Name: stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts
Uploaded: 2018-09-01T13:00:00
Description: Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Gli autori ringraziano il personale dell'ospedale universitario animale impianto (UTE) di Nantes per zootecnia e cura, il cellulare e tessutale, funzione di memoria dell'Università di Nantes (MicroPICell) di imaging per l'imaging e l'impianto di citometria (Cytocell) da Nantes per la loro assistenza tecnica. Questo lavoro è stato finanziato dalla INSERM, CNRS, Université de Nantes, Institut National du Cancer (INCa n #PLBio2014-155), Ligue Nationale contre le Cancer (AO interregionale 2017) e il Consorzio europeo Transcan2 di ERA-Net (Immunoglio). La squadra è finanziata dalla Fondation pour la Recherche Medicale (DEQ20170839118). Questo lavoro è stato realizzato nel contesto di LabEX IGO e i programmi di IHU-Cesti, supportato con la nazionale Ricerca agenzia Investissements d'Avenir tramite i programmi ANR-11-LABX-0016-01 e ANR-10-IBHU-005, rispettivamente. Il progetto di IHU-Cesti è anche supportato da Nantes Métropole e della regione Pays de la Loire. Gli autori ringraziano Chirine Rafia per fornire aiuto nella correzione del manoscritto.

Il seguente procedura che coinvolge animali soggetti è stato eseguito secondo le linee guida istituzionali (contratto n. 00186.02; comitato etico regionale della Pays de la Loire [Francia]). PBMCs umano sono stati isolati da sangue raccolto da donatori sani informati (Etablissement Français du Sang Nantes, Francia). Tutti i passaggi sono eseguiti in condizioni di sterilità.
1. non-specifico espansione dei linfociti T citotossici effettori
<ol><li>Preparare e irradiare le cellule di alimen...

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Un trasferimento adottivo di selezionato nativo o cellule effettrici immuni ingegnerizzati rappresenta un promettente approccio per trattare efficacemente i tumori, quali i tumori al cervello infiltrativo, avendo cura di limitare le reattività contro le cellule non trasformate15, 16,17,18. Tuttavia, il sistema nervoso centrale, che comprende il cervello, ha una particolare condizione immune, ...

GBM (che grado astrocitoma IV), è il più frequente e aggressivo cancro cerebrali primari negli adulti. Nonostante i trattamenti aggressivi che combinano la chirurgia e la radio-chemioterapia, GBM rimane associato con una prognosi estremamente infausta (sopravvivenza mediana di 14,6 mesi e un 2-anno-mortalità > 73%)1. Questo evidenzia che pochi progressi terapeutici efficienti siano stati convalidati sopra gli ultimi dieci anni2. Tra i candidati per la progettazione delle più efficaci strategie terapeutiche3,4,5, immunoterapie6 sono attualmente esplorati per tenere traccia ed eliminare altamente tumore invasivo e radio/chemio-resistenti cellule, ritenuto sospettate per il loro contributo chiave a tumore rapido e fatale ricaduta7. Vari potenziali bersagli immunologici sono stati individuati e proposti per immunoterapie, che coinvolgano naturale o modificate αβ o linfociti T ϒδ quali antigeni tumorali specifici GBM o indotta da stress molecole8,9, 10. La possibilità di amministrare gli GBM-reattiva effettori delle cellule immuni selezionati rappresenta un'opportunità unica per fornire elevate quantità di linfociti effettori direttamente nel sito di malignità residua. Per supportare questa strategia, abbiamo recentemente dimostrato che modelli basati su topi immunodeficienti xenotrapianti GBM umani primari di orthotopic fedelmente ricapitolano lo sviluppo dei tumori cerebrali in pazienti GBM9,11. Inoltre, questi modelli sono stati utilizzati per dimostrare l'efficacia antitumorale forte dei linfociti Vϒ9Vδ2T passivamente trasferiti umani allogenici.
Questo protocollo descrive la procedura sperimentale critica per raggiungere immunoterapie stereotactic dei tumori cerebrali, come GBM, basato sul trasferimento adottivo di linfociti T allogenici. Illustrato nell'articolo: (i) l'amplificazione dei linfociti terapeutici allogenici T effettori immuni, quali linfociti umani di Vϒ9Vδ2T; (ii) la preparazione di questi linfociti T effettori per iniezione; (iii) la procedura per l'amministrazione stereotactic all'interno del cervello, vicino al tumore. Questo articolo fornisce inoltre la comprensione del comportamento di questi effettori cellulari dopo iniezione stereotactic.
L'approccio terapeutico qui presentata si basa sull'iniezione di 20 x 106 cellule effettrici per dose per ogni mouse immunodeficienti di cervello del tumore-cuscinetto. Un passo di espansione iniziale in vitro è necessaria per produrre grandi quantità di cellule del sistema immunitario. Pertanto, espansioni delle cellule aspecifiche sono effettuati usando la fitoemagglutinina (PHA-L) e irradiato le cellule allogeniche alimentatore: cellule mononucleari sangue periferico (PBMCs) da donatori sani e delle cellule B-linfoblastoidi Epstein Barr Virus EBV-trasformati linee (BLCLs), derivati da PBMCs di infezione in vitro con EBV-contenente cultura surnatante dalla linea cellulare Marmoset B95-8, in presenza di 1 µ g/mL ciclosporina-A.
Le cellule immunitarie effettrici reattive GBM sono rispetto e selezionate da in vitro saggi9. Queste cellule effettrici sono attivate e amplificato mediante protocolli standard, secondo la loro natura (ad es., umano Vγ9Vδ2 T linfociti9 o umano anti-herpes virus αβ T linfociti12) con una purezza minima di > 80%, come ordinariamente controllato dall'analisi cytometric. La procedura di espansione delle cellule riportata di seguito si applica a varie sottopopolazioni di linfociti umani.

