Ringraziamo il Single Cell Open Lab (scOpenLab) presso il German Cancer Center (DKFZ) per le discussioni utili. Questa ricerca è stata supportata dal German Cancer Aid, Max-Eder Program grant number 70111964 (S.T.).

Campioni di glioma freschi congelati sono stati ottenuti dalla banca dei tessuti del National Center for Tumor diseases (NCT) di Heidelberg, in Germania. L'uso del materiale del paziente è stato approvato dall'Institutional Review Board presso la Facoltà di Medicina di Heidelberg e il consenso informato è stato ottenuto da tutti i pazienti inclusi nello studio.
1. Preparazione sperimentale
<ol><li>Eseguire tutti i passaggi sul ghiaccio o a 4 °C.</li>
	<li>Tubi pre-raffreddamento, piatti, lamette da rasoio, Douncers e pestelli a 4 °C.</li>
	<li>Preparare tutti i buffer in anticipo. Questi tamponi sono stabili a temperatura ambiente. Si raccomanda la filtrazione sterile, soprattutto per il saccarosio. I preparati di riserva sono modificati da Corces et al.19. Cfr. tabelle 1-7.</li>
	<li>Rimuovere i campioni dall'azoto liquido o dal congelatore a -80 °C e conservare con ghiaccio secco fino al punto 2.1.</li>
</ol>2. Dissezione e dissociazione dei tessuti
<ol><li>Trasferire un campione di tessuto fresco congelato (10-60 mg) in una capsula di Petri pre-refrigerata. Tritare / tritare il tessuto fresco congelato con una lama di rasoio a piccoli pezzi sul ghiaccio.</li>
	<li>Aggiungere 500 μL di tampone di lisi dei nuclei refrigerati a un tubo pre-refrigerato da 1,5 ml. Posizionare i pezzi di tessuto nel tubo da 1,5 ml contenente il tampone di lisi dei nuclei e trasferirli in un Douncer (Tabella dei materiali). NOTA: Esistono due pestelli omogeneizzatori Dounce: pestello "sciolto" o "A" per la riduzione iniziale del campione e pestello "stretto" o "B" per l'omogeneizzazione completa del campione.</li>
	<li>Dounce i pezzi di tessuto con il pestello "sciolto" per circa 20 colpi, fino a quando l'attrito non si riduce. Se sono presenti detriti, il campione può essere pre-eliminato mediante filtrazione con un filtro da 100 mm.</li>
	<li>Dounce con il pestello "stretto" per 20 colpi per ottenere l'omogeneizzazione completa dei tessuti.</li>
	<li>Trasferire l'omogeneo (circa 500 μL) in un tubo pre-refrigerato da 2 mL. Aggiungere 1 mL di tampone di lisi refrigerato. Mescolare delicatamente e incubare sul ghiaccio per 5 minuti. Mescolare delicatamente con una punta di pipetta a foro largo e ripetere 1-2 volte durante l'incubazione.</li>
	<li>Filtrare l'intero omogeneo utilizzando una rete filtrante da 30 μm, raccoglierne in un tubo Falcon da 15 ml e trasferirlo nuovamente in un nuovo tubo pre-refrigerato da 2 ml. Un singolo colino è in genere sufficiente per l'intero omogeneo.</li>
	<li>Controllare al microscopio ottico per verificare la rimozione di detriti di grandi dimensioni e l'integrità della membrana nucleare. I nuclei devono essere rotondi e la membrana nucleare non deve essere distorta. Se sono presenti detriti, i nuclei possono essere rifiltrati.</li>
