We thank the Division of Advanced Research Technologies (DART) at NYU Langone Health, and in particular, the Experimental Pathology Research Laboratory, Genome Technology Center, Cytometry and Cell Sorting Laboratory, Pre-Clinical Imaging Core, which are partially supported by the Perlmutter Cancer Center Support Grant NIH/NCI 5P30CA016087. We thank the NYU Interdisciplinary Melanoma Cooperative Group (PI: Dr. Iman Osman) for providing access to patient-derived melanoma short-term cultures+ (10-230BM and 12-273BM), which were obtained through IRB-approved protocols (Universal Consent study #s16-00122 and Interdisciplinary Melanoma Cooperative Group study #10362). We thank Dr. Robert Kerbel (University of Toronto) for providing 113/6-4L and 131/4-5B1 melanoma cell lines* and Dr. Meenhard Herlyn (Wistar Institute) for providing WM 4265-2, WM 4257s-1, WM 4257-2 melanoma short-term cultures**. E.H. is supported by NIH/NCI R01CA243446, P01CA206980, an American Cancer Society-Melanoma Research Alliance Team Science Award, and an NIH Melanoma SPORE (NCI P50 CA225450; PI: I.O.). Figure 1 was created with Biorender.com.

<ol>
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R., 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+high-throughput+study+in+melanoma+identifies+epithelial-mesenchymal+transition+as+a+major+determinant+of+metastasis.">A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis.</a> Cancer Research. 67 (7), 3450-3460 (2007).</li><li>Rowe, C. J., Khosrotehrani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+biological+determinants+of+melanoma+progression:+Should+all+be+considered+for+clinical+management.">Clinical and biological determinants of melanoma progression: Should all be considered for clinical management.</a> Australasian Journal of Dermatology. 57 (3), 175-181 (2016).</li><li>Plebanek, M. 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The authors have no conflicts of interest to declare.

The aim of this technical report is to offer a standardized, top-to-bottom workflow for the investigation of potential actors in melanoma metastasis. As in vivo experiments can be costly and time-consuming, strategies to maximize efficiency and increase the value of the information obtained are paramount.
It is imperative to use complementary approaches throughout to crossvalidate findings within the same experiment. For example, both NuMA immunohistochemical staining and BLI are comp...