<table border="0" cellpadding="0" cellspacing="0" style="width:825px;" width="825">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PBMCs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:232px;">from 3 different healthy donors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">BLCLs</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td>from 3 different donors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">Roswell Park Memorial Institute medium (RPMI)</td>
			<td style="width:136px;">Gibco</td>
			<td>31870-025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:339px;">FCS</td>
			<td style="width:136px;">Dutscher</td>
			<td style="width:119px;">S1810-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-glutamine</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">25030-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">penicillin/streptomycin</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">IL-2</td>
			<td style="width:136px;">Novartis</td>
			<td style="width:119px;">proleukin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PHA-L</td>
			<td style="width:136px;">Sigma</td>
			<td>L4144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Stereotaxic frame</td>
			<td>Stoelting Co.</td>
			<td align="right">51600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mouse adaptator for stereotaxic frame&#160;&#160;</td>
			<td>Stoelting Co.</td>
			<td align="right">51624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microsyringe pump injector&#160;</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">UMP3-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoFil Syringe</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">NF34BV-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">NSG mice</td>
			<td style="width:136px;">Charles River</td>
			<td style="width:119px;">NSGSSFE07S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ketamine</td>
			<td style="width:136px;">Merial</td>
			<td style="width:119px;">Imalg&#232;ne 1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylazine</td>
			<td style="width:136px;">Bayer</td>
			<td style="width:119px;">Rompur 2%</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Scissors</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">201758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Forceps</td>
			<td style="width:136px;">WPI</td>
			<td style="width:119px;">501215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">OmniDrill 115/230V</td>
			<td style="width:136px;">WPI</td>
			<td align="right">503598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Vicryl 4-0</td>
			<td style="width:136px;">Ethicon</td>
			<td>VCP397H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Xylocaine</td>
			<td style="width:136px;">Astrazeneca</td>
			<td>3634461</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotactic adoptive transfer of cytotoxic immune cells in murine models of orthotopic human glioblastoma multiforme xenografts

