The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

NOTA: approvvigionamento dei tessuti del paziente è stato autorizzato attraverso la revisione bordo istituzionalmente (IRB) -approvato Biospecimen e protocolli clinici (numeri di protocollo 09-121 e 11-041, rispettivamente) al Memorial Sloan Kettering Cancer Center. Procurement 1 Tissue <ol><li> Approvvigionamento di paziente tumore primario / metastasi NOTA: Fino ad oggi, questo protocollo è stata eseguita su pancreatiche, gastriche e tipi di tumore al seno chirurgicamente rimosso, così come, metastasi linfoma. <ol><li> Dirigere il team chirurgico di consegnare il campione di corriere o sistema a tubo pneumatico per il reparto di Patologia in borsa tenuta esemplare di plastica ben chiuso e sterile nel contenitore a prova di fuoriuscita sterile. Dirigere la squadra chirurgica per trasportare il campione allo stato fresco senza formalina o fissante. </li><li> Dirigere l&#39;assistente patologo o patologo per raccogliere il campione fresco con tecnica sterile, che includes uso di guanti e strumenti sterili sotto una cappa a flusso laminare. </li><li> Registrare il tempo di raccolta del campione procurati, che viene tenuto rigorosamente sotto 30 minuti dal completamento della procedura chirurgica. Prendendo in considerazione gli effetti di ischemia fredda, non prelevare campioni da campioni resecati con un tempo di ischemia fredda&gt; 30 min 18. </li><li> Dirigere il personale del Dipartimento di Patologia per rimuovere un campione del tumore primario di circa 0,5 centimetri 3-1,0 cm 3 in una cappa a flusso laminare per mantenere un ambiente sterile. Quando possibile, scegliere il tessuto tumorale dalla periferia della lesione indice per evitare possibili necrosi centrale (morte cellulare) frank. NOTA: Il tessuto necrotico può essere grossolanamente riconoscibile per uno dei seguenti criteri: perdita di colore o pallore del tessuto; perdita di forza in cui il tessuto necrotico è morbido e friabile; una demarcazione netta tra il tessuto necrotico e vitale. </li><li> Dirigere il pathologist o assistente patologo per fornire tessuti periferici che è in eccesso (scarti chirurgica) dopo il prelievo immediato del tumore per la valutazione diagnostica. Porre il campione in un tubo da centrifuga da 15 ml sterile contenente circa 5 ml di supporti essenziale minimo (MEM) contenente 1% antibiotici (penicillina e streptomicina). </li><li> Se disponibile (ad esempio, linfonodi ascellari di coda di campione mastectomia), procurarsi un grossolanamente positivi esemplare linfonodali associato della stessa dimensione (0,5 centimetri 3-1,0 cm 3) e confrontare la risposta ai farmaci del tumore primario. Come il campione del tumore primario, posizionare il campione associati linfonodi in un tubo da centrifuga da 15 ml sterile contenente circa 5 ml di supporto essenziale minimo (MEM) contenente 1% antibiotici (penicillina e streptomicina). </li><li> Se la dimensione dei permessi campioni chirurgici (ad esempio, campioni mastectomia), rimuovere (distante da tumore primario) di un campione di tessuto normale (cioè,normale / parenchima denso fibroso seno) e posto in una provetta da centrifuga conica da 15 ml sterile contenente circa 5 ml di supporti minimi essenziali (MEM) contenenti 1% antibiotici solo a scopo di trasferimento. In seguito trasferimento alle strutture di laboratorio, di trasferire il campione &#39;normale&#39; per un