. A., Pittam, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+spread+by+squamous+carcinomas+of+the+head+and+neck,+a+morphological+study+using+antiaxonal+and+antimyelin+monoclonal+antibodies.">Perineural spread by squamous carcinomas of the head and neck, a morphological study using antiaxonal and antimyelin monoclonal antibodies.</a> J Clin Pathol. 36, 269-275 (1983).</li><li>Beard, C. 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=Perineural+invasion+is+associated+with+increased+relapse+after+external+beam+radiotherapy+for+men+with+low-risk+prostate+cancer+and+may+be+a+marker+for+occult,+high-grade+cancer.">Perineural invasion is associated with increased relapse after external beam radiotherapy for men with low-risk prostate cancer and may be a marker for occult, high-grade cancer.</a> Int J Radiat Oncol Biol Phys. 58, 19-24 (2004).</li><li>Maru, N., Ohori, M., Kattan, M. W., Scardino, P. T., Wheeler, T. 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O., 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+neurotrophic+factor+artemin+promotes+pancreatic+cancer+invasion.">The neurotrophic factor artemin promotes pancreatic cancer invasion.</a> Ann Surg. 244, 274-281 (2006).</li><li>Takahashi, 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=Perineural+invasion+by+ductal+adenocarcinoma+of+the+pancreas.">Perineural invasion by ductal adenocarcinoma of the pancreas.</a> J Surg Oncol. 65, 164-170 (1997).</li><li>Zhu, 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=Nerve+growth+factor+expression+correlates+with+perineural+invasion+and+pain+in+human+pancreatic+cancer.">Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer.</a> J Clin Oncol. 17, 2419-2428 (1999).</li><li>Hirai, I., 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=Perineural+invasion+in+pancreatic+cancer.">Perineural invasion in pancreatic cancer.</a> Pancreas. 24, 15-25 (2005).</li><li>Mitchem, J. B., 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=Targeting+tumor-infiltrating+macrophages+decreases+tumor-initiating+cells,+relieves+immunosuppression,+and+improves+chemotherapeutic+responses.">Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses.</a> Cancer Res. 73, 1128-1141 (2013).</li><li>Kelly, K., 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=Attenuated+multimutated+herpes+simplex+virus-1+effectively+treats+prostate+carcinomas+with+neural+invasion+while+preserving+nerve+function.">Attenuated multimutated herpes simplex virus-1 effectively treats prostate carcinomas with neural invasion while preserving nerve function.</a> FASEB J. 22, 1839-1848 (2008).</li><li>Dai, 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=Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer,+an+in+vitro+approach.">Enhanced survival in perineural invasion of pancreatic cancer, an in vitro approach.</a> Hum Pathol. 38, 299-307 (2007).</li><li>Ayala, G. E., 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=Cancer-related+axonogenesis+and+neurogenesis+in+prostate+cancer.">Cancer-related axonogenesis and neurogenesis in prostate cancer.</a> Clin Cancer Res. 14, 7593-7603 (2008).</li><li>Ayala, G. E., 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=Stromal+antiapoptotic+paracrine+loop+in+perineural+invasion+of+prostatic+carcinoma.">Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.</a> Cancer Res. 66, 5159-5164 (2006).</li><li>Ceyhan, G. O., 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=Neural+invasion+in+pancreatic+cancer,+a+mutual+tropism+between+neurons+and+cancer+cells.">Neural invasion in pancreatic cancer, a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374, 442-447 (2008).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11, 695-707 (2011).</li><li>Ketterer, K., 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=Reverse+transcription-PCR+analysis+of+laser-captured+cells+points+to+potential+paracrine+and+autocrine+actions+of+neurotrophins+in+pancreatic+cancer.">Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer.</a> Clin Cancer Res. 9, 5127-5136 (2003).</li><li>Gil, 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=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13, 6479-6485 (2007).</li><li>Gil, 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102, 107-118 (2010).</li><li>Weizman, 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=Macrophages+mediate+gemcitabine+resistance+of+pancreatic+adenocarcinoma+by+upregulating+cytidinedeaminase.">Macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidinedeaminase.</a> Oncogene. 33, 3812-3819 (2014).</li></ol>

