Name: A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis (Video) | JoVE Journal
Uploaded: 2022-03-08T14:00:00.000+00:00
Duration: 7 min 41 s
Description: Discover a robust workflow for identifying novel mediators of melanoma metastasis using standardized in vivo models and techniques. This method enables reliable target discovery and therapy development through reproducible assessments.

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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.

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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

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...

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 ...

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Research

JoVE Journal

Cancer Research

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

Experimental Metastasis Assay

This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

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

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

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.

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

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.

A 3D Organotypic Melanoma Spheroid Skin Model

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Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

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.

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

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

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.

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

Discover a robust workflow for identifying novel mediators of melanoma metastasis using standardized in vivo models and techniques. This method enables reliable target discovery and therapy development through reproducible assessments.

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

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