Name: a melanoma patient-derived xenograft model
Uploaded: 2019-05-20T14:30:00
Description: Watch this Scientific Journal Video about A Melanoma Patient-Derived Xenograft Model at JoVE.com

The authors thank the Wistar Institute Animal Facility, Microscopy Facility, Histotechnology Facility, and Research Supply Center. This study was funded in part by grants from the U54 (CA224070-01), SPORE (CA174523), P01 (CA114046-07), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and the Melanoma Research Foundation.

The following animal protocols follow the guidelines of The Wistar Institute&#8217;s humane ethics committee and animal care guidelines.
1. Melanoma tumor tissue collection
<ol>
	<li>Collect tumor tissue (termed passage 0) from melanoma patients by one of the following surgery or biopsy methods.
	<ol>
		<li>For surgical excision tissue, maintain a minimum of 1 g of tissue (resect metastases and primary lesions) in transport storage media (RPMI 1640 + 0.1% fungizone + 0.2% gentamicin) at 4 &#...

<ol>
	<li>Garman, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+Genomic+Characterization+of+462+Melanoma+Patient-Derived+Xenografts,+Tumor+Biopsies,+and+Cell+Lines.">Genetic and Genomic Characterization of 462 Melanoma Patient-Derived Xenografts, Tumor Biopsies, and Cell Lines.</a> Cell Reports. 21 (7), 1936-1952 (2017).</li><li>Krepler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+Patient-Derived+Xenograft+Collection+Representing+the+Heterogeneity+of+Melanoma.">A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma.</a> Cell Reports. 21 (7), 1953-1967 (2017).</li><li>Davies, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+the+BRAF+gene+in+human+cancer.">Mutations of the BRAF gene in human cancer.</a> Nature. 417 (6892), 949-954 (2002).</li><li>Paraiso, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+phospho-ERK+activity+allows+melanoma+cells+to+escape+from+BRAF+inhibitor+therapy.">Recovery of phospho-ERK activity allows melanoma cells to escape from BRAF inhibitor therapy.</a> British Journal Of Cancer. 102 (12), 1724-1730 (2010).</li><li>Long, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Outcomes+in+Patients+With+BRAF+V600-Mutant+Metastatic+Melanoma+Who+Received+Dabrafenib+Combined+With+Trametinib.">Long-Term Outcomes in Patients With BRAF V600-Mutant Metastatic Melanoma Who Received Dabrafenib Combined With Trametinib.</a> Journal of Clinical Oncology. 36 (7), 667-673 (2018).</li><li>Hidalgo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discovery. 4 (9), 998-1013 (2014).</li><li>Hausser, H. J., Brenner, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+instability+of+Saos-2+cells+in+long-term+culture.">Phenotypic instability of Saos-2 cells in long-term culture.</a> Biochemical and Biophysical Research Communications. 333 (1), 216-222 (2005).</li><li>Fiebig, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+three+human+small+cell+lung+cancer+models+in+nude+mice.">Development of three human small cell lung cancer models in nude mice.</a> Recent Results In Cancer Research. 97, 77-86 (1985).</li><li>Izumchenko, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenografts+effectively+capture+responses+to+oncology+therapy+in+a+heterogeneous+cohort+of+patients+with+solid+tumors.">Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors.</a> Annals of Oncology. 28 (10), 2595-2605 (2017).</li><li>Shi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+resistance+and+clonal+evolution+in+melanoma+during+BRAF+inhibitor+therapy.">Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy.</a> Cancer Discovery. 4 (1), 80-93 (2014).</li><li>Monsma, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+patient+derived+xenografts+acquire+distinct+Vemurafenib+resistance+mechanisms.">Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms.</a> American Journal of Cancer Research. 5 (4), 1507-1518 (2015).</li><li>Das Thakur, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+vemurafenib+resistance+in+melanoma+reveals+a+strategy+to+forestall+drug+resistance.">Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance.</a> Nature. 494 (7436), 251-255 (2013).</li><li>Meehan, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDX-MI:+Minimal+Information+for+Patient-Derived+Tumor+Xenograft+Models.">PDX-MI: Minimal Information for Patient-Derived Tumor Xenograft Models.</a> Cancer Research. 77 (21), 62-66 (2017).</li><li>Gao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+using+patient-derived+tumor+xenografts+to+predict+clinical+trial+drug+response.">High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.</a> Nature Medicine. 21 (11), 1318-1325 (2015).</li><li>De La Rochere, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+Mice+for+the+Study+of+Immuno-Oncology.">Humanized Mice for the Study of Immuno-Oncology.</a> Trends in Immunology. 39 (9), 748-763 (2018).</li></ol>

We have herein described generating PDX models of melanoma with patient tissue derived from primary and metastatic tumors, core biopsies, and FNAs. When directly engrafted into NSG mice, tumors present similar morphologic, genomic, and biologic properties to those observed in the patient. In the case when only a small quantity of tissue is available to investigators, as often occurs with FNAs, the PDX technique allows for the expansion of the tumor tissue for DNA, RNA, and protein characterization, as well as for therapy...