This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

Questo articolo presenta un modello in vitro che ricapitola il microambiente tumorale nella nicchia neurale, il modello DRG. Il video mostra tutte le fasi a partire da riconoscere punti di riferimento anatomici come il DRG nel topo, la sua estrazione, e, infine, la sua coltura in ECM. è presentato anche co-coltura DRG a fianco con le cellule tumorali. Non ci sono altri modelli in vitro per la ricerca invasione perineurale descritti in letteratura rendendo questo modello indispensabile per studiare la ...

tumori solidi diffondere in tre modi principali: invasione diretta, diffusione linfatica e diffusione hematogenic. Tuttavia, c&#39;è un quarto mezzo di diffusione del cancro che viene spesso ignorata, diffusione lungo i nervi. Cancerose invasione neurale (CNI) è un percorso ben noto di diffusione del cancro, in particolare nei tumori della testa e del collo, della prostata 1 2, 3 e del pancreas. 4-8 CNI si verifica in più del 80% degli individui con adenocarcinoma del pancreas, portando per tumore retroperitoneale diffondersi attraverso i nervi celiaca gangliari. Queste cellule tumorali neurotropic hanno una capacità unica di migrare unidirezionalmente lungo i nervi verso il sistema nervoso centrale (CNS). 9 Questa scoperta suggerisce che il microambiente perineurale può essere sfruttato da cellule tumorali, fornendo fattori che favoriscono la crescita maligna. Uno dei pochi modelli in vitro per la ricerca CNI è gangli delle radici dorsali (DRG) / modello di cellula tumorale. Questo modEL è spesso usato per studiare l&#39;interazione paracrino tra cellule tumorali stroma e neurali. 10-18 In questo modello, mouse o linee cellulari tumorali umane sono cresciuti in matrice extracellulare (ECM) adiacente a preparazioni di DRG colta appena dissociato. In questo articolo il video mostra l&#39;applicazione in vitro CNI in adenocarcinoma del dotto pancreatico.

<table><tbody><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Operating microscope</td><td>Leica</td><td>M205</td><td></td></tr><tr><td>Time Lapse System</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Forceps</td><td>Sigma-Aldrich</td><td>F4142&amp;nbsp;</td><td></td></tr><tr><td>Surgical blade</td><td>Sigma-Aldrich</td><td>Z309036</td><td></td></tr><tr><td>Scissors</td><td>Sigma-Aldrich</td><td>S3271</td><td></td></tr><tr><td>35 mm Petri dishes, glass bottom</td><td>de groot</td><td>60-627860</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Sigma</td><td></td><td></td></tr><tr><td>Cold PBS</td><td>Biological industries</td><td>02-023-1A</td><td></td></tr><tr><td>DMEM</td><td>Biological industries</td><td>01-055-1A</td><td></td></tr><tr><td>FCS</td><td>Rhenium</td><td>10108165</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Biological industries</td><td>01-031-1B</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Biological industries</td><td>03-042-1B</td><td></td></tr><tr><td>L-Glutamine</td><td>Biological industries</td><td>03-020-1B</td><td></td></tr><tr><td>Growth factor-depleted matrigel</td><td>Trevigen</td><td>3433-005-01</td><td></td></tr></tbody></table>

in vitro modeling of cancerous neural invasion: the dorsal root ganglion model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

<html> Utilizzando immagini di microscopia video, il DRG può essere visto germogliare neuriti 5-7 giorni dopo l&#39;impianto, mentre le cellule tumorali migrare lontano dalle loro colonie verso il DRG. Con il 7 ° giorno dopo l&#39;impianto, le cellule tumorali vengono a contatto con neuriti (Figura 2). L&#39;indice di migrazione Forward delle cellule tumorali pancreatiche utilizzati nel protocollo ...

