Gli autori ringraziano i dottori. Fahad Bafakih e Jim Mandell per l&#39;acquisizione e l&#39;identificazione di campioni di pazienti, Garrett F. Beeghly per l&#39;assistenza con immunohistochemistry e il Biorepository and Tissue Research Facility, il Centro di Histologia del Centro Cardiovascolare e l&#39;Analisi Biomolecolare presso l&#39;Università di Virginia per assistenza L&#39;acquisizione di campioni, l&#39;immunohistochemistry e l&#39;imaging.

Questo protocollo identifica i componenti cellulari in campioni embedded (FFPE) fissati in formalina, come è tipico per i campioni pazienti clinici bancati. L&#39;incorporazione di paraffina consente la migliore manutenzione della morfologia cellulare e del tessuto, nonché la migliore longevità delle sezioni. I campioni utilizzati per questa analisi sono stati raggiunti attraverso l&#39;Università di Virginia Biorepository e Tissue Research Facility. I campioni pazienti sono stati selezionati da un neuropatologo basato su una diagnosi definitiva di glioblastoma (astrocitoma, grado IV dell&#39;OMS) che aveva completato resezione tumorale presso l&#39;Università di Virginia tra il 2010 e il 2013 e sono stati identificati prima di questa analisi 11 . 1. Depaffinazione e reidratazione del campione FFPE NOTA: Questa parte del protocollo è specifica per i campioni FFPE. Mentre i campioni embedded con paraffina possono essere più utili per questa analisi a causa della conservazione cellulare e tEmettere morfologia, questa analisi può anche essere fatta con sezioni congelate. Se si usano sezioni congelate, questa parte può essere omessa e procedere direttamente all&#39;immunohistochemistry cromogenico. <ol><li> Eseguire le lavaggi seguenti per 5 min ciascuno: Xilene, Xilene, 100% etanolo, 100% etanolo, 95% etanolo, 95% etanolo, 70% etanolo, acqua deionizzata, acqua deionizzata </li></ol> 2. Recupero di antigeni NOTA: Questa parte del protocollo è necessaria per rompere ponti di metilene formati durante la fissazione di formalina di campioni FFPE e di esporre siti antigene per gli anticorpi da legare. <ol><li> Diluire la soluzione di antigene a pH elevato a base di Tris a raccomandazione del produttore in acqua distillata. </li><li> Eseguire il recupero di antigeni mediato dal calore utilizzando un forno a microonde. Basta anche altre forme di recupero a temperature termiche (come la pentola a pressione, il vapore vegetale o l&#39;acqua bollente). <ol><li> Aggiungere la soluzione diluita a messa a punto in un microfono non sigillatonave. Posizionare le diapositive nella vasca. Posizionare le diapositive in forno a microonde. </li><li> Bollite per 20 minuti ad alta potenza. Controllare i livelli liquidi per l&#39;evaporazione e ricostituire con acqua distillata come necessario. </li></ol></li><li> Lasciare raffreddare i campioni in soluzione per 1 ora a temperatura ambiente. </li></ol> 3. Immunohistochemistry cromogenico <ol><li> Estrarre il campione di tessuto con una penna idrofoba per ridurre al minimo il volume di reagenti necessari per coprire il campione. NOTA: Assicurarsi di mantenere i campioni di tessuto idratati e non lasciarli asciugare, in quanto ciò influirà sull&#39;efficacia della colorazione. </li><li> Pipettare la soluzione di permeabilizzazione sufficiente (soluzione salina a tampone Tris (TBS) + 0.01% Triton-X) per coprire il campione di tessuti (tipicamente circa 100 - 200 μL). </li><li> Rimuovere e scartare la soluzione e ripetere il passo 3.2. </li><li> Incubare i campioni a temperatura ambiente con soluzione di blocco (2,5% cavallo di cavallo + soluzione di permeabilizzazione). </li><li> Incubare i campioni durante la notte in 4 ° C con anticorpi primari diluitiNella soluzione di blocco. NOTA: Tutti gli anticorpi primari utilizzati qui sono diluiti a 1: 200, ma le diluizioni ottimali possono essere determinate usando diluizioni seriali a partire dalla raccomandazione del produttore. Informazioni dettagliate sugli anticorpi utilizzati in questo protocollo sono stati precedentemente pubblicati 11 . </li><li> Rilevare gli anticorpi primari utilizzando un reattore di polimero perossidasi a base di rafano corrispondente all&#39;animale primario di anticorpi, seguendo il protocollo del produttore. </li><li> Pipetta la soluzione permeabilizzazione sufficiente per coprire il campione di tessuto e incubare per 5 min. </li><li> Rimuovere e scartare la soluzione e ripetere il passo 3.7. </li><li> Incubare le diapositive in 0,3% di H 2 O 2 in 1x TBS per 15 minuti. </li><li> Sviluppare campioni con un substrato perossidasi diaminobenzidina (DAB) per 2 - 10 minuti fino a raggiungere la desiderata intensità della macchia. </li><li> Contrassegnare campioni per identificare nuclei cellulari, come ad esempio l&#39;ematossilina, seguendo il protocollo del produttore. </li><li> Campioni di deidrato con 100%Etanolo e xilene. </li><li> Montare i campioni in modo permanente con i supporti di montaggio. </li></ol> 4. Identificazione delle regioni d&#39;interesse <ol><li> Slide di immagini sotto microscopia luminosa in grado di immagini ad alta risoluzione. NOTA: utilizzare i componenti cellulari di imaging con una risoluzione minima di 20x. </li><li> Spostare la fotocamera a determinate aree di interesse in tutti i campioni di tessuto. </li><li> Salvare le immagini come file TIFF per la quantificazione. </li></ol> 5. Analisi delle immagini <ol><li> Apri le immagini in ImageJ per la quantificazione della copertura percentuale. </li><li> Utilizza il plugin di Threshold Color per rimuovere il colore viola dai nuclei colorati da ematossilina. </li><li> Convertire l&#39;immagine a 8 bit. </li><li> Aggiungere soglia senza sfondo scuro. </li><li> Immagine di processo usando uno dei 17 filtri di soglia ImageJ preimpostati ( cioè MaxEntropy), quindi solo le porzioni macchiate DAB sono incluse nella soglia. NOTA: Selezionare il filtro soglia ottimale preimpostato che minimizzaInclusione della colorazione di fondo. Ciò può dipendere dalla qualità della colorazione e dalla specificità dell&#39;anticorpo. Utilizzare la stessa soglia per tutte le repliche tecniche all&#39;interno di ciascun campione del paziente. </li><li> Applica la soglia. </li><li> Misura l&#39;area percentuale di immagine soglia. </li><li> Copertura area percentuale media per più regioni all&#39;interno di ciascun campione. </li></ol>

