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>
	<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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigment+epithelium-derived+factor+blocks+tumor+extravasation+by+suppressing+amoeboid+morphology+and+mesenchymal+proteolysis.">Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis.</a> Neoplasia. 13 (7), 633-642 (2011).</li><li>Ju, R. J., Stehbens, S. J., Haass, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+melanoma+cell-stroma+interaction+in+cell+motility,+invasion,+and+metastasis.">The role of melanoma cell-stroma interaction in cell motility, invasion, and metastasis.</a> Frontiers in Medicine - Dermatology. 5, 307 (2018).</li><li>Wiley, H. E., Gonzalez, E. B., Maki, W., Wu, M. T., Hwang, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CC+chemokine+receptor-7+and+regional+lymph+node+metastasis+of+B16+murine+melanoma.">Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma.</a> Journal of the National Cancer Institute. 93 (21), 1638-1643 (2001).</li><li>Meier, 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=Metastatic+pathways+and+time+courses+in+the+orderly+progression+of+cutaneous+melanoma.">Metastatic pathways and time courses in the orderly progression of cutaneous melanoma.</a> British Journal of Dermatology. 147 (1), 62-70 (2002).</li><li>Turner, N., Ware, O., Bosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+metastasis:+melanoma+and+other+cancers.">Genetics of metastasis: melanoma and other cancers.</a> Clinical &amp; Experimental Metastasis. 35 (5-6), 379-391 (2018).</li><li>Ubellacker, J. 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=Lymph+protects+metastasizing+melanoma+cells+from+ferroptosis.">Lymph protects metastasizing melanoma cells from ferroptosis.</a> Nature. 585 (7823), 113-118 (2020).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Cunningham, C. 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=Actin-binding+protein+requirement+for+cortical+stability+and+efficient+locomotion.">Actin-binding protein requirement for cortical stability and efficient locomotion.</a> Science. 255 (5042), 325-327 (1992).</li><li>Unger, 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=Modeling+human+carcinomas:+physiologically+relevant+3D+models+to+improve+anti-cancer+drug+development.">Modeling human carcinomas: physiologically relevant 3D models to improve anti-cancer drug development.</a> Advanced Drug Delivery Reviews. 79-80, 50-67 (2014).</li><li>Fong, E. L., Harrington, D. A., Farach-Carson, M. C., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heralding+a+new+paradigm+in+3D+tumor+modeling.">Heralding a new paradigm in 3D tumor modeling.</a> Biomaterials. 108, 197-213 (2016).</li><li>Nakamura, K., 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=Characterization+of+mouse+melanoma+cell+lines+by+their+mortal+malignancy+using+an+experimental+metastatic+model.">Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model.</a> Life Science. 70 (7), 791-798 (2002).</li><li>Meeth, K., Wang, J. X., Micevic, G., Damsky, W., Bosenberg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+YUMM+lines:+a+series+of+congenic+mouse+melanoma+cell+lines+with+defined+genetic+alterations.">The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.</a> Pigment Cell Melanoma Research. 29 (5), 590-597 (2016).</li><li>Koya, R. 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=BRAF+inhibitor+vemurafenib+improves+the+antitumor+activity+of+adoptive+cell+immunotherapy.">BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy.</a> Cancer Research. 72 (16), 3928-3937 (2012).</li><li>Jenkins, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+murine+BRaf(V600E)+melanoma+cell+lines+with+sensitivity+to+PLX4032.">Multiple murine BRaf(V600E) melanoma cell lines with sensitivity to PLX4032.</a> Pigment Cell Melanoma Research. 27 (3), 495-501 (2014).</li><li>Tuncer, 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=SMAD+signaling+promotes+melanoma+metastasis+independently+of+phenotype+switching.">SMAD signaling promotes melanoma metastasis independently of phenotype switching.</a> The Journal of Clinical Investigation. 129 (7), 2702-2716 (2019).</li><li>Schwartz, 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=Incipient+Melanoma+Brain+Metastases+Instigate+Astrogliosis+and+Neuroinflammation.">Incipient Melanoma Brain Metastases Instigate Astrogliosis and Neuroinflammation.</a> Cancer Research. 76 (15), 4359-4371 (2016).</li><li>Perez-Guijarro, 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=Multimodel+preclinical+platform+predicts+clinical+response+of+melanoma+to+immunotherapy.">Multimodel preclinical platform predicts clinical response of melanoma to immunotherapy.</a> Nature Medicine. 26 (5), 781-791 (2020).</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>Agrawal, 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=A+systems+biology+approach+identifies+FUT8+as+a+driver+of+melanoma+metastasis.">A systems biology approach identifies FUT8 as a driver of melanoma metastasis.