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.

HINWEIS: Patientengewebebeschaffung wurde durch institutionell Review Board (IRB) zugelassenen -zugelassene Biospecimen und Klinische Protokolle (Protokoll-Nummern 09-121 und 11-041, beziehungsweise) am Memorial Sloan Kettering Cancer Center. 1. Gewebebeschaffung <ol><li> Beschaffung von Patienten Primärtumor / Metastasen HINWEIS: Bis heute hat dieses Protokoll auf chirurgisch entfernt Pankreas, Magen-und Brusttumorarten, sowie, Lymphom Metastasen durchgeführt. <ol><li> Richten Sie die OP-Team, um die Probe per Kurier oder Rohrpostsystem der Pathologie-Abteilung in dicht verschlossenen und sterile dicht Kunststoffbeutel in sterilen Probenspritzwassergeschützte Behälter zu liefern. Richten Sie die OP-Team, um die Probe im frischen Zustand ohne Formalin oder Fixiermittel zu transportieren. </li><li> Richten Sie den Pathologen oder Pathologe Assistent der frischen Probe ernten mit sterilen Technik, die incLudes Verwendung von sterilen Handschuhen und Instrumente unter einer Steril. </li><li> Notieren Sie sich die Erntezeit der beschafften Probe, die streng unter 30 min von der Beendigung des chirurgischen Eingriffs gehalten wird. Unter Berücksichtigung der Effekte kalten Ischämie, Proben nehmen Sie nicht aus reseziert Proben mit einer kalten Ischämiezeit von&gt; 30 min 18. </li><li> Leiten der Pathologie Personal an eine primäre Tumorprobe von etwa 0,5 cm3 bis 1,0 cm 3 in einem Abzug mit laminarer Strömung zu entfernen, um eine sterile Umgebung aufrechtzuerhalten. Wenn möglich, wählen Tumorgewebe von der Peripherie der Läsion Indexpotential Frank zentrale Nekrose (Zelltod) zu vermeiden. HINWEIS: Die nekrotischen Gewebe kann grob durch eines der folgenden Kriterien erkennbar sein: Verlust von Farbe oder Blässe des Gewebes; Verlust von Kraft, in dem nekrotischen Gewebe ist weich und brüchig; eine deutliche Abgrenzung zwischen nekrotischen und lebensfähiges Gewebe. </li><li> Richten Sie die pathologist oder Pathologe Assistent peripheren Gewebe, das im Überschuss (chirurgische Ablage) ist nach sofortiger Abtastung des Tumor für Diagnostik liefern. Die Probe wird in einem 15 ml sterilen konischen Zentrifugenröhrchen mit annähernd 5 ml Minimalmedium (MEM), das 1% Antibiotika (Penicillin und Streptomycin). </li><li> Falls vorhanden (zB Lymphknoten Achsel Schwanz Mastektomie Probe), beschaffen grob positive Lymphknoten assoziiert Probe der gleichen Größe (0,5 cm 3 bis 1,0 cm 3) und zu vergleichen, um das Ansprechen auf Medikamente des Primärtumors. Wie die Primärtumorprobe, legen die zugehörigen Lymphknoten Probe in einem 15 ml sterilen konischen Zentrifugenröhrchen mit annähernd 5 ml Minimalmedium (MEM), das 1% Antibiotika (Penicillin und Streptomycin). </li><li> Wenn die Größe der chirurgischen Probe Genehmigungen (zB Mastektomie Probe), zu entfernen (entfernt von Primärtumor) einer Probe von normalem Gewebe (dhnormalen dicht / Faser Brustparenchym) und in eine 15 ml sterilen konischen Zentrifugenröhrchen mit etwa 5 ml Minimalmedium (MEM), die 1% Antibiotika für nur Übertragungszwecken. Nach dem Transfer auf die Laboreinrichtungen, übertragen Sie die &quot;normalen&quot; Probe einem Kryoröhrchen, &quot;Snap&quot; einzufrieren und an einem -80 ° C Gefrierschrank für zukünftige molekulare Analysen. </li><li> Legen Sie alle Proben auf nassem Eis und sofort in den Laborraum für ex vivo frischem Gewebe Schnitte zu transportieren. Abschnitt nicht die normale Probe, sondern in einem -80 ° C nicht speichern Gefrierschrank für zukünftige molekulare Analysen. Übertragen einen Teil des Primärtumors und der zugehörigen Metastasen in einem Kryoröhrchen, &quot;Snap&quot; einzufrieren und an einem -80 ° C Gefrierschrank für zukünftige molekulare Analysen. </li></ol></li><li> Beschaffung von Gewebe aus klinischen Studien (zB optional Kernnadelbiopsien (CNB)) HINWEIS: Wir erhalten Tumorgewebe von Patientenauf klinische Protokolle, die ihre Zustimmung zu einer CNB einer zugänglichen Tumormetastasierung vor Beginn der Therapie zu unterziehen geben eingeschrieben. CNBs bis heute haben sich auf Patienten mit Brust-, Randzone Lymphom, Mantelzell-Lymphom, Metastasen paraganglioma, des Gebärmutterhalses (Plattenepithelkarzinom) und Eierstockkrebs durchgeführt. <ol><li> Haben klinische Personal aus Interventionelle Radiologie oder Fachabteilung führen eine Vorbehandlung Stanzbiopsie und platzieren Sie die Probe in einem 15 ml konischen sterile Zentrifugenröhrchen mit etwa 5 ml Minimalmedium (MEM), die 1% Antibiotika (Penicillin und Streptomycin). </li><li> Legen Sie die Biopsie auf nassem Eis und sofort auf den Laborraum für ex vivo frischem Gewebe Schnitte zu transportieren. </li></ol></li></ol> 2. Herstellung von Materialien für Schnitte und Vibratom Einstellungen <ol><li> Vorbereitung