Bu çalışmada, Vanderbilt-Ingram Kanser Merkezi Destek Hibe (P30 CA068485), Çocukluk Çağı Beyin Tümörü Vakfı ve Ulusal Sağlık Enstitüsü (NS042205) hibe ile desteklendi.

1. Tümör taşıyan Serebellum ve Tümör Doku ayrılma Micro-diseksiyonu <ol><li> Tümör dokusu alımı <ol><li> Medulloblastom taşıyan Hasta fareler genellikle runted ve hidrosefali ve posterior felç ve devrik zaman duruş yeniden yetmezliği de dahil olmak üzere tipik bir nörolojik semptomlar gösterilecek. Tümör dokusu almak için, karbon dioksit Solunduğunda fareler euthanize. Servikal dislokasyon, posterior kafatasına basınç oluşturur ve tümör dokusu bütünlüğünü tehlikeye düşürebilecek bir prosedür gerçekleştirmek için imporant. </li><li> Decapitation kafatası iyi bir görünüm için saç ve kas dokusu mümkün olduğunca ortadan kaldırarak, bir makas kullanarak ölümünden sonra hemen yapılır. Kafatası yüzeyi,% 95 etanol ile ıslatılmış Kimwipe ile temizleyin. </li><li> Ince makas, kafatasının orta hat boyunca bir açılış kesmek ve kafatası doku tümör taşıyan serebellum da dahil olmak üzere tüm beyin maruz kalmaktadır, bu noktada ince cımbız kullanarak kaldırmak için kullanın. </li><li> Sağlıklı yetişkinlerin cerebella iyi tanımlanmış hemisferlerin ve vermis görüntüler, Tümör taşıyan farelere cerebella genellikle düz bir yüzey ve göze çarpan kan damarları ile amorf, büyümüş. Steril teknikleri kullanarak, buz fosfat cımbız ve yer kullanarak serebellar tümör almak Mg 2 olmadan tamponlu salin (PBS) + ve Ca 2 +. </li></ol></li></ol> Not: tüm araçları kullanmadan önce otoklava% 95 etanol ve buhar dezenfekte edilmektedir. <ol start="2"><li> Tümör dokusu Fototerapisi <ol><li> 4 dakika boyunca oda sıcaklığında 3 dakika 37 inkübasyon tarafından ince makas, ° C ile doku kıyma, PBS den 4 kez tümör dokusu hacmi yaklaşık% 50 Accutase (PBS içinde seyreltilmesi) tümör dokusu transferi sonra doku ilave bir 3 dakika için 1 ml Pipetman tekrarlayan pipeting uğrar. Bu yöntem, tek hücre ve küçük hücresel agrega karışımı boyun eğmek zorundadır. </li><li> Hücresel süspansiyon PBS ve 1000xg pelet hücreleri için 5 dakika süreyle santrifüj ile 3 kat sulandırınız. (Yukarıdaki prosedürler vallahi basın, &quot;medulloblastom kök hücrelerin izolasyonu, zenginleştirme ve bakım&quot; olarak tarif edilmiştir) </li></ol></li></ol> Glutamin, Penisilin-Streptomisin, N2, B27, insan EGF (25 ng / ml) ve temel FGF (25 ng / ml) ile Neurobasal orta oluşan taze hazırlanmış sinir kök hücre orta hücre pelletini tekrar. Çözümün hücre yoğunluğu hesaplayın ve uygun daha fazla dilüsyonları bir ayrışmış tümör hücrelerinin uygun bir numarası, örneğin 5x10 5, steril bir konik uçlu 10μL şırınga içine 4 mcL çözüm yük ki bu tür ek sinir kök hücre orta yapmak . Inoküle hücreleri tam sayısı spesifik deneysel gereksinimlerine uygun olarak araştırmacı tarafından belirlenmelidir. Bir mini 200μL mikrosantrifüj tüp hücre çözümü (PCR tüp) buz üzerinde tutun. 2. Alıcı Fare Anestezik Prosedürleri Cerrahi işlem için, kemirgen cerrahi adanmış bir dezenfekte alan prosedürün süresi için gereklidir. İdeal olarak, hayvan hazırlanması, faaliyet alanı ve hayvan kurtarma için ayrı fakat bitişik alanları ile uzun bir tezgah kurdu. Cerrahi eldivenler, muayene eldivenleri, ameliyat için gerekli. Aseptik teknik, bel üzerinde eldivenli ellerini tutarak muhafaza ve sadece kuru steril bir yüzey steril nesneleri işlemek için kullanılır. <ol><li> Ketamin (fare ağırlığı 1 kg başına 100 mg ketamin) ve xylazine (fare ağırlığı 1 kg başına 10 mg ksilizin) oluşan bir karışım anestezi için kullanılır. Anestezikler intraperitoneal Yönetici. </li><li> Genellikle tam etkili olabilmesi için anestezikler için 3-5 dakika sürer. Bir farenin ayak / kuyruk tutam tam anestezi olup olmadığını belirleyin. Oftalmik merhem fare gözleri küçük bir miktar koyun. Sürekli fare normal nefes olduğundan