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modellazione di cancerose Neural Invasion: La dorsale della radice Ganglio Modello

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

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Research

JoVE Journal

Neuroscience

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

9.0K Views.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

Video: In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Un modello per perineurale Invasion in testa e del collo carcinoma a cellule squamose

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Ricerca

Ricerca sul cancro

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

Un modello In Vivo murino nervo sciatico di invasione perineurale

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Neuroscienze

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Il modello Chick Chorioallantoic Membrane In Vivo per valutare l'invasione perineurale nel cancro alla testa e al collo

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Medicina

Modello avanzato in vitro per lo studio dell'ipersensibilità neuronale nelle colture DRG

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Autore in primo piano: Modelli in vitro e in vivo per migliorare l'efficacia del trattamento del dolore cronico

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

Raccolta, inclusione e coltura dei gangli della radice dorsale in dispositivi multicompartimentali per studiare le caratteristiche neuronali periferiche

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioingegneria

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

Source: Huyett, P. et al. <a href="https://www.jove.com/video/55043" target="_blank">A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the procedure for developing an in vitro perineural cancer cell invasion model by co-culturing mouse dorsal root ganglion and neurotropic cancer cells. The model can be utilized to conduct cancer assays, producing rapid and reproducible results.

Definizione di un modello di co-coltura tridimensionale dei gangli della radice dorsale murina e delle cellule tumorali: un modello in vitro per studiare l'invasione delle cellule tumorali perineurali

Source: Huyett, P. et al. A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (2017)
In this video, we demonstrate the ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

Murine Perineural Invasion Model: Generating an In Vivo Model to Simulate Perineural Invasion in Mouse Sciatic Nerve

Source: Deborde, S. et al. <a href="https://www.jove.com/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse model for perineural invasion by injecting pancreatic cancer cells into the sciatic nerve. This model helps quantify the extent of invasion and study the interactions between cancer cells and nerves.

Modello di invasione perineurale murina: generazione di un modello in vivo per simulare l'invasione perineurale nel nervo sciatico di topo

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse ...

Cancers of the Nervous System

In vivo studies

Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

Source: Na&#39;ara, S. et al. <a href="https://www.jove.com/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an in vitro method for co-culturing DRG and cancer cells to study neurotropism and neural invasion.

Modellazione dell'invasione neurale cancerosa in vitro: un metodo per la co-coltura dei gangli della radice dorsale e delle cellule tumorali

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

In Vitro Modellazione di cancerose Neural Invasion: La dorsale della radice Ganglio Modello

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un modello per perineurale Invasion in testa e del collo carcinoma a cellule squamose

Un modello In Vivo murino nervo sciatico di invasione perineurale

Radice dorsale Ganglion iniezione e dorsale radice Lesioni Crush come un modello per sensoriale Axon Regeneration

Il modello Chick Chorioallantoic Membrane In Vivo per valutare l'invasione perineurale nel cancro alla testa e al collo

Raccolta, inclusione e coltura dei gangli della radice dorsale in dispositivi multicompartimentali per studiare le caratteristiche neuronali periferiche

Raccolta, inclusione e coltura dei gangli della radice dorsale in dispositivi multicompartimentali per studiare le caratteristiche neuronali periferiche

Raccolta, inclusione e coltura dei gangli della radice dorsale in dispositivi multicompartimentali per studiare le caratteristiche neuronali periferiche

Definizione di un modello di co-coltura tridimensionale dei gangli della radice dorsale murina e delle cellule tumorali: un modello in vitro per studiare l'invasione delle cellule tumorali perineurali

Modello di invasione perineurale murina: generazione di un modello in vivo per simulare l'invasione perineurale nel nervo sciatico di topo

Modellazione dell'invasione neurale cancerosa in vitro: un metodo per la co-coltura dei gangli della radice dorsale e delle cellule tumorali