	<li>Centrifugare i nuclei a 500 x g per 5 min a 4 °C su una centrifuga da banco. Rimuovere il surnatante, lasciando circa 50 μL con pellet contenente i nuclei. Risuscisciare delicatamente il pellet in un altro 1 mL di tampone di lisi dei nuclei e incubare per 5 minuti sul ghiaccio.</li>
	<li>Centrifugare i nuclei a 500 x g per 5 min a 4°C. Rimuovere il surnatante senza disturbare il pellet, aggiungere 500 mL di 1x tampone di omogeneizzazione (HB)(Tabella 4)e incubare per 5 minuti senza sosuscire. Quindi, risuscisci i nuclei in altri 1,0 mL di 1x HB.</li>
	<li>Centrifugare i nuclei a 500 x g per 5 min a 4 °C. Rimuovere il surnatante e riaspentare delicatamente i nuclei in 200 μL di 1x HB in un nuovo tubo da 2,0 mL.</li>
</ol>3. Centrifugazione a gradiente
<ol><li>Aggiungere 200 μL di soluzione di iodixanolo al 50% (Tabella 5) per dare una concentrazione finale di iodixanolo al 25%. Mescolare bene 10 volte con pipetta impostata su 300 ml.</li>
	<li>Aggiungere 300 μL di soluzione di iodixanolo al 29% (Tabella 6) sotto la miscela al 25%. Utilizzare una punta fine P1000 per evitare la miscela degli strati.</li>
	<li>Aggiungere 300 μl di soluzione di iodixanolo al 35% (Tabella 7) sotto la miscela al 29%. Utilizzare una punta fine P1000 per evitare la miscela degli strati. Attenzione: questo passaggio richiede la rimozione graduale della punta della pipetta durante il pipettaggio per evitare un eccessivo spostamento del volume.</li>
	<li>Posizionare i campioni in una centrifuga a secchio oscillante, girare per 20 minuti a 3.500 x g a 4 °C con il freno spento.</li>
	<li>Rimuovere delicatamente i campioni senza agitare e osservare sotto la luce. Una banda bianca chiara di nuclei puri al 95% dovrebbe essere visibile tra il secondo e il terzo strato (Figura 1).</li>
</ol>4. Isolamento dei nuclei
<ol><li>Aspirare gli strati superiori fino alla banda dei nuclei bianchi all'interfase del 29%-35%.</li>
	<li>Raccogliere la banda dei nuclei in un volume di 200 ml, trasferirla in un tubo fresco e filtrare con un filtro da 20 μm (Tabella dei materiali). NOTA: i nuclei non devono essere risuscisi prima della filtrazione.</li>
	<li>Controllare al microscopio ottico per verificare la rimozione di detriti di grandi dimensioni e l'integrità della membrana nucleare. I nuclei devono essere rotondi e la membrana nucleare non deve essere distorta.</li>
	<li>Conta i nuclei usando la colorazione blu di Trypan su un emocitometro e i nuclei aliquoti per snRNA-seq/snATAC-seq.</li>
</ol>5. Nuclei singoli RNA e ATAC seq
<ol><li>Elaborare immediatamente i nuclei utilizzando l'espressione genica monocellulare e i kit di reagenti ATAC a singola cellula (Tabella dei materiali). NOTA: Le concentrazioni di campioni di nuclei per il sistema di genomica 10x sono 1500-3000 nuclei per μL per snRNA-seq e 3500-7000 nuclei per m μL per snATAC-seq. I nuclei possono essere diluiti utilizzando 1x PBS.</li>
	<li>Sequenziare le librerie risultanti presso la Genomics Core Facility.</li>
	<li>Eseguire l'analisi del controllo qualità dei dati. I nuclei sono inclusi per ulteriori analisi se contengono identificatore molecolare univoco (UMI) >1000, numero di geni >500 e percentuale di geni mitocondriali <5% e sono all'interno della media + tre deviazioni standard delle IMI e dei geni.</li>
</ol>