emin olmak için izleme, işletim alanı için tam anestezi fare getirin. </li></ol> 3. Tümör Hücrelerinin Aşılama İntrakranial <ol><li> , Orta beyin ve beyincik yukarıdaki posterior kafatası ortaya çıkarmak için bir saç kesme makinesi kullanarak fare dorsal arka baş bölgesinde, tıraş. </li><li> Alkol ped ile silerek bir pamuk uçlu aplikatör kullanarak maruz kalan kafa derisi Betadine çözümü uygulayın. Bu iki sterilizasyon adımları iki kez tekrarlayın. </li><li> Steril bir neşter kullanılarak posterior kafa derisi çeyrek inç kesi olun. Matkap sağ 0,5 mm burr hole 2 mm ve 2 mm steril bir diş matkap kullanarak lambda posterior. </li><li> Fare çerçevesinde tutun üzerine kesiciler çengel stereotaktik çerçeve üzerine yerleştirin. Dikkatle ineceği ve çapak deliğe tümör hücre çözümü 4 mcL yüklü konik uçlu 10 mcL şırınga pozisyon. Syri kez eğimnge iğne kafatası yüzeyi altında, başka bir 3 mm alçalma ve daha sonra 0.5 mm yükselmek. Yavaşça alıcının beyincik, 30 saniyelik bir zaman dilimi içinde sabit bir güçle, tümör hücrelerinin istenilen miktarda enjekte. Onun yerine iğne, enjeksiyon tamamlanmasından sonra ek bir iki dakika bırakın. Daha sonra iğne ve şırınga çıkarın. AutoCLIP kullanarak yara Klip. </li><li> Kendi vücut ısısını korumaya yardımcı olmak için, ameliyat sonrası bir ısınma battaniye üzerinde fare tutun. Hareketlilik ve solunum desenler sürekli izlenecektir. Fare anestezi kurtarır sonra, steril bir konut kafesine geri koyun. Gıda ve su alımı, davranış, tımar ve gözleri kırmızı boyanması için günlük izleyin. Kesi yerinde enfeksiyon belirtileri için kontrol edin. Gün 7 cerrahi sonrası autoclips çıkarın. 2-6 ay ortalama fareler korumak ve ileri çalışmalar için tipik medulloblastom belirtileri gösteren bu fedakarlık. </li></ol> 4. Temsilcisi Sonuçlar İkincil medülloblastomlar alıcı farelerde geliştirilen yüksek birincil tümörlerin benziyordu. Cerebella ikincil tümörleri taşıyan tüm bağlar olarak bakıldığında genellikle belirgin ektopik kan damarları ile amorf, büyümüş. Ikincil tümör dokusu immünohistokimyasal analizleri, tümör hücrelerinin, açık, güçlü SmoM2 YFP ve Math1 ve Pax6 gibi serebellar granül nöron öncüleri, belirteçleri yüksek proliferatif olduğunu ortaya çıkardı. Ayrışmış ve kültür rapor yöntemleri kullanarak (&quot;İzolasyon, zenginleştirme ve medulloblastom kök hücreleri bakım&quot; tuşuna basın Jüpiter), ikincil medulloblastom tümör hücreleri hızla genişledi olabilir zaman, açık birden fazla kök hücre belirteçleri, clonogenic ve daha fazla tümör oluşumunu başlatabilir ek immün sistemi baskılanmış alıcıları naklediliyor. <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="Şekil 1" /> Şekil 1. Tipik tüm montaj ve ikincil medulloblastom doku kesit 5x10 5 primer tümör hücrelerinin enjeksiyonundan 26 gün sonra gelişen ikincil medulloblastom dokusu tipik bir örneği. Hematoksilen boyama yüksek sitoplazmik / nükleer oranı ve nükleer polimorfizm de dahil olmak üzere, tipik hücresel morfoloji medulloblastom ortaya koymaktadır. Ki67 ifade belirtildiği gibi bu ikincil tümör hücrelerinin yüksek proliferatif ve aynı zamanda güçlü bir membranöz SmoM2-YFP ifade. <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="Şekil 2" /> Şekil 2. İkincil medulloblastom hücrelerin in vitro kültürü ve genişletilebilir Birincil fare medulloblastom hücreleri (&quot;İzolasyon, zenginleştirme ve medulloblastom kök hücreleri bakım&quot; tuşuna basın Jüpiter) açıklanan protokol kullanarak, bu ikincil medulloblastom hücrelerinin kültürlü ve birden fazla geçit için genişletilmiş. Resim, 4 gün ve 14 gün kültüre aynı temsilcisi tümör koloni gösterir. Kültür tümör hücrelerini bipolar, uzun hücresel agrega yoğun bir çekirdek genellikle yetersizdir sitoplazma ve yayar. Bu büyüme temas inhibisyonu göstermezler. Küçük yuvarlak hücreler kırmızı kan hücreleri vardır.