esageratamente, congelare &#39;scatto&#39; e conservare in un freezer -80 ° C per le analisi molecolari futuri. </li><li> Mettere tutti i campioni su ghiaccio bagnato e trasportare immediatamente allo spazio laboratorio per il sezionamento ex vivo tessuto fresco. Non tratto il campione normale, ma piuttosto conservare in un freezer -80 ° C per le analisi molecolari futuri. Trasferire una parte del tumore primitivo e metastasi associato ad una esageratamente, congelare &#39;scatto&#39; e conservare in freezer -80 ° C per le analisi molecolari futuri. </li></ol></li><li> Approvvigionamento di tessuto da studi clinici (ad esempio, centrali opzionale biopsie (CNB)) NOTA BENE: Otteniamo tessuti tumorali da pazientiiscritti ai protocolli clinici che danno il loro consenso a sottoporsi ad un CNB di una metastasi tumorale accessibili prima dell&#39;inizio della terapia. CNB, ad oggi, sono stati effettuati su pazienti con mammella, linfoma della zona marginale, linfoma a cellule del mantello, paraganglioma metastatico, cervice (cellule squamose) e tumori ovarici. <ol><li> Disporre di personale clinici di Radiologia Interventistica o servizio competente di eseguire un pre-trattamento nucleo ago biopsia e posizionare il campione in un 15 ml sterile provetta da centrifuga conica contenente circa 5 ml di supporti essenziale minimo (MEM) contenente 1% antibiotici (penicillina e streptomicina). </li><li> Posizionare il campione di biopsia su ghiaccio bagnato e trasportare immediatamente allo spazio laboratorio per il sezionamento ex vivo tessuto fresco. </li></ol></li></ol> 2 Preparazione dei materiali per le impostazioni sezionamento e vibratome <ol><li> Preparazione dei materiali per il sezionamento <ol><li> Fare una soluzione di agarosio al 4% con l&#39;aggiunta di 4 g di agasalito a 100 ml di PBS. Autoclave (Impostazioni ciclo: liquidi, 121 ° C, 30 min, a 15 libbre per pollice quadrato) la soluzione prima del primo utilizzo. Per gli usi successivi, posizionare l&#39;agarosio 4% in un forno a microonde per liquefare (Alto calore per intervalli di 30 sec) prima di ogni utilizzo e raffreddare adeguatamente a ~ 25 ° C (cioè, caldo al tatto), consapevoli che agarosio solidifica a temperatura ambiente. </li><li> Utilizzare l&#39;agarosio 4% come un supporto di tessuto embedding non permanente. Quando l&#39;agarosio 4% si è raffreddato a sufficienza ed è ancora in uno stato liquido, con una pinza sterile posizionare il campione di tessuto in uno stampo embedding e versare il agarosio 4%. </li><li> Posizionare il campione di tessuto stretti (~ 2 mm di distanza), ma non direttamente sulla parete superficie di taglio dello stampo embedding. L&#39;agarosio 4% inizierà a solidificarsi in fretta, quindi opportunamente posizionare il campione di tessuto in fretta. Durante il sezionamento, mantenere l&#39;agarosio 4% in un bagno d&#39;acqua a 55 ° C per mantenere in soluzione. </li><li> Per una piastra a 24 pozzetti per l&#39;analisi di sezione, aggiungere 1 ml of multimediale completo (ad esempio, MEM + 10% di siero fetale bovino + 1% antibiotici) in ciascun pozzetto. Mantenere la piastra a 24 pozzetti a temperatura ambiente (~ 25 ° C) per tutta la durata di sezionamento. </li></ol></li><li> Preparazione delle impostazioni vibratome <ol><li> Utilizzando il microtomo vibrante, impostare la velocità con cui la lama si avvicina il campione [0 (0,00 mm / sec) a 10 (2,5 mm / sec)] e la frequenza