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Immunol. 189, 444-453 (2012).</li><li>Yuan, J. X., Bafakih, F. F., Mandell, J. W., Horton, B. J., Munson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+the+Cellular+Microenvironment+of+Glioblastoma+to+Develop+Predictive+Statistical+Models+of+Overall+Survival.">Quantitative Analysis of the Cellular Microenvironment of Glioblastoma to Develop Predictive Statistical Models of Overall Survival.</a> J. Neuropathol. Exp. Neurol. , (2016).</li><li>Yuan, Y., 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=Quantitative+Image+Analysis+of+Cellular+Heterogeneity+in+Breast+Tumors+Complements+Genomic+Profiling.">Quantitative Image Analysis of Cellular Heterogeneity in Breast Tumors Complements Genomic Profiling.</a> Sci. Transl. Med. 4 (157), (2012).</li><li>Yi, E. S., 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=Distribution+of+Obstructive+Intimal+Lesions+and+Their+Cellular+Phenotypes+in+Chronic+Pulmonary+Hypertension.">Distribution of Obstructive Intimal Lesions and Their Cellular Phenotypes in Chronic Pulmonary Hypertension.</a> Am. J. Respir. Crit. Care Med. 162 (4), 1577-1586 (2000).</li><li>Turcan, S., 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=IDH1+mutation+is+sufficient+to+establish+the+glioma+hypermethylator+phenotype.">IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype.</a> Nature. 483 (7390), 479-483 (2012).</li><li>Songtao, Q., 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=IDH+mutations+predict+longer+survival+and+response+to+temozolomide+in+secondary+glioblastoma.">IDH mutations predict longer survival and response to temozolomide in secondary glioblastoma.</a> Cancer Sci. 103 (2), 269-273 (2012).</li><li>Shinojima, 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=Prognostic+Value+of+Epidermal+Growth+Factor+Receptor+in+Patients+with+Glioblastoma+Multiforme.">Prognostic Value of Epidermal Growth Factor Receptor in Patients with Glioblastoma Multiforme.</a> Cancer Res. 63, 6962-6970 (2003).</li><li>Karpel-Massler, G., Schmidt, U., Unterberg, A., Halatsch, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+inhibition+of+the+epidermal+growth+factor+receptor+in+high-grade+gliomas:+where+do+we+stand?.">Therapeutic inhibition of the epidermal growth factor receptor in high-grade gliomas: where do we stand?.</a> Mol. Cancer Res. 7 (7), 1000-1012 (2009).</li><li>Badie, B., Schartner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microglia+in+glioma+biology.">Role of microglia in glioma biology.</a> Microsc. Res. Tech. 54 (2), 106-113 (2001).</li><li>Watters, J. J., Schartner, J. M., Badie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+function+in+brain+tumors.">Microglia function in brain tumors.</a> J. Neurosci. Res. 81 (3), 447-455 (2005).</li><li>Alves, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+cells:+A+heterogeneous+and+fatal+tumor+interacting+with+the+parenchyma.">Glioblastoma cells: A heterogeneous and fatal tumor interacting with the parenchyma.</a> Life Sci. 89 (15), 532-539 (2011).</li><li>Hambardzumyan, D., Gutmann, D. H., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microglia+and+macrophages+in+glioma+maintenance+and+progression.">The role of microglia and macrophages in glioma maintenance and progression.</a> Nat. Neurosci. 19 (1), 20-27 (2015).</li><li>Zhai, H., Heppner, F. L., Tsirka, S. 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Il nostro metodo proposto qui è un approccio quantitativo per l&#39;analisi dei campioni istologici macchiati usando l&#39;immunohistochemistry tradizionale cromogeno. La metodologia attuale per questo tipo di analisi comprende protocolli di colorazione similari seguiti da classificazione da patologi indipendenti. Questo metodo è stato affidabile, tuttavia per una serie di applicazioni è necessaria una comprensione più precisa del trucco cellulare, come una migliore comprensione dell&#39;eterogeneità associata ai t...