The high mortality associated with metastatic melanoma combined with an increasing incidence of melanoma worldwide1 (an estimated 7.86% increase by 2025) calls for new treatment approaches. Advances in target discovery hinge upon reproducible models of metastasis, a highly complex process. Throughout the steps of the metastatic cascade, melanoma cells must overcome countless barriers to achieve immune system evasion and colonization of distant tissues2. The resilience and adaptability of melanoma cells arise from a multitude of factors, including their high genetic mutational burden3 and their neural crest origin, which confer crucial phenotypic plasticity3,4,5. At each step, transcriptional programs allow metastasizing melanoma cells to switch from one state to another based on cues from the crosstalk with the microenvironment, comprising the immune system6, the extracellular milieu7,8, and the cellular architecture of physical barriers9 with which they come in contact. For example, melanoma cells escape immune surveillance by downregulating the expression of important immune-priming tumor-secreted factors6.
Studies describe a &#34;premetastatic niche&#34;, wherein melanoma cells secrete chemokines and cytokines to prime the distant &#34;target&#34; organ for metastasis10. These findings raise important questions about the organ tropism of metastatic melanoma cells and the anatomic route they take to access distant tissues. After intravasation, melanoma cells are known to metastasize through lymphatics (lymphatic spread) and blood vessels (hematogenous spread)2,11. While most patients present with localized disease, a small subset of cases presents with distant metastatic disease and no lymphatic dissemination (negative lymph node involvement)11, suggesting the existence of alternative metastatic pathways for melanoma.
When they colonize a metastatic site, melanoma cells undergo epigenetic and metabolic adaptations12,13. To access and invade new compartments, melanoma cells employ proteases14 and cytoskeletal modifications11,15, which enable them to traverse to and grow in their new location. The difficulty in targeting melanoma cells resides in the complexity and number of such adaptations; thus, the field should make efforts to recreate experimentally as many steps and adaptations as possible. Despite numerous advances in in vitro assays such as organoids and 3D cultures16,17, these models only incompletely recapitulate the in vivo metastatic cascade.
Murine models have shown value by striking a balance between reproducibility, technical feasibility, and simulation of human disease. Intravascularly, orthotopically and heterotopically implanted melanoma cells from patient-derived xenografts or short-term cultures into immune-compromised or humanized mice represent the backbone of target discovery in metastatic melanoma. However, these systems often lack a crucial biological constraint on metastasis: the immune system. Syngeneic melanoma metastasis models that possess this constraint are relatively scarce in the field. These systems, developed in immunocompetent mice, including B16-F1018, the YUMM&#160;family of cell lines19, SM120, D4M321, RIM322 or more recently, the RMS23 and M1 (Mel114433), M3 (HCmel1274), M4 (B2905)24 melanoma cell lines, facilitate the investigation of the complex role of the host immune response in melanoma progression.
Here, a pipeline for melanoma metastasis target identification is presented. With increasing and larger &#39;omics&#39; datasets being generated from melanoma patient cohorts, we postulate that studies holding the most clinical promise are those that stem from big data integration, leading to meticulous functional and mechanistic interrogation25,26,27,28. By using mouse models to study potential targets in the metastatic process, one can account for in vivo-specific events and tissue interactions, thus increasing the probability of clinical translation. Multiple methods to quantify metastatic burden are outlined, providing complementary data on the results of any given experiment. A protocol for single-cell isolation from tumors in various organ is described to aid the unbiased characterization of gene expression in metastatic cells, which may precede single-cell or bulk RNA sequencing.

