Name: development and maintenance of a preclinical patient derived tumor xenograft model for the investigation of novel anti-cancer therapies
Uploaded: 2016-09-30T13:00:00
Description: Watch this Scientific Journal Video about Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies at JoVE.com

This work was supported by grant 1R01CA152303-01.

Declaración de Ética: las muestras de tumor de adenocarcinoma colorrectal derivados de los pacientes se obtuvieron de pacientes que consienten en la Universidad de Colorado Hospital, de acuerdo con un protocolo aprobado por la Junta de Revisión Institucional múltiple Colorado (08 a 0439). Todo el trabajo de los animales se realizó en animales protocolos aprobados por la Universidad de Colorado en Denver Institucional Cuidado de Animales y el empleo Comisión (IACUC, Protocolo # 51412 (06) 1E y 96813 (04) 1E). <...

<ol>
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La plataforma de descubrimiento de fármacos PDTX ofrece un modelo mejorado de las deficiencias de otros modelos preclínicos que son poco fiables para predecir la actividad clínica de nuevos compuestos. Es importante destacar que los tumores en este modelo son biológicamente estable, retener potencial metastásico, y muestran la capacidad de respuesta droga similar de generación en generación. En este modelo, los tumores de pacientes derivada se inyectan en ratones desnudos atímicos, pases, y posteriormente utiliz...