Il glioblastoma (GBM), il cancro al cervello più comune e maligno, è caratterizzato da un&#39;invasione altamente diffusa dalla massa tumorale primaria nel cervello sano, parenchima 1 , 2 . Questa invasione diffusa rende particolarmente difficile il respiro del tumore, e le cellule tumorali invasive che rimangono post-terapia sono il motivo più comune per la ricorrenza inevitabile 2 , 3 , 4 . In precedenza, abbiamo trovato inibendo l&#39;invasione di cellule diffuse degli gliomi per essere terapeuticamente vantaggiosa 5 , tuttavia poco si sa circa i complessi meccanismi che contribuiscono all&#39;invasione GBM. Il microambiente del tumore, o il tessuto che circonda il cancro, è stato implicato nella progressione dei tumori nei tumori multipli 6 , 7 . Il microambiente del tumore di glioblastoma, in pArticolare, è relativamente poco caratterizzato ed è univoco complesso, composto da cellule gliali multipli, come astrociti, microglia e oligodendrociti, così come matrice extracellulare, fattori solubili e fattori biofisici. Sperimentalmente, gli astrociti e la microglia sono stati dimostrati per aumentare la progressione dell&#39;loma e l&#39;invasione 8 , 9 , 10 , ma la composizione di tutte le cellule gliali nel cervello umano nativo microambiente è sconosciuta. Abbiamo precedentemente mostrato che i componenti microambientali possono prevedere la sopravvivenza del paziente analizzando quantitativamente i componenti cellulari del microambiente glioblastoma e incorporando le nostre analisi in un modello di rischio proporzionale 11 . Qui descriviamo il metodo di analisi quantitativa per identificare le popolazioni delle cellule gliali all&#39;interno della massa tumorale e delle regioni adiacenti di resezione tumorale del paziente glioblastomaS. Abbiamo utilizzato immunohistochemistry cromogeno per identificare le popolazioni di cellule gliali e ImageJ per analizzare la copertura percentuale di colorazione per ogni popolazione gliale. La valutazione della percentuale di copertura crea una misurazione semplice per determinare le differenze morfologiche delle cellule, in particolare quelle colpite dalle interazioni con le cellule tumorali. Precedenti studi per la quantificazione della colorazione istopatologica usano la colorazione standard come l&#39;ematoxilina e l&#39;eosin 12 o il tricromo 13 di Masson, che non approfittano della specificità della colorazione immunohistochemica a base di anticorpi. Il nostro metodo è stato sviluppato per quantificare direttamente le popolazioni gliali nelle resezioni tumorali del paziente glioblastoma, che intendiamo utilizzare per chiarire il microambiente complesso di glioblastoma.