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Il campo dell'eterogeneità intratumorale è in una fase entusiasmante, con nuovi saggi e piattaforme in fase di sviluppo per sfidare ed espandere le conoscenze esistenti. L'eterogeneità intratumorale è un fattore cruciale che contribuisce alla progressione della malattia e alla resistenza alle attuali modalità di trattamento nei gliomi28. Recenti studi sui tumori cerebrali si sono concentrati su questo importante aspetto utilizzando saggi trascrittomici ed epigenomici monocellulari per caratte...

I gliomi diffusi di grado inferiore (LGG), il tumore cerebrale primario più comune negli adulti, sono neoplasie infiltrative che spesso si presentano nell'emisfero cerebrale. Gli LCG mostrano eterogeneità sia inter- che intra-tumorale, che non è guidata solo dalla popolazione tumorale, ma anche dalle cellule non maligne coinvolte in modo complesso nello sviluppo e nella progressione del tumore1,2,3,4,5.
Nell'ultimo decennio, c'è stata una valanga di dati genomici raccolti nel campo dei gliomi. Questi dati provengono principalmente da studi di sequenziamento dei tumori di massa e hanno contribuito immensamente alla caratterizzazione molecolare e all'attuale classificazione dei tumori cerebrali5,6,7,8,9,10,11. Tuttavia, anche se questi studi hanno rivelato l'ampio panorama molecolare associato ai gliomi, c'è ancora una deludente mancanza di progressi per quanto riguarda l'intervento terapeutico. Uno degli ostacoli alla resistenza al trattamento nei tumori cerebrali è l'eterogeneità intratumorale. Per affrontare questo problema, vari studi si sono concentrati sull'eterogeneità genomica, trascrittomica, proteomica ed epigenetica presente all'interno di un tumore a livello di singola cellula12,13,14,15,16,17.
Sebbene ci siano stati notevoli progressi tecnologici nel campo delle singole cellule negli ultimi due anni, uno dei principali fattori limitanti è la disponibilità di campioni freschi necessari per isolare le cellule ed eseguire questi esperimenti. Per superare questa limitazione, ci sono stati diversi tentativi riusciti di eseguire saggi, come snRNA-seq e snATAC-seq da tessuti congelati, utilizzando nuclei piuttosto che cellule18,19. La maggior parte di questi metodi si basa su strategie di selezione cellulare attivate dalla fluorescenza (FACS) o di filtrazione. Sia gli approcci a singola cellula che a singolo nucleo hanno i loro punti di forza e svantaggi. Gli approcci a singola cellula mantengono i trascritti mitocondriali, che sebbene possano essere informativi, possono anche ridurre la copertura del trascrittoma a causa della loro elevata abbondanza. Gli approcci di isolamento dei singoli nuclei eliminano un'alta percentuale della frazione mitocondriale, consentendo così una copertura più approfondita dei trascritti nucleari20.
Esistono varie piattaforme disponibili in commercio che sono state utilizzate negli ultimi anni per saggiare i dati di genomica a singola cellula, tra cui RNA-seq e ATAC-seq. Una delle piattaforme più importanti è la piattaforma 10x Genomics Chromium per l'espressione genica a singola cellula e la profilazione ATAC a singola cellula. Poiché la piattaforma funziona con l'aiuto di camere microfluidiche, eventuali detriti o aggregati possono ostruire il sistema portando alla perdita di dati, reagenti e preziosi campioni clinici. Pertanto, il successo degli studi su singole cellule dipende in gran parte dall'accurato isolamento di singole cellule / nuclei.
Il protocollo che dimostreremo qui è una combinazione leggermente modificata dei protocolli DroNc-seq e Omni-ATAC-seq e utilizza un approccio simile a studi recenti che utilizzano snRNA-seq per comprendere disturbi neurologici e tipi di cellule neuronali nel cervello umano18,19,21,22,23,24. Il protocollo utilizza una combinazione di dissociazione enzimatica/meccanica di campioni congelati seguita da filtrazione e centrifugazione a gradiente e consente un isolamento rapido e accurato di singoli nuclei da tessuti di glioma freschi congelati. Abbiamo utilizzato con successo questo protocollo per generare dati snRNA-seq e snATAC-seq dalla stessa preparazione dei nuclei da campioni di tumore al cervello.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>Active motif</td>
			<td>40401</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>MACs Smart Strainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-463</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Ac)2</td>
			<td>Sigma</td>
			<td>63052-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma</td>
			<td>NUC101-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (20 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell ATAC</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Gene Expression</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

nuclei isolation from fresh frozen brain tumors for single-nucleus rna-seq and atac-seq