El cáncer colorrectal (CCR) es un importante contribuyente a las muertes por cáncer en los Estados Unidos. En 2015, había un estimado de 132.700 nuevos casos de CCR con 49.700 muertes 1. Aunque el pronóstico en pacientes con enfermedad localizada es excelente, los pacientes con enfermedad avanzada tienen resultados pobres, haciendo de esta una de las principales prioridades en el desarrollo de nuevas terapias. A pesar de los regímenes estándar de cuidado quimioterapéuticos y biológicos más nuevos que se implementan contra esta enfermedad, sólo ha habido un aumento gradual en la supervivencia global. En consecuencia, hay un esfuerzo significativo en la comprensión de las vías conductor implicado en facilitar el crecimiento del tumor en esta enfermedad. La Red Atlas del Genoma del Cáncer ha identificado recientemente numerosas vías principales que están implicados en la desregulación y CRC incluyen: WNT, fosfoinosítido 3-quinasa (PI3K), RAS, factor de crecimiento transformante-β (TGF-β) y TP53 2. Juntos, con la descripción de las investigaciones otsus vías que potencien el crecimiento en el CCR han encendido el desarrollo de nuevas terapias destinadas a mejorar significativamente la supervivencia en esta población de pacientes 3-5. Utilizando modelos preclínicos en el desarrollo de fármacos oncológicos han sido esenciales en este proceso en la predicción de la actividad clínica de estos nuevos compuestos. Varios modelos preclínicos se han utilizado en el proceso de desarrollo de fármacos. Teniendo en cuenta que los modelos animales preclínicos transgénicas y líneas celulares inmortalizadas no han tenido éxito en la determinación de la actividad clínica de nuevas terapias de oncología, en gran parte debido a su incapacidad para reflejar la complejidad de los tumores humanos, se han establecido derivado del paciente de xenoinjertos de tumores (PDTX) modelos. La mayor ventaja de este modelo es que la heterogeneidad del tumor se mantiene intacto y refleja fielmente las características moleculares y clonalidad del tumor del paciente 6-9 originario. PDTX modelos proporcionan una excelente in vivoplataforma preclínico para el estudio de nuevos agentes, las vías de resistencia a fármacos, estrategias combinatorias, y la biología de células madre del cáncer 10. Una visión general del proceso de PDTX se ilustra en la Figura 1. Se inicia en la clínica, consintiendo pacientes para permitir que parte de su exceso de tejido tumoral que se utilizará para esta investigación. A continuación, en la cirugía, un pedazo del tumor se ganó en total por un patólogo y puesto en los medios de comunicación para ser transportado a personal de investigación. Inmediatamente después de esto, una sección del tumor se corta en trozos pequeños y trasplantado en ratones inmunodeficientes subcutáneamente. Una vez que el tumor crece, es pasado en diferentes generaciones de ratones con el fin de mantener el tumor 10. Normalmente, después de la generación F3 del tumor se puede ampliar en un estudio de tratamiento donde se evalúan nuevos compuestos y / o terapias combinadas. Utilizando Next Gen SEQ (Exoma Seq, ARN SEQ y SNP array) potenciales biomarcadores predictivos son descubrired que ayudan en la selección de pacientes que podrían beneficiarse de la de un tratamiento particular. Los objetivos generales de la utilización de modelos PDTX son los siguientes: 1) evaluar la eficacia de las terapias innovadoras como agente único o en combinación y 2) identificar biomarcadores predictivos de sensibilidad o resistencia antes de la investigación clínica. En este manuscrito, proporcionamos la metodología en la iniciación y mantenimiento de un banco CRC PDTX y proporcionar las ventajas y limitaciones de este modelo de desarrollo descubrimiento de fármacos. <img alt="Figura 1" src="/files/ftp_upload/54393/54393fig1.jpg" /> Figura 1. Descripción general del modelo de protocolo CRC PDTX. Un tumor derivado del paciente se recibe de la cirugía e inmediatamente se inyecta en ratones desnudos atímicos por vía subcutánea. Una vez que el tumor crece, se expande en las generaciones posteriores y, finalmente, se expandió por los estudios de tratamiento. tratamiento RESPOns son evaluados y biomarcadores predictivos son identificados que pueden ayudar en la selección de los pacientes. <a href="https://www.jove.com/files/ftp_upload/54393/54393fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI or DMEM</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:499px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Non-essential Amino Acids</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">35-010-CV</td>
			<td>Thaw in -4 &#176;C, then activate for 30 minutes at 60 &#176;C water bath</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CPT blood tube</td>
			<td style="width:81px;">BD vacutainer</td>
			<td style="width:119px;">362761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube</td>
			<td style="width:81px;">Surelock</td>
			<td style="width:119px;">A-7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phospate-Buffered Saline</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cyrogenic vials</td>
			<td style="width:81px;">Cyroking</td>
			<td style="width:119px;">C0732901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic tumor cutting dish</td>
			<td style="width:81px;">Trueline</td>
			<td style="width:119px;">TR4001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:81px;">Roboz</td>
			<td style="width:119px;">RS-5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:81px;">Roboz</td>
			<td style="width:119px;">RS-5135</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Matrigel (gelatinous protein mixture)</td>
			<td style="width:81px;">Corning</td>
			<td style="width:119px;">354234</td>
			<td>Store at -20 or -80 &#176;C, then thaw on ice, do not leave at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Formalin cups</td>
			<td style="width:81px;">Protocol</td>
			<td style="width:119px;">032-059</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Liquid Nitrogen Dewar Storage</td>
			<td style="width:81px;">Thermolyne</td>
			<td style="width:119px;">CY50900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable liquid nitrogen dewar</td>
			<td style="width:81px;">Nalgene</td>
			<td style="width:119px;">4150-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:81px;">Fischer</td>
			<td style="width:119px;">67-68-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Freezing container: Mr Frosty</td>
			<td style="width:81px;">Nalgene</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropyl Alcohol</td>
			<td style="width:81px;">Decon</td>
			<td style="width:119px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Trocars</td>
			<td style="width:81px;">Innovative Research of America</td>
			<td style="width:119px;">MP-182</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anesthesia machine</td>
			<td style="width:81px;">Patterson Veterinary</td>
			<td style="width:119px;">none</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anesthesia box</td>
			<td style="width:81px;">Patterson Veterinary</td>
			<td style="width:119px;">none</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:81px;">Vet one</td>
			<td style="width:119px;">1038005</td>
			<td></td>
		</tr>
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development and maintenance of a preclinical patient derived tumor xenograft model for the investigation of novel anti-cancer therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Similitudes de mutaciones comunes en los modelos CRC PDTX y el TCGA Hemos investigado si el porcentaje de mutaciones comunes (KRAS, las ANR, BRAF, PIK3CA, APC, CTNNB1 y TP53) en el banco CRC PDTX eran representativas de la frecuencia de mutación se observa en la población de pacientes de CRC. Como se muestra en la Figura 2A (TCGA) y B (CRC banco PDTX), la frecuencia...