der Materialien für die Schnitte <ol><li> Stellen Sie einen 4% igen Lösung durch Zugabe von 4 g agastieg auf 100 ml PBS. Autoklaven (Zykluseinstellungen: flüssig, 121 ° C, 30 min, 15 Pfund pro Quadratzoll) die Lösung vor dem ersten Gebrauch. Für nachfolgende Verwendungen, legen Sie die 4% Agarose in der Mikrowelle auf (hohe Hitze für 30 Sekunden-Intervallen) vor jeder Verwendung zu verflüssigen und kühlen angemessen auf ~ 25 ° C (dh warm an) bewusst, dass Agarose erstarrt bei RT. </li><li> Verwenden Sie die 4% Agarose als nicht-ständiges Einbettung Gewebe Medien. Wenn die 4% Agarose ausreichend abgekühlt ist und sich noch in einem flüssigen Zustand mit einer sterilen Pinzette Platzieren der Gewebeprobe in einem Einbettmedium Form gießen und in 4% Agarose. </li><li> Die Gewebeprobe in der Nähe (ca. 2 mm Abstand), aber nicht direkt in der Schneidfläche Wand der Einbettung Form. Die 4% Agarose beginnt schnell zu verfestigen, damit entsprechend der Gewebeprobe zu positionieren schnell. Während des Schneidens, halten Sie die 4% Agarose in einem 55 ° C Wasserbad in Lösung zu halten. </li><li> Zu einem 24-Well-Platte für Schnittanalyse, 1 ml of Vollmedien (dh MEM + 10% fötales Rinderserum + 1% Antibiotika) in jede Vertiefung. Halten Sie die 24-Well-Platte bei RT (~ 25 ° C) während der Dauer der Schneiden. </li></ol></li><li> Vorbereitung der Vibratom Einstellungen <ol><li> Verwendung der vibrierenden Mikrotom, stellen Sie die Geschwindigkeit, mit der die Klinge nähert die Probe [0 (0,00 mm / s) auf 10 (2,5 mm / sec)] und die Frequenz der Klinge [0 (0 Hz) bis 10 (100 Hz) ]. Passen Sie die Einstellungen nach der Beschaffenheit des Gewebes (dh Dichte / Festigkeit). </li><li> Für einen primären Brusttumor von invasiven duktalen Karzinom, passen Sie die Einstellungen bis 6 und 3, Geschwindigkeit und Häufigkeit auf. Für weniger dicht / Firma Tumorgewebe, reduziert sich die Geschwindigkeit entsprechend zu erhöhen und Frequenz. </li><li> Stellen Sie die Schnittdicke innerhalb der Grenzen Instrument (1-999 um). Für diese Analysen, stellen Sie die Schnittdicke auf 200 um. </li><li> Montieren Sie die Agarose eingebettete Probe mit einer dünnen Schicht aus flüssigem Klebstoff auf der Objektplatte that in einem Reservoir von Medien, die in nassem Eis eingeschlossen ist taucht. </li></ol></li></ol> 3. Gewebe Schnitte, Behandlung &amp; Analysen <ol><li> Sofort nach Schneiden mit einer sterilen Pinzette und in eine Multi-Well-Platte mit 1 ml Vollmedium entfernen Sie die präzise geschnittenen Abschnitte. Wenn genügend Abschnitte sind für die Antwort und korrelativen Analysen erworben, legen Sie die Multi-Well-Platte zur Behandlung Reaktion in einem Gewebekulturbrutschrank bei Standardgewebekulturbedingungen eingestellt. </li><li> Nehmen mehrere Steuerabschnitte, die die behandelten Bereiche in dem Bemühen, eine dosisabhängige Reaktion zu bestätigen, im Gegensatz zu kultivierten Artefakte Buchstütze. Führen Schneiden schnell Gewebe (~ 30 - 40 min), der in jedes nachteiligen Auswirkungen der kalten Ischämiezeit (~ 2-4 h), 18. Führen Behandlung der Abschnitte erst nach einer Eingewöhnungszeit 1 Stunde. </li><li> Nach der Eingewöhnungszeit 1 Stunde, fügen steigenden Dosen (therapeutisch relevant ist; <em.&gt; ZB 0 - 10 uM Konzentrationen) von Arzneimittel zu den Abschnitten und Haus unter Standardzellkulturbedingungen für die Behandlungsdauer. Für diese Studie verwenden 24 und / oder 48 Stunden Veranlagungszeiträume. Jedoch wird die Kontrolle (nur Vehikel) Gewebeschnitte lebensfähig bleiben, wie in dieser Studie getestet, für Zeiträume bis zu einschließlich 96 Stunden Beurteilungszeiträume. </li><li> Nach der Behandlung entfernt alle Abschnitte steril und Formalin-fix, indem der Gewebeabschnitt in einer Röhre, die entweder 10% gepuffertem Formalin und Stelle bei RT O / N oder in einem Rohr, enthaltend 4% Paraformaldehyd (Verdünnung 16% Paraformaldehyd aus einem Fläschchen mit PBS für eine endgültige Konzentration von 4%) und Platz bei RT für 2 Stunden. <ol><li> Fix größeren Gewebeschnitten (zB., ~ 6 mm x 4 mm) 200 um dick in 10% Formalin O / N bei RT. Beheben kleinere Abschnitte (beispiels., 1 mm x 1 mm), 200 um dick, in 4% Paraformaldehyd 2 h bei RT. HINWEIS: Für eine effiziente Penetration der Fixierungs das Volumen von fixative sollte das Volumen des Gewebes 20x werden. Angesichts, dass dieses Protokoll zu extrem kleinen Gewebe Größen (zB 6 mm 3 Gewebe = 6 ul Volumen), für Sicherung der richtige Fixierung Verwendung von 1 ml Fixiermittel für alle Gewebe Größen. </li><li> Nach der Fixierung müssen die Gewebeproben in Paraffin durch die Pathologie-Abteilung Personal eingebettet. Abschnitt Probe Paraffinblöcke auf geladene Objektträger zu Hämatoxylin und Eosin gefärbt und bewertet immunhistochemisch (zB Apoptose) sein. </li></ol></li></ol>