</a> Cell. 31 (6), 804-819 (2017).</li><li>Hanniford, D., 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=Epigenetic+silencing+of+CDR1as+drives+IGF2BP3-mediated+melanoma+invasion+and+metastasis.">Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis.</a> Cancer Cell. 37 (1), 55-70 (2020).</li><li>Kim, 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=PRMT5+control+of+cGAS/STING+and+NLRC5+pathways+defines+melanoma+response+to+antitumor+immunity.">PRMT5 control of cGAS/STING and NLRC5 pathways defines melanoma response to antitumor immunity.</a> Science Translational Medicine. 12 (551), (2020).</li><li>de Miera, E. V., Friedman, E. B., Greenwald, H. S., Perle, M. A., Osman, I. <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+five+new+melanoma+low+passage+cell+lines+representing+the+clinical+and+genetic+profile+of+their+tumors+of+origin.">Development of five new melanoma low passage cell lines representing the clinical and genetic profile of their tumors of origin.</a> Pigment Cell Melanoma Research. 25 (3), 395-397 (2012).</li><li>Morsi, A., 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+and+characterization+of+a+clinically+relevant+mouse+model+of+melanoma+brain+metastasis.">Development and characterization of a clinically relevant mouse model of melanoma brain metastasis.</a> Pigment Cell Melanoma Research. 26 (5), 743-745 (2013).</li><li>Huynh, 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=Efficient+in+vivo+microRNA+targeting+of+liver+metastasis.">Efficient in vivo microRNA targeting of liver metastasis.</a> Oncogene. 30 (12), 1481-1488 (2011).</li><li>Zou, 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=Experimental+variables+that+affect+human+hepatocyte+AAV+transduction+in+liver+chimeric+mice.">Experimental variables that affect human hepatocyte AAV transduction in liver chimeric mice.</a> Molecular Therapy Methods and Clinical Development. 18, 189-198 (2020).</li><li>Kleffman, K., 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-secreted+Amyloid+Beta+Suppresses+Neuroinflammation+and+Promotes+Brain+Metastasis.">Melanoma-secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis.</a> bioRxiv. , 854885 (2019).</li><li>Curtis, A., Calabro, K., Galarneau, J. R., Bigio, I. J., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+skin+pigmentation+in+C57BL/6+mice+affect+optical+bioluminescence+quantitation.">Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.</a> Molecular Imaging and Biology. 13 (6), 1114-1123 (2011).</li><li>Sil, P., Wong, S. W., Martinez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+skin+deep:+autophagy+is+vital+for+skin+barrier+function.">More than skin deep: autophagy is vital for skin barrier function.</a> Frontiers in Immunology. 9, 1376 (2018).</li><li>Chen, S., 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=Genome-wide+CRISPR+screen+in+a+mouse+model+of+tumor+growth+and+metastasis.">Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.</a> Cell. 160 (6), 1246-1260 (2015).</li><li>Hart, T., 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-resolution+CRISPR+screens+reveal+fitness+genes+and+genotype-specific+cancer+liabilities.">High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities.</a> Cell. 163 (6), 1515-1526 (2015).</li><li>Wang, T., 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=Identification+and+characterization+of+essential+genes+in+the+human+genome.">Identification and characterization of essential genes in the human genome.</a> Science. 350 (6264), 1096-1101 (2015).</li><li>Edgar, R., Domrachev, M., Lash, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+Omnibus:+NCBI+gene+expression+and+hybridization+array+data+repository.">Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.</a> Nucleic Acids Research. 30 (1), 207-210 (2002).</li><li>Lappalainen, I., 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=The+European+Genome-phenome+Archive+of+human+data+consented+for+biomedical+research.">The European Genome-phenome Archive of human data consented for biomedical research.</a> Nature Genetics. 47 (7), 692-695 (2015).</li><li>Cerami, 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=The+cBio+cancer+genomics+portal:+an+open+platform+for+exploring+multidimensional+cancer+genomics+data.">The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.</a> Cancer Discovery. 2 (5), 401-404 (2012).</li><li>Grossman, R. 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=Toward+a+shared+vision+for+cancer+genomic+data.">Toward a shared vision for cancer genomic data.</a> New England Journal of Medicine. 375 (12), 1109-1112 (2016).</li></ol>

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 ...

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

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

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

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 ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...