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

Çıkar çatışması ilan etti.

Verim ikincil tümör oluşumu aşağıdaki gibidir allografting başarılı medulloblastom hücre sağlamak için birkaç kritik faktörler: Birincisi, sadece doku disosiasyon için% 50 Accutase kullanımı ve uzun süreli enzim tedavisi hücre canlılığını anlamlı bir azalma yol olarak doku üzerinde sindirimi yoktur. Ayrıca sadece 1 mL boyutu Pipetman küçük ipuçları da ayrışmış hücreleri fiziksel hasar oluşturabilir kullanın. Allografting için tek hücreler ve küçük agrega karışımı ince. İk...

<table><tbody><tr><td>Neurobasal medium</td><td>Invitrogen</td><td>21103049</td><td></td></tr><tr><td>hEGF</td><td>Invitrogen</td><td>PHG0311</td><td>25ng/ml</td></tr><tr><td>bFGF</td><td>Invitrogen</td><td>PHG0023</td><td>25ng/ml</td></tr><tr><td>N2</td><td>Invitrogen</td><td>17502048</td><td>1X</td></tr><tr><td>B27(-RA)</td><td>Invitrogen</td><td>12587010</td><td>1X</td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>A1110501</td><td>50% </td></tr><tr><td>Glutamine</td><td>Invitrogen</td><td>25030081</td><td>2mM</td></tr><tr><td>Stereotaxic instrument</td><td>Stoelting Co.</td><td>51730</td><td></td></tr><tr><td>Foredom flexible shaft drill, hang-up style</td><td>Stoelting Co.</td><td>58650</td><td></td></tr><tr><td>Large probe holder</td><td>Stoelting Co.</td><td>51633</td><td></td></tr><tr><td>10 &amp;mu;L syringe, 26 ga., bevel tip</td><td>Stoelting Co.</td><td>51105</td><td></td></tr><tr><td>Drill bit .75mm</td><td>Stoelting Co.</td><td>51455-3</td><td></td></tr></tbody></table>

intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

Medulloblastom, sinir sistemi, en sık görülen pediatrik tümördür. Hayvan çalışmalarında büyük bir vücut medulloblastom 1-4 hücre kökenli olarak serebellar granül nöron öncüleri (CGNPs) odaklanmıştır. Ancak, insan hasta (nodüler desmoplastik, klasik ve hücre / anaplastik büyük) ve medulloblastom ektopik CGNP işaretleyici 5 herhangi bir ifade ile insan hastaların bir alt kümesi bulundu olduğu gerçeğini, medulloblastom alt tiplerinin farklı klinik sunumlar öneririz. medulloblastom hücresel ve moleküler kökenleri, çok daha karmaşık ve tamamen deşifre. Bu nedenle, hücre tipi özel bir tedavi yöntemi geliştirdi dayalı alternatif medulloblastom tümör hücre-kökenli olup olmadığını belirlemek için esastır. Bu amaçla, alıcıların ikincil tümör gelişiminin sonraki analizlerde takip genetik olarak işaretlenmiş tümör hücre tiplerinin allografting intrakranial tümör başlatan hücreleri hücresel kökenli ortotopik belirlenmesini sağlayacaktır. Burada intrakranial ortotopik medulloblastom primer tümör dokusundan elde edilen hücreler, allografting için deney protokolünde tanımlamak ve bu prosedür de kurulan hücre hatları hücrelerin nakli için kullanılabilir.

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Bu protokol, fare medulloblastom doku izolasyon ve ayrılma açıklar ve ikincil medulloblastom başlatmak için immün sistemi baskılanmış alıcı farelere tümör hücrelerinin sonraki allografting.

Medulloblastom Hücre bağışıklık Fare İntrakranial Ortotopik Allografting

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

intracranial-orthotopic-allografting-medulloblastoma-cells

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Neuroscience

12.1K Görüntüleme.  Vanderbilt University. Bu protokol, fare medulloblastom doku izolasyon ve ayrılma açıklar ve ikincil medulloblastom başlatmak için immün sistemi baskılanmış alıcı farelere tümör hücrelerinin sonraki allografting.

Video: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Biyoparlaklık Görüntüleme kullanarak Tümör Büyüme ve Terapi yanıt sonraki Analizi ile İntrakranial Beyin Tümörü Xenografts kurulması

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Nörobilim

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Bir Ortotopik Fare Beyin Tümörü Modelinde Kombinasyon Radyoterapi

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Tıp

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Primer Ortotopik Glioma Xenografts Infiltratif Büyüme ve İsocitrat Dehidrogenaz I Mutasyonu Rekapitulate

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Ortotopik Glioblastoma Fare Modeli Beyin parankim fiziksel kısıtlamaları bakımı ve Intravital İki foton Mikroskopi için uygundur

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Glioblastoma multiforme'un Translasyonel Ortotopik Modelleri

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Kanser Araştırmaları

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelleme Astrositom Patogenez<em&gt; İn Vitro</em&gt; Ve<em&gt; In Vivo</em&gt; Kortikal Astrositler veya Şartlı, Genetiği fareler ile Nöral Kök Hücreler kullanma

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

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

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

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

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

İmmünoloji ve Enfeksiyon

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Bir Fare Modelinde Tedavi Edici Kök Hücrelerin Glioblastomaya İntranazal Dağıtımı

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Görüntü güdümlü rezeksiyon Glioblastoma ve kafa içi tedavi kök hücre numaralı seribaşı iskele implantasyonu

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...