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No conflicts of interest disclosed.

Krebs Biologen vor großen Herausforderungen, wenn man versucht, wirksame therapeutische Strategien zu entwickeln. Testen von Medikamenten in der Entwicklung auf etablierte Krebszelllinien nicht genau in vivo Reaktion und in vivo Versuchen an PDX Modelle sind arbeitsintensiv und sehr teuer. Angesichts des Vorstehenden wird die Anwendung der Ex-vivo-Techniken der Primärtumoren Patienten 14, 15 wird nun neben der molekularen Analyse von etablierten Tumorzelllinien und von einem Patie...

Entwicklung wirksamer Krebstherapeutika hat sich als äußerst schwierig. Krebszelllinien und Tumor Explantate - ebenso wie Heterotransplantate wurden in der Krebsforschung seit über einem halben Jahrhundert 1,2,3 verwendet. Bis heute ist die molekulare Analyse von Arzneimittelempfindlichkeit und Widerstand in beiden etablierten Krebszelllinien und Patienten abgeleitete Xenografts (PDX) unverzichtbar. Jedoch ist der Test-Verbindungen in etablierte Krebszelllinien oft nicht prädiktiv für die Wirksamkeit in vivo, und die entsprechenden in vivo-Untersuchungen an Tieren, insbesondere in PDX Modelle, ist sehr teuer und zeitaufwendig. Die Grenzen dieser Modellsysteme, nämlich der Unfähigkeit, über den Einfluss der nativen Mikroumgebung der Tumorprogression und der Reaktion auf therapeutische Strategien zu informieren, hat das Forschungsfeld, um zusätzliche Methoden, um diese Analysen zu entwickeln Kompliment geführt. Der letzten, ist erhöhte Aufmerksamkeit in Richtung Ex-vivo-Analyse von Patienten TUM bezahltoder Explantaten 4, 5 aufgrund der besseren Verständnis, dass Krebs therapeutische Antwort nicht exklusiv ist, um die inhärente molekulare Zusammensetzung von Krebszellen, sondern wird stark durch die Tumorzelle Mikro 6, 7 eine Funktion, die nicht durch herkömmliche Kultivierungsverfahren und rekapitulieren beeinflusst / oder PDX. Ex-vivo-Analyse in der obigen Kontext (dh., Einfluss der benachbarten Umgebung von Tumorzellen Mikroumgebung) impliziert Bewertung von lebensfähigen primären Tumor / Metastasen Abschnitte, statt ex vivo Analyse zellulärer Isolate 8, 9. Wir berichten hier über einen Ex-vivo-Technik (dh., Präzise geschnittenen frischen Gewebeschnitten) der beiden Patienten Primärtumoren und Metastasen assoziiert (dh Lymphknoten), die treu informiert über Antwort (IC50), Off-Target-Effekte und ermöglicht für die molekulare Analyse von Widerstand und Feedback-Mechanismen. Zusätzlich wird ein Korrelat Analyse therapeUTIC Empfindlichkeit / Resistenz gegen Biomarker und Genexpressionsprofil kann in dem Bemühen, Patienten eher zu dem experimentellen Medikament von Interesse reagieren zu identifizieren durchgeführt werden (dh., hohe Medikamentenantwort passt Patienten mit bestimmten biologischen Profil). Anwendung der Ex-vivo-Technik und Bewertung in einer Multi-Parameter-Mode ist Bewegung in Richtung Patientenauswahl und die allgemeine Verbesserung der klinischen Ergebnisse. Ex-vivo-Behandlung Response-Analyse konnte ein Standardwerkzeug in der präklinischen und klinischen Entwicklung von Krebstherapeutika zu werden und wird als ein Schritt hin zu einer personalisierten Medizin Ansatz in der therapeutischen Entwicklungsstrategien in Betracht gezogen.