<table><tbody><tr><td>Xylene</td><td>Fisher Chemical</td><td>X3P</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>High pH antigen unmasking solution</td><td>Vector Labs</td><td>H-3301</td><td></td></tr><tr><td>TBS</td><td></td><td></td><td></td></tr><tr><td>Triton-X</td><td>Amresco</td><td>9002-93-1</td><td></td></tr><tr><td>Horse serum</td><td></td><td></td><td></td></tr><tr><td>Anti-ALDH1L1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab56777</td><td></td></tr><tr><td>Anti-Iba1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab5076</td><td></td></tr><tr><td>Anti-Oligodendrocyte Specific Protein1&amp;nbsp;</td><td>abcam&amp;nbsp;</td><td>ab53041</td><td></td></tr><tr><td>ImmPRESS anti-goat</td><td>Vector Labs</td><td>MP-7405</td><td></td></tr><tr><td>ImmPRESS Universal (anti-mouse/rabbit)</td><td>Vector Labs</td><td>MP-7500</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>216763</td><td></td></tr><tr><td>ImmPACT DAB substrate</td><td>Vector Labs</td><td>SK-4105</td><td></td></tr><tr><td>Hematoxylin counterstain</td><td>ThermoScientific</td><td>72404</td><td></td></tr><tr><td>Histochoice Mounting Media</td><td>Amresco</td><td>H157-475</td><td></td></tr><tr><td>Aperio Scanscope</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Image Scanscope</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>Super HT PAP Pen</td><td>Research Products International</td><td>195506</td><td></td></tr></tbody></table>

quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

Con il crescente interesse per il microambiente del tumore, abbiamo deciso di sviluppare un metodo per determinare in modo specifico i componenti del microambiente nei campioni dei pazienti di glioblastoma, il cancro al cervello più letale e più invasivo. Non solo metodi quantitativi utili per descrivere in modo preciso i tessuti malati, possono anche contribuire alla prognosi, alla diagnosi e allo sviluppo di sistemi e sostituzioni sintetici. Nel glioblastoma, le cellule gliali, come microglia e astrociti, sono state correlate indipendentemente con una prognosi scadente basata sulla classificazione patologica. Tuttavia, lo stato di queste cellule e di altri componenti di cellule gliali non è stato ben descritto in modo quantitativo. Ciò può essere difficile a causa dei grandi processi che segnano queste cellule gliali. Inoltre, la maggior parte delle analisi istologiche si concentrano sul campione globale del tessuto o solo all&#39;interno della maggior parte del tumore, al contrario di delineare quantificazioni basate sulle regioniIl tessuto molto eterogeneo. Qui descriviamo un metodo per identificare e analizzare quantitativamente le popolazioni delle cellule gliali all&#39;interno della massa tumorale e delle regioni adiacenti di resezione tumorale da pazienti con glioblastoma. Abbiamo utilizzato immunohistochemistry cromogeno per identificare le popolazioni di cellule gliali nelle resections del tumore del paziente e ImageJ per analizzare la copertura percentuale di colorazione per ogni popolazione gliale. Con queste tecniche possiamo descrivere meglio le cellule gliali in tutte le regioni del microambiente tumorale degli amiomi.