Watch this Scientific Journal Video about Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies at JoVE.com

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

La utilización de los tumores derivados del paciente en un modelo preclínico subcutánea es una manera excelente para estudiar la eficacia de nuevas terapias, el descubrimiento de biomarcadores predictivos y vías resistentes a los fármacos. Este modelo, en el proceso de desarrollo de fármacos, es fundamental para determinar el destino de muchas nuevas terapias contra el cáncer antes de la investigación clínica.

Desarrollo y mantenimiento de un tumor del paciente preclínicos Derivado modelo de xenoinjerto para la Investigación de Novela Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

The overall goal of this subcutaneous Patient Derived Tumor Xenograft procedure is to the study the efficacy of novel therapies predictive biomarker discovery and drug resistant pathways in tumors. This method can help answer key questions in the oncology field about whether a particular drug exhibits activity against a specific tumor type. The main advantage of this technique is that Patient Derived Tumor Xenographs maintain the molecular, genetic and histologist heterogeneity typical of the tumors of origin, making the model more clinically relevant.Demonstrating the procedure will be Stacey Bagby, a professional research assistant and certified veterinary technician from our laboratory. After collecting one to two milliliters of blood in a blood cell separation tube containing sodium citrate, centrifuge the samples and transfer the peripheral blood mononuclear cell layer into a 1.5 milliliter micro-centrifuge tube. Fill the tube to the top with sterile PBS.Then, spin down the cells again. Followed by a quick wash with one milliliter of PBS, being careful not to disturb the PMBC pellet. Then, use a pipette to transfer the plasma from the blood cell collection tube to a 1.5 milliliter sterile cryogenic vial, and store the plasma and the PBMC pellet at negative 80 degrees Celsius.Transfer the human tumor tissue to the animal facility in a sterile cup containing complete medium and place the cup in a laminar flow hood. As soon as possible, after harvesting, transfer the tissue into a sterile plastic cutting dish in a small amount of the retrieval medium. Then, using autoclave sterilized small scissors and forceps, cut the tissue into approximately three by three by three milliliter pieces and place 10 to 12 pieces into an autoclave sterilized 1.5 milliliter micro-centrifuge tube containing 300 microliters of gelatinous protein mixture solution on ice for the implantation.For flash frozen storage, transfer the appropriate number of tumor pieces into a new 1.5 milliliter micro-centrifuge tube and place the vial in a liquid nitrogen doer. Then, place in a minus 80 degrees Celsius freezer. For formal infixing and paraffin embedding, place three small tumor pieces into a 10 milliliter 10%formalin cup for 24 hours.After the tissues are processed into paraffin embedded blocks, transfer any remaining tissue pieces into a cryogenic tube. Then, add one milliliter of complete medium supplemented with 10%DMSO to the tissue and store the tubes on ice until they can be transferred into an isopropyl alcohol freezing container. And put that container in a minus 80 degrees Celsius freezer.To inject the tumor tissue, load the pieces of tumor stored in the gelatinous protein mixture solution into autoclaved 12-gauge trocars, taking care that each tumor piece is completely inserted into the trocar. Next, confirm a lack of response to toe pinch in a six to eight week old athymic, nude mouse and transfer the mouse onto a clean field in a sterile laminar flow hood. Deliver the F0 tumor subcutaneously to the dorsal neck region and to each side of the mouse's flank, followed by the subcutaneous administration of analgesia distal to one of the flank sites of tumor injection.Then, place the mouse back in its cage with monitoring until it is fully recovered. Monitor the growth and health of the xenograph-implanted mice at least once per week, recording the tumor sizes, tumor generation, date of tumor injection, and health of the mice in an appropriate tracking system. When a tumor reaches approximately 1, 500 to 2, 000 cubic millimeters in size, use autoclaved scissors and forceps to excise the tumor and passage the F1 tumor into the next generation of five animals, as just demonstrated, taking care to collect flash frozen formalin and viable tumor samples as demonstrated for each tumor.When the remaining mice from the F0 tumor group exhibit tumors approximately 1, 500 to 2, 000 cubic millimeters in size, collect these tumors as just demonstrated for collection. When the stage F15 tumor generation is reached, use the earliest generation of viable tumor pieces available in liquid nitrogen storage for the next series of tumor injections. When the desired tumor is very large, 1, 500 to 2, 000 cubic millimeters, follow the procedures above to collect the tumor to expand it into the desired amount of mice.Dose the mice with the drug and weigh and measure them twice per week. Randomize the mice into treatment groups with 10 tumors per group, and a group tumor volume average of 100 to 300 cubic millimeters within a few standard deviations of each other. Then, sort the mice into the appropriate groups for treatment.Dual color fluorescence in situ hybridization for the human and mouse caught one DNA demonstrates that the human stromal cells in the F0 tumor are replaced by the mouse stromal cells in the F1 tumor. Whereas, all of the human-derived hepatocyte growth factor in the F0 generation is replaced with mouse hepatocyte growth factor in F1 generation, the vascular endothelial growth factor ligand consists of both human and mouse protein in the F1 generation. The treatment of different generations of the same tumor does not change the treatment response.With the resistance or sensitivity of the tumor appearing to be tumor strain rather than tumor generation specific. Further, the frequency of mutations in these commonly mutated genes is very similar between the cancer genome atlas and the colorectal patient-derived tumor xenograph bank. Suggesting that the common mutations observed in the colorectal patient population are well represented in the colorectal patient-derived xenotransplant model.Once mastered, this technique can be completed in one to two hours, if performed properly. While attempting this procedure, it is important to have a cohesive clinical team for identifying and obtaining consent from the cancer patients and for the removal and grossing of the tumor tissue. Following this procedure, other methods, like the investigation of anti-cancer compounds as single or combination agents, can be performed to answer additional questions about the viability of these strategies for patients with a particular tumor type.After its development, this technique paved the way for researchers in the field of oncology to explore novel cancer therapeutics, tumor heterogeneity, treatment resistant mechanisms, cancer stem cell biology, and tumor stromal interactions. After watching this video, you should have a good understanding on how to develop and maintain a patient derived tumor xenograph model for evaluating novel cancer therapeutics.