Multiforme di glioblastoma (GBM), il cancro primario del cervello più frequenti e più aggressivo negli adulti, è generalmente associato con una prognosi difficile, e sono state proposte terapie efficienti scarse nell'ultimo decennio. Tra i candidati promettenti per la progettazione di nuove strategie terapeutiche, immunoterapie cellulari sono stati designati per eliminare altamente invasive e chemio-radioresistenti cellule del tumore, probabilmente coinvolti in una ricaduta rapida e mortale di questo cancro. Così, somministrazioni di trapianto allogeneic GBM-reattiva effettori delle cellule immuni quali linfociti T Vϒ9Vδ2 umani, in prossimità del tumore sarebbe rappresenta un'opportunità unica per fornire efficiente e altamente concentrato agenti terapeutici direttamente nella sito di tumori maligni del cervello. Qui, presentiamo un protocollo per la preparazione e la somministrazione stereotassica di linfociti umani allogenici in topi immunodeficienti portatori di tumori cerebrali primari umano ortotopico. Questo studio fornisce un proof-of-concept preclinici per sia la fattibilità e l'efficacia antitumorale di questi cellulari immunoterapie che si basano sulle iniezioni di stereotactic dei linfociti umani allogenici all'interno letti tumore intrabrain.

Questo studio descrive la strategia del trasferimento adottivo di cellule effettrici immuni cellulari all'interno del cervello di topi portatori di tumore, basato su stereotactic iniezioni eseguite direttamente all'interno della base del tumore.
Per ridurre al minimo qualsiasi rischio di ferita di cervello connessa con un volume di grande iniezione, la sospensione di cellule effettrici deve essere concentrato (20 x 106</sup...

Watch this Scientific Journal Video about Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts at JoVE.com

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Trasferimento adottivo stereotactic di citotossici cellule immunitarie nei modelli murini di xenotrapianti Multiforme di Glioblastoma umano ortotopico