<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.

Für diese Studie wurde die Ex-vivo-Technik in einem korrelativen Analyse der therapeutischen Empfindlichkeit / Widerstand eines Hitzeschockprotein 90-Inhibitor (Hsp90i) verwendet. In einer präklinischen Bewertung dieses Hsp90i, den Primärtumor Brustkrebs, einem ER + invasive duktale Karzinom (IDC) und die zugehörigen Lymphknoten-Metastasen wurden ex vivo zur Behandlung Reaktion analysiert &#160; (Abbildung 1). Mehrere 200 um serielle Schnitte wurden mit Vehikel all...

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

Gegründet Krebszelllinien und Heterotransplantate waren die Hauptstütze der Krebsforschung in den letzten Jahrzehnten. Jedoch schlägt letzten Beweis, dass therapeutische Reaktion wird durch die Tumorzelle Mikro beeinflusst. Daher haben wir eine ex vivo-Analyse des Primärtumorproben für die Arzneimittelentwicklung Zwecke entwickelt.

Ex-vivo-Ansprechen auf die Behandlung von Primärtumoren und / oder assoziierten Metastasen für die präklinische und klinische Entwicklung von Therapeutika

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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Research

JoVE Journal

Medicine

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

14.7K Aufrufe.  Memorial Sloan Kettering Cancer Center.  Gegründet Krebszelllinien und Heterotransplantate waren die Hauptstütze der Krebsforschung in den letzten Jahrzehnten. Jedoch schlägt letzten Beweis, dass therapeutische Reaktion wird durch die Tumorzelle Mikro beeinflusst. Daher haben wir eine ex vivo-Analyse des Primärtumorproben für die Arzneimittelentwicklung Zwecke entwickelt. 

Serie: 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.

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

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

Forschung

Medizin

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.

Generierung von orthotopischen Pankreastumoren und Ex-vivo-Charakterisierung der tumorinfiltrierenden T-Zell-Zytotoxizität

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

Krebsforschung

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.

Testen von gezielten Krebstherapien mit struktureller DNA-Alterationsanalyse und patientenabgeleiteten Xenografts

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.

Kombinierte Verwendung von Tail Vein Metastasis Assays und Real-Time In Vivo Imaging zur Quantifizierung der metastasierenden Kolonisation und Belastung von Brustkrebs in der Lunge

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.

Messung der Echtzeit-Arzneimittelreaktion in organotypischen Tumorgewebescheiben

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.

Orthotope Transplantation von Brusttumoren als präklinische Modelle für Brustkrebs

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.

Patienten-abgeleitete Tumor-Explantate als "Live" präklinische Plattform zur Vorhersage von Arzneimittelresistenzen bei Patienten

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.

Profiling der Sensitivität gegenüber zielgerichteten Therapien in EGFR-mutierten NSCLC-patientenabgeleiteten Organoiden

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.

Multiparametrisches Tumor-Organoid-Wirkstoff-Screening mit Weitfeld-Live-Cell-Imaging für die Bulk- und Einzelorganoid-Analyse

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.

Bewertung der Wirksamkeit der Krebsmedikament Sensibilisierung<em&gt; In-vitro-</em&gt; Und<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-Ansprechen auf die Behandlung von Primärtumoren und / oder assoziierten Metastasen für die präklinische und klinische Entwicklung von Therapeutika

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