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

Yazarlar personeli sitometresi tesisi (Cytocell) ve Üniversite Hastanesi hayvan tesis (UTE) Nantes hayvan yetiştirme ve bakım, hücresel ve tissular çekirdek tesis Nantes Üniversitesi (MicroPICell) görüntüleme için görüntüleme için Nantes teşekkür uzman teknik yardım. Bu eser INSERM, CNRS, Université de Nantes, Institut National du kanser (Inca #PLBio2014-155), Ligue Nationale contre le kanser (AO Bölgelerarası 2017) ve Avrupa konsorsiyumu dönemi-Net Transcan2 tarafından finanse edildi (Immunoglio). Takım Fondation pour la Recherche Medicale (DEQ20170839118) tarafından finanse edilmektedir. Bu eser IGO LabEX bağlamında gerçekleşmiştir ve IHU Cesti programları, desteklenen Ulusal Araştırma Ajansı Investissements d'Avenir yolu ile tarafından programları ANR-11-LABX-0016-01 ve ANR-10-IBHU-005, anılan sıraya göre. IHU Cesti projesi de Nantes Metropole ve Pays de la Loire bölgesi tarafından desteklenmektedir. Yazarlar Chirine Rafia el yazması düzeltmek yardım sağlamak için teşekkür ederiz.

Aşağıdaki yordam içeren hayvan konular (anlaşma #00186.02; Pays de la Loire [Fransa] bölgesel Etik Komitesi) Kurumsal kurallarına göre gerçekleştirilen. İnsan PBMCs bilinçli sağlıklı bağış (Etablissement Français du Sang Nantes, Fransa) toplanan kan gelen izole edildi. Tüm adımlar steril koşullarda gerçekleştirilir.
1. non-spesifik sitotoksik efektör T lenfositler genişlemesi
<ol><li>Hazırlamak ve besleyici hücreler, 35 Gy ışınlatayım. 2 x 105 - 4 x...

<ol>
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Bir evlat edinen transferini yerli seçilen veya mühendislik bağışıklık efektör hücreleri temsil eden verimli tümör hücreleri dönüştürülmüş15karşı ayrıntıları sınırlayıcı dikkat çekici infiltrative beyin kanser gibi tedavisi için umut verici bir yaklaşım, 16,17,18. Ancak, beyin oluşur, merkezi sinir sistemi, kan - beyin bariyerini varlığını ve klasik lenfati...

(Kim IV tümörü grade) GBM erişkinlerde en sık ve agresif birincil beyin kanseri var. Cerrahi ve radyo-kemoterapi birleştirir agresif tedaviler rağmen GBM son derece kötü prognoz (medyan sağkalım 14.6 ay ve % 2-yılı-ölüm > 73)1ile ilişkili olarak kalır. Bu kaç etkili tedavi gelişmeler üzerinde son on yılda2doğrulandıktan kanıtlamaktadır. Daha etkili tedavi stratejileri3,4,5tasarımı için adaylar arasında immunotherapies6 Şu anda incelenmiştir izlemek ve son derece invaziv ve radyo/kemoterapi dayanıklı tümör ortadan kaldırmak için hücreleri, hızlı ve ölümcül tümörün nüks7anahtar katkılarından dolayı şüpheli. Çeşitli potansiyel immünolojik hedefleri belirlendi ve için önerilen immunotherapies, doğal içeren veya değiştirilmiş αβ veya ϒδ T lenfositler GBM özgü tümör antijenleri veya stres kaynaklı molekülleri8,9gibi olduğunu, 10. Seçili GBM reaktif bağışıklık hücre effectors yönetmek için olasılık efektör lenfositler yüksek miktarlarda kalıntı malignite siteye doğrudan sunmak için eşsiz bir fırsat temsil eder. Bu strateji desteklemek için biz son zamanlarda orthotopic birincil insan GBM xenografts sadakatle taşıyan immünyetmezligi fareler üzerinde temel modelleri beyin tümörleri GBM hastaların9,11' deki gelişimi özetlemek göstermiştir. Ayrıca, bu modeller adoptively transfer edilen allojeneik insan Vϒ9Vδ2T lenfosit güçlü antitümör etkinliğini göstermek için kullanılmıştır.
Bu iletişim kuralı GBM, allojeneik T lenfositler evlat edinen transferinde dayalı gibi beyin tümörlerinin stereotaktik immunotherapies ulaşmak için kritik deneysel adımları açıklar. Makale gösterir: (i) gibi insan Vϒ9Vδ2T lenfosit; tedavi allojeneik bağışıklık efektör T lenfositler amplifikasyon (ii) enjeksiyon için bu efektör T lenfositler hazırlanması; (iii) içinde beyin tümörü yakınındaki stereotaksik yönetim için yordam. Bu makalede ayrıca bu hücresel effectors davranışını içgörü sonra stereotaksik enjeksiyon sağlar.
Burada sunulan tedavi yaklaşımı 20 x 106 efektör hücreleri başına doz her beyin tümörü taşıyan immünyetmezligi fare için enjeksiyon temel alır. Bağışıklık hücreleri büyük miktarlarda üretmek için bir ilk vitro genişleme adım gereklidir. Bu nedenle, belirsiz hücre açılımları phytohemagglutinin (PHA-L) kullanarak gerçekleştirilir ve allojenik besleyici hücreler ışınlanmış: periferik kan mononükleer hücreler (PBMCs) sağlıklı bağış ve Epstein Barr virüsü EBV dönüştürülmüş B-lymphoblastoid hücre vitro enfeksiyon EBV içeren kültür Marmoset B95-8 hücre satırından 1 µg/mL cyclosporin-A. huzurunda süpernatant ile PBMCs elde satırları (BLCLs),
GBM reaktif efektör bağışıklık hücreleri karşılaştırılır ve vitro deneyleri9' dan seçilmiş. Bu efektör hücreler aktif hale gelir ve standart iletişim kuralları kullanarak kendi doğası göre (Örneğin, insan Vγ9Vδ2 T lenfositler9 veya insan anti-herpes virüsü αβ T lenfositler12) > %80, rutin olarak en az bir saflığı ile güçlendirilmiş sitometrik analiz tarafından kontrol. Aşağıda verilen hücre genişletme yordam çeşitli insan lenfosit alt kümeleri için geçerlidir.