<table><tbody><tr><td>#15 Scapel Blade&amp;nbsp;</td><td>WPI</td><td>500242</td><td>For surgical procedures</td></tr><tr><td>#3 Scapel Handle</td><td>WPI</td><td>500236</td><td>For surgical procedures</td></tr><tr><td>1 mL Tuberculin syringe, slip tip&amp;nbsp;</td><td>BD</td><td>309626</td><td>Injections</td></tr><tr><td>10 mL syringe, slip tip&amp;nbsp;</td><td>BD</td><td>301029</td><td>Perfusion</td></tr><tr><td>10% Formalin Sodium Buffered</td><td>EK Industries</td><td>4499-20L</td><td>For perfusion/tissue fixative</td></tr><tr><td>15 mL Conical</td><td>Corning&amp;nbsp;</td><td>430052</td><td>Cell culture</td></tr><tr><td>15 mL Conical Polypropylene Centrifuge Tubes</td><td>Falcon</td><td>352196</td><td>Cell culture</td></tr><tr><td>200 Proof Ethanol</td><td>Deacon Labs</td><td>04-355-223</td><td>Histology</td></tr><tr><td>22G &amp;ndash; 22mm needle</td><td>BD</td><td>305156</td><td>Perfusion</td></tr><tr><td>4-0 Vicryl Suture</td><td>Ethicon</td><td>J464G</td><td>Suture</td></tr><tr><td>4% Carson&#039;s phosphate buffered paraformaldehyde&amp;nbsp;</td><td>EMS</td><td>15733-10</td><td>For perfusion/tissue fixative</td></tr><tr><td>40&amp;micro;m</td><td>Corning</td><td>431750</td><td>Tissue processing</td></tr><tr><td>5-0 Absorbable Suture&amp;nbsp;</td><td>Ethicon</td><td>6542000</td><td>Closure</td></tr><tr><td>50 mL Conical&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>430828</td><td>Cell culture</td></tr><tr><td>50mL Conical Polypropylene Centrifuge Tubes</td><td>Falcon</td><td>352070</td><td>Cell culture</td></tr><tr><td>7-0 Silk suture&amp;nbsp;</td><td>FST</td><td>18020-70</td><td>Ligature</td></tr><tr><td>70&amp;micro;m</td><td>Corning</td><td>431751</td><td>Tissue processing</td></tr><tr><td>Anti-fade mounting media&amp;nbsp;&amp;nbsp;</td><td>Vector Labs</td><td>H-1000-10</td><td>Immunofluorescence</td></tr><tr><td>Approximator applying Forceps, 10cm&amp;nbsp;</td><td>WPI</td><td>14189</td><td>For microsurgical procedures</td></tr><tr><td>Avance</td><td>Bruker</td><td>3 HD</td><td>NMR Console&amp;nbsp;</td></tr><tr><td>Biospec 7030&amp;nbsp;</td><td>Bruker</td><td>7030</td><td>Micro MRI</td></tr><tr><td>BSA</td><td>Bioreg</td><td>A941</td><td>NuMA Staining</td></tr><tr><td>Castroviejo suturing forceps, straight tips 5.5mm tying platform, 11cm&amp;nbsp;</td><td>WPI</td><td>WP5025501</td><td>For microsurgical procedures</td></tr><tr><td>Coplin Staining Jar</td><td>Bel-Art&amp;nbsp;</td><td>F44208-1000</td><td>Histology</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542-1MG</td><td>Immunofluorescence</td></tr><tr><td>dCas9-KRAB</td><td>Addgene</td><td>110820</td><td>Genetic manipulation</td></tr><tr><td>DNase I</td><td>NEB</td><td>M0303L</td><td>Tissue processing</td></tr><tr><td>DPBS</td><td>Corning</td><td>21-030-CM</td><td>Tissue processing</td></tr><tr><td>Extra Sharp Uncoated Single Edge Blade</td><td>GEM</td><td>62-0167</td><td>Tissue processing</td></tr><tr><td>Extracellular Matrix Substrate&amp;nbsp;</td><td>Corning</td><td>354234</td><td>Consider the Growth Factor Reduced ( as alternative&amp;nbsp;</td></tr><tr><td>FBS</td><td>Cytiva</td><td>SH30910.03</td><td>Cell culture</td></tr><tr><td>Fiji Image J</td><td>Fiji Image J</td><td>Software</td><td>Immunofluorescence</td></tr><tr><td>Goat anti-rabbit HRP conjugated multimer&amp;nbsp;</td><td>Thermo Fisher</td><td>A16104</td><td>NuMA Staining</td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>PCN5000</td><td>Immunofluorescence</td></tr><tr><td>HBSS</td><td>Corning</td><td>21-020-CV</td><td>Tissue processing</td></tr><tr><td>Hematoxylin&amp;nbsp;</td><td>Richard-Allan Scientific&amp;nbsp;</td><td>7231</td><td>Histology</td></tr><tr><td>Illumina III&amp;nbsp;</td><td>PerkinElmer</td><td>CLS136334</td><td>BLI Instrument</td></tr><tr><td>Insulin syringe 28G - 8mm needle</td><td>BD</td><td>329424</td><td>Injections</td></tr><tr><td>Insulin syringe 31G - 6mm needle&amp;nbsp;</td><td>BD</td><td>326730</td><td>Injections</td></tr><tr><td>Iris Forceps, 10.2cm, Full Curve, serrated</td><td>WPI</td><td>504478</td><td>For perfusion and surgical procedures</td></tr><tr><td>Isoflurane USP</td><td>Covetrus</td><td>11695067772</td><td>Anesthesia</td></tr><tr><td>Jewelers #7 Forceps Titanium 11 cm 0.07 x 0.01 mm Tip</td><td>WPI</td><td>WP6570</td><td>For microsurgical procedures</td></tr><tr><td>Ketamine HCl 100mg/mL</td><td>Mylan Ind.