Preclinical models are critical for all aspects of translational cancer research, including disease characterization, discovery of actionable vulnerabilities unique to cancer versus normal cells, and the development of efficacious therapies that exploit these vulnerabilities to increase the overall survival of patients. In the melanoma field, tens of thousands of cell line models have been heavily utilized for drug screening, with &#62;4,000 contributed by our group alone (WMXXX series). These cell line models were derived from melanoma patients with various forms of cutaneous melanoma (i.e., acral, uveal, and superficial spreading) and diverse genotypes (i.e., BRAFV600-mutant and neuroblastoma RAS viral oncogene homolog [NRASQ61R-mutant]), which span the spectrum of disease present in the clinic1,2.
Unequivocally, the most successful, targeted therapy strategy in the melanoma field has emerged from 1) the genomic characterization of patients&#8217; tumors identifying BRAF mutations in ~50% of melanomas3 and from 2) preclinical investigation leveraging melanoma cell line models4. The BRAF/MEK inhibitor combination was Food and Drug Administration (FDA)-approved in 2014 for the treatment of patients whose melanomas harbor activating BRAFV600E/K mutations and boasts a &#62;75% response rate5. Despite this initial efficacy, resistance rapidly arises in nearly every case due to multifarious intrinsic and acquired resistance mechanisms and intratumoral heterogeneity. Unfortunately, cell line models do not recapitulate representative biological heterogeneity when grown in two-dimensional culture in plastic vessels, which masks their clinically predictive potential when investigators attempt to experimentally determine therapies that might be effective in patients with a specific form or genotype of melanoma6. Understanding how to best model patient intratumoral heterogeneity will allow investigators to better develop therapeutic modalities that can kill therapy-resistant subpopulations that drive failure to current standard-of-care therapies.
Paramount to the limited predictive value of cell line models is how they are initially established. Irreversible alterations occur in the tumor clonal landscape when a single-cell suspension of a patients&#8217; tumor is grown on two-dimensional, plastic tissue culture vessels, including changes in proliferative and invasive potential, the elimination of specific subpopulations, and the alteration of genetic information7. Xenografts into mice of these melanoma cell line models represent the most frequently used in vivo platform for preclinical studies; however, this strategy also suffers from the poor recapitulation of complex tumor heterogeneity observed clinically. To overcome this shortcoming, there has been a growing interest in incorporating more sophisticated preclinical models of melanoma, including the PDX model. PDX models have been utilized for &#62;30 years, with seminal studies in lung cancer patients demonstrating concordance between the patients&#39;&#160;response to cytotoxic agents and the response of the PDX model derived from the same patient8. Recently, there has been a drive to utilize PDX models as the tool of choice for preclinical investigations both in the industry and in academic centers. PDX models, because of their superior recapitulation of tumor heterogeneity in human patients, are more clinically relevant to use in therapy optimization efforts than cell line xenografts9. In melanoma, there are immense hurdles that blunt the therapeutic management of advanced disease10. Clinically relevant PDX models have been used to model clinical resistance and identify therapeutic strategies with clinically available agents to treat therapy-resistant tumors11,12. Briefly, the protocol presented here to generate PDX models requires the subcutaneous implantation of fresh tissue from primary or metastatic melanomas (collected by biopsy or surgery) into NOD/scid/IL2-receptor null (NSG) mice. Different variations in methodological approach are used by different groups; however, a fundamental core exists13.

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			<td>SIGMA-ALDRICH CORPORATION</td>
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			<td>SIGMA-ALDRICH CORPORATION</td>
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			<td>Invitrogen</td>
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			<td>The Wistar Institute, animal facility</td>
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			<td>breeding</td>
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a melanoma patient-derived xenograft model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Tumor tissue for melanoma PDX models can come from a variety of different sources and can also be processed per the growth dynamics of individual models and the desired use of the PDX tissue. The priority when establishing a PDX model is to have sufficient material to bank for future use and DNA for characterization (Figure 1).
Once sufficient material is banked, tumor tissue can be expanded in one of three main methods to grow enough tumor to perform a formal the...