Qui, presentiamo un protocollo per la preparazione e la somministrazione stereotassica di linfociti umani allogenici in topi immunodeficienti portatori di tumori cerebrali primari umano ortotopico. Questo studio fornisce un proof-of-concept per sia la fattibilità e l'efficacia antitumorale di immunoterapie cellulari intrabrain-consegnato.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Glioblastoma multiforme remains the most frequent and aggressive primary brain tumor in adults. Despite aggressive treatments based on surgery followed by radio and chemotherapy, GBM is associated with an extremely poor prognosis and a median survival lower than 15 months. Since few therapeutic advances have been made over the last decade, cellular immunotherapies are currently explored to eliminate highly-invasive and resistant GBM cells likely involved in rapid tumor relapse.administrations of selected GBM-reactive cytotoxic immuno factors in the vicinity of the tumor could represent a unique opportunity to deliver concentrated cellular immunotherapy, directly into the site of residual malignancy. Our group recently showed that immunodeficient NSG mice carrying orthotopic primary human GBM xenograft recapitulate GBM to more development in patients. These GBM models were used to evaluate the efficacy of cytotoxic immune effectors on intratumoral injections.This protocol describes our therapeutical process based on first the isolation and amplification of GBM-reactive immune effector cells. Second, the propagation of these effector cells for intercranial injection. And third, their stereotactic injection at the tumor site within the mouse brain.The behavior of the immune effector after orthotopic injection was also investigated. Three weeks after PHA fetus stimulation, effector cells, which form cell clump in the bottom of the flask should have returned to rest state and are ready to be used. After checking effector cell count, resuspend and collect cells in 115 milliliters tube and centrifuge them.Carefully and completely remove a supernatant. And resuspend cells in sterile PBS and centrifuge to wash them. Carefully and completely take off the supernatant.And then resuspend cells in one milliliter of sterile PBS. Transfer cells in a microtube for centrifugation. Again, carefully and completely take off the supernatant by pipetting slowly.The next step is critical, resuspend cells in half of the final volume of sterile PBS and homogenize softly and carefully. Verify the final volume with micropipette, final volume per mouse has to be between 15 and 20 microliter for 20 million cells. Be sure it will not exceed 20 microliter per mouse.Keep cells on ice until injection. Ensure that the animal is adequately anesthetized by a toe pinch and then remove fur from the surgical site, between the two ears and up to the nose. Resuspend cells slowly to prevent cell clumping and load cell suspension into syringe with great care to avoid the presence of bubbles.Then, place the syringe into the adapted syringe pump. Disinfect the surgical site with salt and povidone iodine 5%solution and place a lubricating ophthalmic ointment in the mouse's eyes to prevent drying of the cornea. Then, position the anesthetized mouse on the stereotactic frame on a warm block.Appropriately position mouse's nose and teeth above the tooth bar. Tighten ear bars firmly in the mouse ears to immobilize the head. Be careful not to damage the eardrums, compromise the respiration, and make sure that head is well immobilized.Make a midline sagittal skin incision with sterile scissors along the upper part of the cranium to expose the skull. Identify the intersection of the sagittal and coronal structures to serve as landmarks for stereotactic localization, and place the syringe above this point. Move the syringe at predetermined coordinates.Two millimeter right lateral, and 0.5 millimeter anterior of the Using a microdrill, make a small hole in the skull with sterile drill bit. Be careful to remain superficial in order to avoid brain injury. Insert syringe carefully into the drilled hole.Move slowly forward the needle three millimeter down in the dura, and then backward 0.5 millimeter to a final depth of 2.5 millimeter cells injection. Run the cell injection at two or three microliter per minute, and monitor the mice all along the injection time. Once injection is complete, withdraw the needle for only one millimeter and keep syringe in place for an additional minute before slowly withdrawing completely the syringe to prevent any leakage from the infusion site.Remove carefully the animal from the stereotactic frame. Close skin with appropriate surgical suture and apply 2%Lidocaine gel directly on the wound. Transfer anesthetized mouse to its respective cage above heating pad set to 37 degrees Celsius to maintain appropriate mouse body temperature until full recovery from anesthesia.As the suspension is very concentrated, the cell viability and reactivity were checked. Effector cells were loaded in syringe in order to mimic in vivo injection. Cells were collected immediately or 10 minutes later and were analyzed by flow cytometry for propidium iodide staining.As you can see, the preparation and the loading did not affect the viability of effector cells at 24 hours. The reactivity against Glioblastoma tumor cells were also checked using either unprepared cells or effectors prepared and stayed three hours on the eyes. Reactivity is monitored by positive staining of the activation marker CD107A.These results demonstrate that the effector cells'reactivity were not affected by the preparation. Seven days after effector cells'injection, mice brains were collected, sectioned, and stained for hematic coloration to allow the identification of tumor structure. Then anti-human CD3 staining allowed localization of effector cells.Interestingly, effector cells were found around the tumor and in the tumor core, but also in contralateral hemisphere. Human effector T-cells isolated from mouse brain 48 hours after the injection represent 2%of brain cells. They were still able to proliferate in presence of PHA, feeders and IL2 stimulation, with a 99%purity after amplification.Thanks to this protocol, resting human effector cells can survive and patrol with a mouse brain parenchyma for several days following the stereotaxic injection. In conclusion, adoptive transfer of human effector cells by stereotactic injections represent promising approaches to efficiently treat infiltrative malignant brain tumors with limited deleterious effects on healthy cells. This route of administration takes advantage that human cells are delivered closely to the tumor site, limiting their dilution within the tissues and the organism.Here we described the procedure following the delivering of a large amount of immune cells within the brain tumor without harmful effects on effector cells and, importantly, without any visible deleterious effect on mice. Moreover, locally-injected immune cells in mouse brain are not only able to survive and keep their ability to be activated and amplified ex vivo, but also to and eliminate disseminated tumor cells in the surrounding brain tissue. Collectively, these features are essential to allow the elimination of strong infiltrative tumor cells that characterize GBM tumors.This protocol opens new opportunity for the establishment of adoptive transfer procedure in mice model of brain tumor. Essential step before considering clinical CD.

Research

JoVE Journal

Immunology and Infection

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Metastasi sperimentale e CTL adottivi Immunoterapia modello di trasferimento del mouse

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Ricerca

Immunologia e infezioni

Seven Steps to Stellate Cells

Sette passi per Stellate Cellule

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Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Superare blocco nel encefalomielite autoimmune sperimentale (EAE) ceppi di topi resistenti dal trasferimento adottivo e uno stimolo antigenico

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Accelerated diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Valutare lo sviluppo delle cellule murine plasmocitoidi dendritiche in placche del Peyer Utilizzando trasferimento adottivo di ematopoietiche Progenitori

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Generazione di cellule CAR T per adottiva terapia nel contesto di Glioblastoma standard di cura

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...