</td><td>1049007</td><td>Anesthesia</td></tr><tr><td>lentiCRISPRv2</td><td>Addgene</td><td>98290</td><td>Genetic manipulation</td></tr><tr><td>Lycopersicon Esculentum (Tomato) Lectin, DyLight 649</td><td>Invitrogen</td><td>L32472</td><td>Vascular endothelial cells marker</td></tr><tr><td>MEM non-essential amino acids X 100</td><td>Corning</td><td>25-025-CI</td><td>Cell culture</td></tr><tr><td>Metzenbaum Scissors</td><td>WPI</td><td>503269</td><td>For surgical procedures</td></tr><tr><td>Microinjection Unit</td><td>KOPF</td><td>5000</td><td>Intracardiac injections</td></tr><tr><td>NaCl</td><td>Fisher</td><td>S25877&amp;nbsp;</td><td>NuMA Staining</td></tr><tr><td>Needle 30G x 25mm</td><td>BD</td><td>305128</td><td>Intracardiac Injection</td></tr><tr><td>Needle 33G x 15mm</td><td>Hamilton</td><td>7747-01</td><td>Intracarotid Injection</td></tr><tr><td>Needle holder, Castroviejo, 14cm, with lock, 1.2mm Serrated Jaws</td><td>WPI</td><td>14137-G</td><td>For microsurgical procedures</td></tr><tr><td>NOD.Cg-Prkdc&lt;sup&gt;scid&lt;/sup&gt; Il2rg&lt;sup&gt;tm1Wjl&lt;/sup&gt;/SzJ mice</td><td>The Jackson Laboratory</td><td>005557</td><td>Murine model</td></tr><tr><td>NU/J mice</td><td>The Jackson Laboratory</td><td>002019</td><td>Murine model</td></tr><tr><td>Nuclear Mitotic Apparatus Protein polyclonal rabbit anti-human&amp;nbsp;</td><td>Abcam</td><td>97585</td><td>NuMA Staining</td></tr><tr><td>Penicillin-Streptomycin 10000U/mL</td><td>Gibco</td><td>15140122</td><td>Cell culture</td></tr><tr><td>Percoll</td><td>GE</td><td>0891-01</td><td>density separation solution&amp;nbsp;</td></tr><tr><td>PI Classic Surgical Gloves</td><td>Cardinal Health</td><td>2D72PT75X</td><td>Surgery</td></tr><tr><td>pLKO Tet-On</td><td>Addgene</td><td>21915</td><td>Genetic manipulation</td></tr><tr><td>Povidone-Iodine 10% Solution</td><td>Medline</td><td>MDS093943</td><td>Surgery</td></tr><tr><td>Proparacaine Drops 0.5%</td><td>Akorn Pharma</td><td>AX0501</td><td>Opthalmic local anesthetic</td></tr><tr><td>Puralube Petrolatum Opthalmic Ointment</td><td>Dechra</td><td>83592</td><td>Anesthesia</td></tr><tr><td>Razor Blade Double Edge Blades&amp;nbsp;</td><td>EMS</td><td>72000</td><td>Shaving and Vibrotome Brain Slicing&amp;nbsp;</td></tr><tr><td>Reflex 9mm EZ Clip&amp;nbsp;</td><td>Braintree</td><td>EZC-&amp;nbsp;KIT</td><td>Wound closure</td></tr><tr><td>RPMI 1640&amp;nbsp;</td><td>Corning</td><td>10-040-CM</td><td>Cell culture</td></tr><tr><td>Scissors, Spring 10.5cm Str, 8mm Blades</td><td>WPI</td><td>501235</td><td>For microsurgical procedures</td></tr><tr><td>Semi-Automatic Vibrating Blade Microtome</td><td>Leica</td><td>VT1200</td><td>Brain Slice Immunofluorescence</td></tr><tr><td>Single Channel Anesthesia Vaporizer System</td><td>Kent Scientific</td><td>VetFlo-1210S&amp;nbsp;</td><td>Anesthesia</td></tr><tr><td>Smartbox Tabletop Chamber System and Exhaust Blower</td><td>EZ Systems</td><td>TT4000</td><td>CO2 Euthanasia</td></tr><tr><td>Sterile Fenestrated Disposable Drape</td><td>Medline</td><td>NON21002</td><td>Surgery</td></tr><tr><td>Sterile Non-Reinforced Aurora Surgical Gowns with Set-In Sleeves</td><td>Medline</td><td>DYNJP2715</td><td>Surgery</td></tr><tr><td>T25 Flask</td><td>Corning&amp;nbsp;</td><td>430639</td><td>Cell culture</td></tr><tr><td>Tris</td><td>Corning</td><td>46-031-CM</td><td>NuMA Staining</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td>Immunofluorescence</td></tr><tr><td>Troutman tying forceps, 10cm, Curved G pattern, 0.52mm tip with tying platform</td><td>WPI</td><td>WP505210</td><td>For microsurgical procedures</td></tr><tr><td>Vessel clips 10G Pressure 5x 0.8mm Jaws, 5/pkg</td><td>WPI</td><td>15911</td><td>For microsurgical procedures</td></tr><tr><td>Visiopharm</td><td>Visiopharm</td><td>Visiopharm</td><td>NuMA Staining Quantification Software</td></tr><tr><td>Xylasine 100mg/mL</td><td>Akorn Pharma</td><td>59399-111-50</td><td>Anesthesia</td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P-1GAL</td><td>Histology</td></tr></tbody></table>