Per questa analisi sono state identificate due regioni di interessi all&#39;interno delle nostre rese tumorali - la massa tumorale primaria e le regioni adiacenti, composta principalmente da tessuti sani con cellule tumorali invasive diffuse ( Figura 1A , 1B ) - sono stati identificati collaborando neuropatologi con pazienti affetti da ematossilina ed eosina campioni. All&#39;interno di ogni campione di pazienti è stata identificata una col...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Immunohistochemia Quantitativa del Microambiente Cellulare in Riapplicazioni del Glioblastoma Paziente

Questo protocollo è stato sviluppato per identificare quantitativamente i componenti del microambiente del tumore nelle resezioni dei pazienti con glioblastoma utilizzando immunohistochemistry cromogene e ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

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9.6K Views.  University of Virginia.  Questo protocollo è stato sviluppato per identificare quantitativamente i componenti del microambiente del tumore nelle resezioni dei pazienti con glioblastoma utilizzando immunohistochemistry cromogene e ImageJ. 

Video: Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Coculture saggi per studiare macrofagi e microglia stimolazione del glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Ricerca

Ricerca sul cancro

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

Arricchimento e caratterizzazione dei tumore immuni e Non immuni microambienti in tumori murini sottocutanei stabiliti

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Valutazione di biomarcatori in Glioma mediante immunoistochimica su paraffina 3D Glioma Neurosphere culture

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Ottimizzazione della coltura cellulare glioma di alto grado da campioni chirurgici per l'uso in modelli animali clinicamente rilevanti e immunochimica 3D

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicina

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Profilazione spaziale digitale per la caratterizzazione del microambiente in gliomi diffusamente infiltranti di tipo adulto

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

Immunofluorescenza multiplex e analisi di immagini spaziali del microambiente tumorale

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Autore in primo piano: Immunofluorescenza multiplex combinata con l'analisi di immagini spaziali per la valutazione clinica e biologica del microambiente tumorale  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

Immunofluorescenza multipla combinata con analisi di immagini spaziali per la valutazione clinica e biologica del microambiente tumorale

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e analisi citofluorimetrica di-Glioma infiltrazione sangue periferico mononucleari Cellule

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Immunologia e infezioni

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.

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

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

Immunohistochemia Quantitativa del Microambiente Cellulare in Riapplicazioni del Glioblastoma Paziente

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Coculture saggi per studiare macrofagi e microglia stimolazione del glioblastoma Invasion

Arricchimento e caratterizzazione dei tumore immuni e Non immuni microambienti in tumori murini sottocutanei stabiliti

Valutazione di biomarcatori in Glioma mediante immunoistochimica su paraffina 3D Glioma Neurosphere culture

Ottimizzazione della coltura cellulare glioma di alto grado da campioni chirurgici per l'uso in modelli animali clinicamente rilevanti e immunochimica 3D

Profilazione spaziale digitale per la caratterizzazione del microambiente in gliomi diffusamente infiltranti di tipo adulto

Immunofluorescenza multipla combinata con analisi di immagini spaziali per la valutazione clinica e biologica del microambiente tumorale

Immunofluorescenza multipla combinata con analisi di immagini spaziali per la valutazione clinica e biologica del microambiente tumorale

Immunofluorescenza multipla combinata con analisi di immagini spaziali per la valutazione clinica e biologica del microambiente tumorale

Isolamento e analisi citofluorimetrica di-Glioma infiltrazione sangue periferico mononucleari Cellule

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