Research

JoVE Journal

Cancer Research

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

Etiquetado de los xenoinjertos derivados de los pacientes con cáncer de mama Trazables Reporteros para el crecimiento tumoral y la metástasis Estudios

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Investigación

Investigación sobre el cáncer

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Un carcinoma de células singénicas modelo de ratón de la renales metastásico para la evaluación cuantitativa y longitudinal de preclínicos sobre Terapias

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

Engraftment de tumor en un modelo de ratón de xenoinjerto de linfoma de manto humano

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Establecimiento de un cultivo primario de Sarcoma de tejidos blandos derivados del paciente

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

Digital de la reacción en cadena de polimerasa para la variación genética en un paciente de la poliposis adenomatosa familiar esporádico con el formato de Chip en tubo

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Investigación de las dependencias genéticas usando análisis CRISPR Cas9 basado en competencia

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Modelos de xenoinjerto ortotópico derivados del paciente para el carcinoma de células uroteliales humanas y el crecimiento tumoral del cáncer colorrectal y la metástasis espontánea

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Modelo de xenoinjerto heterogéneo derivado del paciente del cáncer de páncreas utilizando larvas de pez cebra como anfitriones para la evaluación comparativa de fármacos

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

Un modelo de xenoinjerto derivado del paciente de melanoma

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

Establecimiento de modelos de xenoinjerto saqueos derivados del paciente y líneas celulares primarias

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...