della lama [0 (0 Hz) a 10 (100 Hz) ]. Regolare le impostazioni in base alla consistenza del tessuto (ad esempio, densità / consistenza). </li><li> Per un tumore mammario primario del carcinoma duttale invasivo, regolare le impostazioni a 6 e 3, la velocità e la frequenza rispettivamente. Per tessuto tumorale meno denso / ditta, diminuire la velocità e aumentare la frequenza di conseguenza. </li><li> Impostare lo spessore di taglio entro i limiti dello strumento (1-999 micron). Per queste analisi, impostare lo spessore della sezione a 200 micron. </li><li> Montare il campione agarosio-embedded utilizzando un sottile strato di adesivo liquido sul tha disco dei campionit è immerso in un serbatoio di media che è racchiuso nel ghiaccio bagnato. </li></ol></li></ol> 3 Tissue sezionamento, trattamento e analisi <ol><li> Rimuovere le sezioni di precisione-affettato subito dopo il sezionamento con pinza sterile e posto in un piatto multi-bene con 1 ml di completo supporto. Quando le sezioni sono acquisite sufficienti per la risposta e le analisi correlative, posizionare la piastra multi-bene per la risposta al trattamento in un tessuto culturale incubatore fissato a condizioni di coltura dei tessuti normali. </li><li> Prendere più sezioni di controllo che fermalibri sezioni trattate nel tentativo di confermare la risposta dose-dipendente al contrario di manufatti in coltura. Eseguire tessuto sezionamento rapidamente (~ 30 - 40 minuti), ben al di eventuali effetti negativi del freddo tempo ischemico (~ 2 - 4 ore) 18. Eseguire il trattamento delle sezioni solo dopo un periodo di acclimatazione 1 ora. </li><li> Dopo il periodo di acclimatazione 1 ora, aggiungere dosi crescenti (terapeuticamente rilevanti; <em.&gt; Ad esempio, 0 - 10 micron) concentrazioni di farmaco alle sezioni e la casa in condizioni di coltura di tessuti standard per il periodo di trattamento. Per questo studio, utilizzare i periodi di valutazione hr 24 e / o 48. Tuttavia, il controllo (solo veicolo) sezioni di tessuto rimarranno vitali, come testato in questo studio, per periodi fino al periodi di valutazione 96 hr. </li><li> Dopo il trattamento, rimuovere tutte le sezioni in condizioni sterili e formalina-fix posizionando la sezione di tessuto in una provetta contenente sia il 10% formalina tamponata e posto a RT O / N o in una provetta contenente 4% paraformaldeide (diluito 16% paraformaldeide da un flaconcino con PBS per una concentrazione finale di 4%) e posto a temperatura ambiente per 2 ore. <ol><li> Fissare sezioni di tessuto di grandi dimensioni (ad es., ~ 6 mm x 4 mm) 200 micron di spessore in 10% formalina tamponata O / N a RT. Fissare sezioni più piccole (ad es. 1 mm x 1 mm) 200 micron di spessore in paraformaldeide al 4% per 2 ore a temperatura ambiente. NOTA: Per una penetrazione efficace del fissativo del volume di fixative opportuno 20X il volume del tessuto. Dato che il presente protocollo si traduce in dimensioni estremamente piccole di tessuto (ad esempio 6 mm 3 di tessuto = 6 ml di volume), per la garanzia di un uso corretto fissaggio 1 ml di fissativo per tutte le dimensioni del tessuto. </li><li> Dopo fissazione hanno i campioni di tessuto incluso in paraffina da parte del personale del Dipartimento di Patologia. Sezione esemplare blocchi di paraffina su vetrini caricati per essere ematossilina e eosina macchiato e valutati immunoistochimica (ad esempio, apoptosi). </li></ol></li></ol>

