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.

NOTE: The animal procedures involved in the following protocol were approved by the New York University Institutional Animal Care and Use Committee (IACUC). All the procedures are conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Figure 1 depicts the general experimental approach.
1. Patient-derived melanoma short-term cultures (STCs)
<ol>
	<li>Place the tissue ...

<ol>
	<li>Sung, 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=Global+Cancer+Statistics+2020:+GLOBOCAN+Estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).</li><li>Adler, N. R., Haydon, A., McLean, C. A., Kelly, J. W., Mar, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+pathways+in+patients+with+cutaneous+melanoma.">Metastatic pathways in patients with cutaneous melanoma.</a> Pigment Cell Melanoma Research. 30 (1), 13-27 (2017).</li><li>Platz, A., Egyhazi, S., Ringborg, U., Hansson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cutaneous+melanoma;+a+review+of+NRAS+and+BRAF+mutation+frequencies+in+relation+to+histogenetic+subclass+and+body+site.">Human cutaneous melanoma; a review of NRAS and BRAF mutation frequencies in relation to histogenetic subclass and body site.</a> Molecular Oncology. 1 (4), 395-405 (2008).</li><li>Alonso, S. 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. 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=Pre-metastatic+cancer+exosomes+induce+immune+surveillance+by+patrolling+monocytes+at+the+metastatic+niche.">Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche.</a> Nature Communications. 8 (1), 1319 (2017).</li><li>Orgaz, J. L., 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=Loss+of+pigment+epithelium-derived+factor+enables+migration,+invasion+and+metastatic+spread+of+human+melanoma.">Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma.</a> Oncogene. 28 (47), 4147-4161 (2009).</li><li>Ladhani, O., Sanchez-Martinez, C., Orgaz, J. L., Jimenez, B., Volpert, O. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >#15 Scapel Blade&#160;</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&#160;</td>
			<td >BD</td>
			<td >309626</td>
			<td>Injections</td>
		</tr>
		<tr >
			<td >10 mL syringe, slip tip&#160;</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&#160;</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 &#8211; 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&#39;s phosphate buffered paraformaldehyde&#160;</td>
			<td >EMS</td>
			<td >15733-10</td>
			<td>For perfusion/tissue fixative</td>
		</tr>
		<tr >
			<td >40&#181;m</td>
			<td >Corning</td>
			<td >431750</td>
			<td>Tissue processing</td>
		</tr>
		<tr >
			<td >5-0 Absorbable Suture&#160;</td>
			<td >Ethicon</td>
			<td >6542000</td>
			<td>Closure</td>
		</tr>
		<tr >
			<td >50 mL Conical&#160;</td>
			<td >Corning&#160;</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&#160;</td>
			<td >FST</td>
			<td >18020-70</td>
			<td>Ligature</td>
		</tr>
		<tr >
			<td >70&#181;m</td>
			<td >Corning</td>
			<td >431751</td>
			<td>Tissue processing</td>
		</tr>
		<tr >
			<td >Anti-fade mounting media&#160;&#160;</td>
			<td >Vector Labs</td>
			<td >H-1000-10</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr >
			<td >Approximator applying Forceps, 10cm&#160;</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&#160;</td>
		</tr>
		<tr >
			<td >Biospec 7030&#160;</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&#160;</td>
			<td >WPI</td>
			<td>WP5025501</td>
			<td>For microsurgical procedures</td>
		</tr>
		<tr >
			<td >Coplin Staining Jar</td>
			<td >Bel-Art&#160;</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&#160;</td>
			<td >Corning</td>
			<td>354234</td>
			<td>Consider the Growth Factor Reduced ( as alternative&#160;</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&#160;</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&#160;</td>
			<td>Richard-Allan Scientific&#160;</td>
			<td >7231</td>
			<td>Histology</td>
		</tr>
		<tr >
			<td >Illumina III&#160;</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&#160;</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&#160;</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-Prkdcscid Il2rgtm1Wjl/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&#160;</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&#160;</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&#160;</td>
			<td >EMS</td>
			<td >72000</td>
			<td>Shaving and Vibrotome Brain Slicing&#160;</td>
		</tr>
		<tr >
			<td >Reflex 9mm EZ Clip&#160;</td>
			<td >Braintree</td>
			<td>EZC-&#160;KIT</td>
			<td>Wound closure</td>
		</tr>
		<tr >
			<td >RPMI 1640&#160;</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&#160;</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&#160;</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 ...