I gliomi diffusi adulti mostrano eterogeneità inter- e intra-tumorale. Fino a poco tempo fa, la maggior parte degli sforzi di profilazione molecolare su larga scala si sono concentrati su approcci di massa che hanno portato alla classificazione molecolare dei tumori cerebrali. Negli ultimi cinque anni, gli approcci di sequenziamento a singola cellula hanno evidenziato diverse caratteristiche importanti dei gliomi. La maggior parte di questi studi ha utilizzato campioni di tumore cerebrale fresco per isolare singole cellule utilizzando la citometria a flusso o metodi di separazione basati su anticorpi. Andando avanti, l'uso di campioni di tessuto appena congelati provenienti da biobanche fornirà una maggiore flessibilità alle applicazioni a singola cellula. Inoltre, con l'avanzare del campo a singola cellula, la prossima sfida sarà quella di generare dati multi-omici da una singola cellula o dalla stessa preparazione del campione per svelare meglio la complessità del tumore. Pertanto, protocolli semplici e flessibili che consentono la generazione di dati per vari metodi come il sequenziamento dell'RNA a nucleo singolo (snRNA-seq) e il test a nucleo singolo per la cromatina accessibile alla trasposasi con sequenziamento ad alto rendimento (snATAC-seq) saranno importanti per il campo.
I recenti progressi nel campo delle singole cellule, insieme a strumenti microfluidici accessibili come la piattaforma di genomica 10x, hanno facilitato le applicazioni a singola cellula nei laboratori di ricerca. Per studiare l'eterogeneità dei tumori cerebrali, abbiamo sviluppato un protocollo avanzato per l'isolamento di singoli nuclei da gliomi freschi congelati. Questo protocollo unisce i protocolli a cella singola esistenti e combina una fase di omogeneizzazione seguita dalla filtrazione e dalla centrifugazione a gradiente mediata dal buffer. I campioni risultanti sono sospensioni di nuclei singoli puri che possono essere utilizzati per generare dati sull'espressione genica del singolo nucleo e sull'accessibilità della cromatina dalla stessa preparazione dei nuclei.

La genomica a nucleo singolo è un campo in evoluzione con dati e protocolli limitati. Un fattore critico che influenza l'esito dei test dei singoli nuclei è l'isolamento dei nuclei puri e intatti. Abbiamo combinato due protocolli pubblicati (protocolli DroNc-seq e Omni-ATAC-seq) per isolare nuclei puri e di alta qualità da blocchi di tessuto di glioma congelato fresco in un tempo relativamente breve, mantenendo così la stabilità dei trascritti (Figura 1).
L'u...

Watch this Scientific Journal Video about Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq at JoVE.com

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

L'eterogeneità intratumorale è una caratteristica intrinseca dei tumori, compresi i gliomi. Abbiamo sviluppato un protocollo semplice ed efficiente che utilizza una combinazione di tamponi e centrifugazione a gradiente per isolare singoli nuclei da tessuti di glioma freschi congelati per studi di sequenziamento di RNA a nucleo singolo e ATAC.

Isolamento dei nuclei da tumori cerebrali freschi congelati per RNA-seq a nucleo singolo e ATAC-seq

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus

Research

JoVE Journal

Cancer Research

11.9K Views.  University Hospital Heidelberg. L'eterogeneità intratumorale è una caratteristica intrinseca dei tumori, compresi i gliomi. Abbiamo sviluppato un protocollo semplice ed efficiente che utilizza una combinazione di tamponi e centrifugazione a gradiente per isolare singoli nuclei da tessuti di glioma freschi congelati per studi di sequenziamento di RNA a nucleo singolo e ATAC.

Video: Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

Successivo sequenziamento di nuova generazione per il rilevamento di Actionable mutazioni nei tumori solidi e liquidi

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Ricerca

Ricerca sul cancro

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Il trapianto di tumori del cervello pediatrico di Zebrafish in ospedali immunitario-competenti per uno studio a lungo termine del comportamento della cellula tumorale e della risposta ai farmaci

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Sviluppo di un modello preclinico umano di osteoclastogenesi dai monociti del sangue periferico co-coltivati con linee cellulari di cancro al seno

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

Rilevamento di una variante rara trascrizione CDH1 in tessuti di cancro gastrico fresco congelato mediante PCR digitale basata su Chip

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Ottenere le sfere delle cellule staminali tumorali da tumori ginecologici e tumori del cancro

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generazione di tumori pancreatici ortotopici e caratterizzazione ex vivo della citotossicità delle cellule T infiltranti di tumore

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Trapianto ortotopico di tumori al seno come modelli preclinici per il cancro al seno

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

Un flusso di lavoro di screening rapido per identificare la potenziale terapia combinata per GBM utilizzando cellule staminali di glioma derivate dal paziente

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

Ottimizzazione ATAC-Seq per la ricerca sull'epigenetica del cancro

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

Organoidi tumorali e fibroblasti derivati dal cancro del pancreas da tessuto fresco

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Autore in primo piano: Istituzione di organoidi tumorali e fibroblasti derivati dal cancro del pancreas da tessuto fresco  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Costituzione di organoidi e fibroblasti tumorali derivati dal cancro del pancreas da tessuto fresco

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...