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No conflicts of interest disclosed.

Biologi cancro devono affrontare sfide significative nel tentativo di sviluppare efficaci strategie terapeutiche. Test farmaci in sviluppo su linee cellulari di cancro stabilite non può riflettere con precisione in risposta vivo e in vivo su modelli PDX sono laborioso e molto costoso. Considerato quanto sopra, l&#39;applicazione di tecniche ex vivo di paziente primaria tumori 14, 15 è ora posizionato accanto alla analisi molecolare di consolidate linee cellulari tumorali ...

Lo sviluppo di terapie efficaci cancro ha dimostrato di essere estremamente impegnativo. Linee cellulari tumorali e espianti tumorali - così come xenotrapianti sono stati utilizzati nella ricerca sul cancro per oltre mezzo secolo 1,2,3. Ad oggi, l&#39;analisi molecolare della sensibilità di droga e resistenza in entrambe le linee di cellule di cancro affermati e xenotrapianti di derivazione paziente (PDX) è indispensabile. Tuttavia, i test di composti in linee cellulari di cancro stabiliti spesso non è predittivo di efficacia in vivo, e corrispondenti studi in vivo negli animali, soprattutto nei modelli PDX, è molto costoso e richiede tempo. I limiti di questi sistemi modello, vale a dire l&#39;impossibilità di informare l&#39;influenza del microambiente nativo nella progressione tumorale e la risposta alle strategie terapeutiche, ha portato il campo di ricerca per sviluppare metodi aggiuntivi per complimentarmi con queste analisi. Di recente, accresciuta attenzione viene rivolta verso ex vivo analisi di tum pazienteo espianti 4, 5 a causa della maggiore comprensione che il cancro risposta terapeutica non è esclusiva alla composizione molecolare intrinseca delle cellule tumorali, ma piuttosto è fortemente influenzato dal microambiente delle cellule tumorali 6, 7, una caratteristica che non può essere riassunta con metodi di coltura tradizionali e / o PDX. ex vivo analisi nel contesto di cui sopra (ad esempio., influenza adiacente tumorale circostante microambiente cellulare) implica la valutazione delle sezioni tumore / metastasi primarie vitali, piuttosto che ex vivo analisi degli isolati cellulari 8, 9. Riportiamo qui su una tecnica ex vivo (ad esempio., Sezioni di precisione-fette fresche di tessuto) di entrambi i tumori primari del paziente e metastasi associate (ad esempio, linfonodi) che segnala fedelmente alla risposta (IC50), gli effetti off-target e consente molecolare analisi dei meccanismi di resistenza e di feedback. Inoltre, l&#39;analisi correlativa di therapeUTIC sensibilità / resistenza contro biomarker e profilo di espressione genica può essere eseguita in uno sforzo per identificare i pazienti più probabilità di rispondere al farmaco sperimentale di interesse (ad esempio., la risposta alle alte farmaco corrisponde paziente con particolare profilo biologico). Applicazione della tecnica ex vivo e la valutazione in modo multi-parametro è movimento verso la selezione dei pazienti e il miglioramento complessivo dei risultati clinici. Trattamento ex vivo analisi di risposta potrebbe diventare uno strumento standard per lo sviluppo preclinico e clinico di terapie del cancro ed è immaginato come un passo verso un approccio di medicina personalizzata nelle strategie di sviluppo terapeutiche.

<table><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr></tbody></table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Per questo studio, la tecnica ex vivo è stato utilizzato in un&#39;analisi correlativa terapeutico sensibilità / resistenza di un inibitore della heat shock protein 90 (Hsp90i). In una valutazione preclinica di questo Hsp90i, il cancro al seno tumore primario, un ER + invasivo carcinoma duttale (IDC), e associata metastasi linfonodali sono stati analizzati ex-vivo per la risposta al trattamento &#160; (Figura 1). Più di 200 micron sezioni seriali sono stati trattati...

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Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Fondata linee cellulari tumorali e xenotrapianti sono stati il ​​cardine della ricerca sul cancro negli ultimi decenni. Tuttavia, recenti evidenze suggeriscono che la risposta terapeutica è fortemente influenzata dal microambiente delle cellule tumorali. Di conseguenza, abbiamo sviluppato una analisi ex vivo di campioni tumorali primarie per scopi di sviluppo della droga.

Ex Vivo risposta al trattamento di tumori primari e / o associati per metastasi preclinici e sviluppo clinico di Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

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Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.7K Views.  Memorial Sloan Kettering Cancer Center.  Fondata linee cellulari tumorali e xenotrapianti sono stati il ​​cardine della ricerca sul cancro negli ultimi decenni. Tuttavia, recenti evidenze suggeriscono che la risposta terapeutica è fortemente influenzata dal microambiente delle cellule tumorali. Di conseguenza, abbiamo sviluppato una analisi ex vivo di campioni tumorali primarie per scopi di sviluppo della droga. 

Video: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Vivo Imaging di risposte droga nel microambiente tumorale in modelli murini di cancro al seno

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Ricerca

Medicina

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 ...

Ricerca sul cancro

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

Test delle terapie mirate nel cancro utilizzando l'analisi dell'alterazione strutturale del DNA e gli Xenogrini derivati dal paziente

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Uso combinato dei saggi di metastasi della vena della coda e dell'imaging in vivo in tempo reale per quantificare la colonizzazione metastatica del cancro al seno e il carico nei polmoni

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Misurazione della risposta dei farmaci in tempo reale nelle fette di tessuto tumorale organotipico

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

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. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

Espianti tumorali derivati dal paziente come piattaforma preclinica "live" per prevedere la resistenza ai farmaci nei pazienti

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Profilazione della sensibilità alle terapie mirate negli organoidi derivati dal paziente NSCLC EGFR-Mutant

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Screening multiparametrico di farmaci organoidi tumorali mediante imaging a cellule vive a campo ampio per analisi di massa e organoidi singoli

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Valutare l&#39;efficacia delle Cancer Drug Sensibilizzazione<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Ex Vivo risposta al trattamento di tumori primari e / o associati per metastasi preclinici e sviluppo clinico di Therapeutics

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