Watch this Scientific Journal Video about A Melanoma Patient-Derived Xenograft Model at JoVE.com

A Melanoma Patient-Derived Xenograft Model

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

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

The PDX has revolutionized the cancer experimental pre-clinical studies. We can now mimic, in the laboratory, what is happening in the patients. And now we can work on how to overcome the resistance and how, on the longterm, we can increase survival of patients.Demonstrating the procedure will be Min Xiao. To begin, transfer previously collected melanoma tissue to a sterile Petri dish. Working with surgical scissors, scalpel blade, and forceps, first remove normal tissue from the normal tissue as much as possible.And then remove the necrotic tissue identified by pale white-ish tissue located centrally within the tumor. Then use a scalpel to subdivide an initial tumor chunk into approximately equal pieces for surgical mouse implantation. If the tumor tissue is too hard for mechanical dissociation, use a scalpel to mince the tumor chunks as finely as possible to form a slurry.Transfer the slurry into a 50 milliliter tube with cold Hank's Balanced Salt Solution without calcium and magnesium ions. Centrifuge at 220 times g for four minutes at four degrees Celsius. After removing the supernatant, re-suspend the slurry in 10 milliliters of warmed, fresh digest media per one gram of tumor tissue.Place the tube in a 37 degrees Celsius water bath for 20 minutes and mix vigorously every five minutes with a disposable pipette. Wash the slurry using up to 50 milliliters of HBSS, centrifuge at 220 times g for four minutes at four degrees Celsius, and then remove the supernatant. Add five milliliters of pre-warmed TEG buffer per one gram of tumor tissue, shake to gently re-suspend, and place the tube at 37 degrees Celsius for two minutes, without mixing.To quench the trypsin, add at least one equal volume of cold staining media and centrifuge at 220 times g for four minutes at four degrees Celsius. After removing the supernatant, add 10 milliliters of staining media per one gram of tumor tissue, and re-suspend the pellet by pipetting up and down. Filter it through a 40 micrometer cell strainer to get a single cell suspension for mouse injection.To implant surgical excision or surgical biopsy tissue, first shave hair from the lower back of NSG six to eight week old anesthetized mice, leaving an approximately 1.5 centimeters to three centimeters area without hair. Prepare tumor chunks, and divide tumor slurry in a Petri into individual mounds for surgical implantation. Using a scalpel blade, make an approximately five millimeter long incision on the center of the back of the mouse.With one pair of forceps, life up the skin on the side of the incision opposite the operator. With scissors in the other hand, separate the skin from the muscle layer by gently cutting the fascial membrane with small scissor cuts, thereby creating a pocket for the tumor tissue. Use a scalpel blade to pick up one tumor chuck, as well as one individual mound of tumor slurry tissue, and gently place the tissue into the created pocket.Then add 100 microliters of artificial extracellular matrix on the tumor tissue mound in the pocket. Using two pairs of forceps, pull up the incision on both ends so that the wound edges come close together. Then apply one or two wound clips to close the wound.Subcutaneously inject one to five milligrams per kilogram of analgesic. When healing is completely, after approximately seven days, remove the wound clips. Monitor mice once weekly to check for palpable tumors.Once tumors reach a desired volume, use surgical forceps to lift the skin adjacent to the tumor of the euthanized mouse and make a horizontal cut using curved scissors. Use a blunt separation technique to mobilize the skin on both sides of the tumor and over the tumor, exposing the tumor. Use scissors and a scalpel blade to separate the tumor from the fascia.Cut out the tumor and transfer it to a sterile Petri dish. Cut the tumor into small pieces and remove necrotic tissue from the tumor. To bank tumor tissue for future implantation, take two to three small tumor pieces and mince them into pieces smaller than one by one millimeter.Transfer all minced tissue to a two milliliter cryogenic vile. Add one milliliter of freezing media and mix well. Place the vials into a pre-cooled isopropanol-based cell freezing container on dry ice, store the container in a minus 80 degrees Celsius freezer overnight, and then transfer the vials to liquid nitrogen storage.To snap freeze tissue for downstream assays, place the tumor tissue pieces into a cryogenic vial, put the cryogenic vile in liquid nitrogen immediately, and store the vials in a minus 80 degrees Celsius freezer. When three different methods of growing tumor tissue were compared, single cell suspension of tumor cells with the use of enzymatic digestion was most successful. Besides allowing for more rapid tumor growth, it was also sufficient for one initial tumor to be expanded into 10 to 20 mice, whereas the tumor chuck and slurry method can only be expanded into five to 10 mice.Melanoma patient-derived xenograft models often reflect the drug sensitivity the patient displayed when on therapy. A xenograft derived from melanoma patient who initially responded to a BRAF inhibitor, but ultimately relapsed, also displayed initial sensitivity to BRAF protein inhibition, plus MEK kinase inhibitor, but the tumors ultimately relapsed. It's critical to take care when preparing PDX tissue for banking, as this will ensure success in future PDX expansions, therapy trials, as well as characterization.With PDX material, single cell RNA sequencing, whole exome sequencing, and proteomic characterization, are among the many methods our lab leverages to dissect therapy resistance. The PDX technique allows researchers to investigate therapy resistance using tumor cells that better recapitulate the tumor biology in a human patient relative to traditional cancer models grown on plastic. This advantage allows for more predictive insights.Researchers should always take care and use biosafety level two precautions when handling tumor tissue derived from a human patient.

Research

JoVE Journal

Cancer Research

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

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

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

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A 3D Organotypic Melanoma Spheroid Skin Model

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

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

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

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

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Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...