a robust discovery platform for the identification of novel mediators of melanoma metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

The following figures illustrate how the described workflow has been applied for the identification of novel drivers of melanoma metastasis. Figure 2 summarizes the results of a published study in which the effects of silencing the fucosyltransferase FUT8 in in vivo melanoma metastasis were studied26. Briefly, analysis of human patient glycomic data (obtained by lectin arrays) and transcriptomic profiling revealed increased levels of alpha-1,6-fucose associat...

Watch this Scientific Journal Video about A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis at JoVE.com

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

a-robust-discovery-platform-for-identification-novel-mediators

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

2.3K Views.  NYU Grossman School of Medicine. This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action. 

Video: A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicine

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Murine Model for Studying Leptomeningeal Disease Using Circulating Tumor Cells

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma&#45;Associated Leptomeningeal Disease

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Author Spotlight: Assessing the Potential of Circulating Tumor Cells in Leptomeningeal Disease Research

This article describes a protocol for the propagation of cerebral spinal fluid-circulating tumor cells (CSF-CTCs) collected from patients with melanoma-associated leptomeningeal disease (M-LMD) to develop preclinical models to study M-LMD.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the ...

Patient-Derived Melanoma Organoids: A Frontier in Cancer Immunotherapy

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

Author Spotlight: Recreating Melanoma Complexity with Patient-Derived Organoids for Immunotherapy Evaluation

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Experimental Metastatic Melanoma Mouse Model: A Platform to Study the Metastatic Potential of Melanoma Cells in the Lungs

Source: Joshua J. Timmons&#160;et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/54039"> Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs</a>. J. Vis. Exp. (2016)
In this video, melanoma cells are intravenously injected through the tail vein in the mouse model. The melanoma cells further travel through the circulatory system, adapting to the distant microenvironment and causing metastasis in organs like lungs.

Source: Joshua J. Timmons&#160;et al.  Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Skin Cancer

Animal model studies

Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Source: Zink, K. E. et. al. <a href="https://jove.unicartagenaproxy.elogim.com/t/59490">Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry.</a> J. Vis. Exp. (2019)
This protocol describes a technique for sample preparation by co-culturing ovarian explants with the fallopian tube cancer cells. These samples are visualized using imaging mass spectroscopy, or IMS,&#160; to detect small molecule exchange between the cells and tissues leading to tumor development.

Source: Zink, K. E. et. al. Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry. J. Vis. Exp. (2019)
This ...

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

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