This protocol describes a series of techniques within a continuous workflow that can be used for assessing the in vivo effects of candidate regulators of melanoma metastasis, but also of metastasis of other solid malignancies. The main advantage of our systematic workflow is increased reproducibility of the data due to robust and standardized in vivo models and techniques. This pipeline offers a pathway for target discovery and therapy development by testing candidates inferred from Omics data or in vitro assays in an in vivo setting.The learning curve associated with the techniques presented in this protocol is shortened by using the materials provided and following the detailed description when practicing the steps. Helping to demonstrate the procedure will be Orlando Aristizabal, a research scientist in The Preclinical Imaging Core and Ran Moubarak, an instructor in my lab. For intradermal injections, anesthetize and shave an eight to 10 week old mouse inside a biosafety cabinet while maintaining aseptic conditions.Then grasp and retract the skin backwards against the trajectory of the needle stab and using a six millimeter long, 31 gauge insulin syringe needle, gently puncture the skin at an acute angle with the bevel facing upward. Inject 30 microliters of the tumor cell suspension slowly, until a dome shaped wheel is observed. Monitor the progression of the tumor growth, weight loss, and overall health status.During these monitoring sessions, take measurements with calipers and use the length and width dimensions of the tumor to calculate the volume. For intracardiac injections, transfer an anesthetized mouse onto the heated platform of the ultrasound machine. Then using hypoallergenic tape, secure the mouse to the nose cone.Shave the thorax with a straight razor blade or a scalpel blade, tilted at a 30 degree angle and clean the skin around the procedure area with 10%povidone iodine. Then, position the ultrasound probe in the middle of the thorax on the left side of the mouse to capture a horizontal window oriented to obtain a cross-sectional view of the left ventricle. With the long axis of the probe facing upwards, fix the probe at a 50 degree angle and the heated platform at a 20 degree angle, then lock the probe and the support frame in the position.While working inside the safety cabinet, draw up the cell suspension in a tuberculin one milliliter syringe with a 30 gauge one inch needle. Remove any air bubbles present in the syringe. Lock the syringe in the stereotactic injector.And then under ultrasound guidance, advance the needle through the thoracic wall into the left ventricle of the heart and inject 100 to 250 microliters of the tumor cell suspension slowly. For staged survival surgery, place the anesthetized mouse on the warming pad and clean the skin around the procedure area with a 10%povidone iodine solution. After donning sterile personal protection equipment and gloves, lay a sterile drape over the animal's body.Next, using Iris scissors or a scalpel, incise the skin, maintaining a five to seven millimeter resection margin from the edge of the tumor. In case of intradermal tumors, resect the tumor along with the circumferential skin. For subcutaneous tumors, dissect and remove the tumor under the skin.After tumor resection, close the wound with a nine millimeter stapling device. For in vivo imaging, administer d-luciferin substrate to the mouse by intraperitoneal injection with a one milliliter insulin syringe and a 28 gauge needle then anesthetize the mouse and place it into a nose cone inside the bioluminescence imaging scanner. Start the instrument by pressing initialize and set the exposure time to auto.For capturing a blank image for background subtraction, click acquire and save the image after the acquisition sequence is completed. For data analysis and the same in vivo imaging software, navigate to the folder where the images are saved and open the images of all the mice from one experiment. Set the units to radiance and ensure that the checkbox indicating individual is not checked as this will preclude normalization of signal across groups.Next, using the region of interest or ROI drawing tool, draw circular ROI's for the brain region and rectangular ROI's for the body, exclude the ears and nose in the brain ROI as they tend to emit unspecific luminance. To minimize bias, draw ROI's on the photographs of the mice without the luminescent signal overlaid, then select measure ROI's to quantify the signal and export the data to a spreadsheet. Analyze differences between groups by plotting total luminescent flux in body regions of interest.After a NuMA staining using software, draw an ROI to include all NuMA stained cells within the organ tissue, except other organ parenchyma and empty spaces. Adjust the settings to categorize the NuMA positive and NuMA negative cells while using appropriate positive and negative controls for each organ. Use an established software algorithm to quantify the total number of percentage of NuMA positive cells for each sample.Bioluminescence, bright field, ex vivo fluorescence and hematoxylin and eosin staining images illustrate the multi-pronged approach for the analysis of candidate genes effects on melanoma metastasis. Fucosyltransferase silencing impaired the metastatic dissemination of melanoma cells. The NuMA stained lung sections of melanoma cells infected with control Linda virus and with a metastasis suppressor expressing Linda virus are shown here.The metastatic melanoma cells are labeled green and the organ area is delimitated by a green hatched line. Maintaining proper tension in the skin when performing intradermal injections, avoiding air embolism when preparing syringes and obtaining adequate resection margins that should not interfere with the animal's locomotion or predispose it to wound complications, help improve the success rate and reproducibility. Further employment of multiplex imaging to examine immune filtration, single cell RNA sequencing for gene expression analysis and spatial transcriptomics could all provide complimentary avenues to examine intratumoral heterogeneity.The combination of techniques described in this protocol helped us identify novel regulators in melanoma brain metastasis, such as the role of melanoma secreted amyloid beta in suppressing neuroinflammation and promoting brain metastasis.

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JoVE Journal

Cancer Research

2.3K visualizações.  NYU Grossman School of Medicine. Este protocolo descreve uma série de técnicas dentro de um fluxo de trabalho contínuo que pode ser usado para avaliar os efeitos in vivo dos reguladores candidatos da metástase de melanoma, mas também de metástases de outras malignidades sólidas. A principal vantagem do nosso fluxo de trabalho sistemático é o aumento da reprodutibilidade dos dados devido a modelos e técnicas in vivo robustas e padronizadas. Este pipeline oferece um caminho para o desenvolvimento de descoberta e tera...