<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

Glioblastoma multiforme (GBM), Yetişkin, en sık ve agresif birincil beyin kanseri genellikle kötü prognoz ile ilişkilidir ve kıt etkili tedaviler son on yılda önerilmiştir. Roman tedavi edici stratejiler tasarlamak için umut verici adaylar arasında hücresel immunotherapies son derece invaziv ortadan kaldırmak için hedef almış ve kemoterapi-olup tümör hücreleri, büyük olasılıkla bu kanser bir hızlı ve ölümcül nüks dahil. Böylece, tümör çevresinde insan Vϒ9Vδ2 T lenfositler gibi allojeneik GBM reaktif bağışıklık hücre effectors administration(s) verimli sunmak için eşsiz bir fırsat temsil eder ve son derece konsantre doğrudan içine terapötik ajanlar beyin maligniteler sitesi. Burada, orthotopic insan birincil beyin tümörü taşıyan immünyetmezligi farelerde hazırlık ve allojenik insan lenfosit stereotaksik yönetim için bir iletişim kuralı mevcut. Bu çalışmada fizibilite ve allojenik insan lenfosit intrabrain tümör yataklar içinde stereotaksik enjeksiyonları itimat bu hücresel immunotherapies antitümör etkinliğini preklinik bir kanıtı-of-concept sağlar.

Bu çalışma içinde beyin tümörü taşıyan fareler stereotaksik enjeksiyonları tümör yatağın içinde gerçekleştirilen temel hücresel bağışıklık efektör hücrelerin evlat edinen transferlerin strateji açıklar.
Herhangi bir büyük enjeksiyon birimle ilişkili beyin hasarı riskini en aza indirmek için efektör hücre süspansiyon (15-20 µL PBS, 20 x 106 hücreler) konsantre gerekir. Bu hücre top...

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

Orthotopic insan Glioblastoma Multiforme Xenografts fare modelleri sitotoksik bağışıklık hücrelerinin stereotaksik evlat edinen Transfer

Burada, orthotopic insan birincil beyin tümörü taşıyan immünyetmezligi farelerde hazırlık ve allojenik insan lenfosit stereotaksik yönetim için bir iletişim kuralı mevcut. Bu çalışmada bir kanıtı-of-concept fizibilite ve hücresel immunotherapies intrabrain teslim antitümör etkinliği için sağlar.

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

Deneysel metastaz ve CTL Kabul edilmiş Transfer İmmünoterapi Fare Modeli

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

İmmünoloji ve Enfeksiyon

Seven Steps to Stellate Cells

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

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

Kabul edilmiş Transfer ve antijenik Challenge Deneysel Otoimmün Encephalomyelitis (EAE) Dayanıklı Fare Suşlarında yanıtsızlık Aşmak

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

Diyabetojenik CD4 Kabul edilmiş Transfer ile Farelerde Hızlandırılmış Tip 1 Diyabet İndüksiyon + T Hücreleri

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

Hematopoetik progenitörlerin Evlatlık Transferi kullanarak Peter Parçacıklannda Murin plazmasitoid dendritik hücreleri Gelişimini değerlendirilmesi

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

Bakım Glioblastoma Standardı Kapsamında Adoptif Tedavisi için CAR T hücrelerinin üretilmesi

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

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

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