{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:48:29Z","timestamp":1785408509659,"version":"3.56.0"},"reference-count":52,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T00:00:00Z","timestamp":1781222400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100023078","name":"State of North Carolina","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100023078","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000005","name":"Department of Defense","doi-asserted-by":"publisher","award":["W911NF2120265"],"award-info":[{"award-number":["W911NF2120265"]}],"id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"publisher"}]},{"award":["W911NF2120265"],"award-info":[{"award-number":["W911NF2120265"]}],"id":[{"id":"https:\/\/ror.org\/0447fe631","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Melanoma is the most aggressive type of skin cancer, driven by early invasion, phenotypic plasticity, and frequent resistance to targeted therapies. Although genomic profiling informs treatment selection, genotype alone often fails to predict therapeutic response, underscoring the need for rapid and physiologically relevant functional testing platforms. Here, we present a three-dimensional melanoma\u2013skin organoid (mSO) model that integrates primary skin cells with melanoma cell lines in a self-assembling, high-throughput format. The spherical mSOs recapitulate native human skin architecture, including a stratified epidermis and a dermal\u2013hypodermal core, while supporting melanoma growth within an appropriate tissue microenvironment. In this niche, melanoma cells display epidermal spreading in radial growth-like patterns, outward invasion, and transcriptional shifts toward a pro-invasive phenotype. Using live confocal imaging coupled with a custom automated image analysis pipeline, we quantitatively measured tumor growth, migration beyond the organoid boundary, and interactions between melanoma cells and normal melanocytes. The mSOs also captured genotype-specific drug responses: BRAF-mutant melanoma cells were sensitive to BRAF and MEK inhibition, whereas NRAS-mutant, BRAF\u2013wild-type cells were resistant to BRAF inhibition but remained responsive to MEK inhibition. Altogether, our mSO platform combines architectural and functional complexity with experimental scalability, providing a robust framework for modeling melanoma progression and evaluating targeted therapeutic responses within a relevant skin microenvironment. In the future, adaptation of this system to include patient-derived tumor cells could support personalized therapeutic decision-making in melanoma.<\/jats:p>","DOI":"10.3390\/ijms27125314","type":"journal-article","created":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T06:53:10Z","timestamp":1781247190000},"page":"5314","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Rapid 3D Melanoma\u2013Skin Organoid for High-Throughput Assessment of Tumor Dynamics and Drug Response"],"prefix":"10.3390","volume":"27","author":[{"given":"Gemma","family":"Nomdedeu-Sancho","sequence":"first","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"Edenhoffer","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasiya","family":"Gorkun-Roeder","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ola A.","family":"Gaser","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8788-4221","authenticated-orcid":false,"given":"Carlos","family":"Kengla","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allie","family":"Benton","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2644-350X","authenticated-orcid":false,"given":"David W.","family":"Mullins","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony","family":"Atala","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shay","family":"Soker","sequence":"additional","affiliation":[{"name":"Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27101, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,12]]},"reference":[{"key":"ref_1","first-page":"17","article-title":"Cancer statistics, 2023","volume":"73","author":"Siegel","year":"2023","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Eddy, K., Shah, R., and Chen, S. (2021). Decoding Melanoma Development and Progression: Identification of Therapeutic Vulnerabilities. Front. Oncol., 10.","DOI":"10.3389\/fonc.2020.626129"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2413","DOI":"10.1038\/onc.2013.194","article-title":"Melanoma metastasis: New concepts and evolving paradigms","volume":"33","author":"Damsky","year":"2014","journal-title":"Oncogene"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1535","DOI":"10.1056\/NEJMoa1910836","article-title":"Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma","volume":"381","author":"Larkin","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3998","DOI":"10.1200\/JCO.22.01599","article-title":"Seven-Year Follow-Up of the Phase III KEYNOTE-006 Study: Pembrolizumab Versus Ipilimumab in Advanced Melanoma","volume":"41","author":"Robert","year":"2023","journal-title":"J. Clin. Oncol."},{"key":"ref_6","first-page":"42","article-title":"Exploring resistance to immune checkpoint inhibitors and targeted therapies in melanoma","volume":"7","author":"Jalil","year":"2024","journal-title":"Cancer Drug Resist."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1038\/s43018-024-00794-1","article-title":"Determinants of resistance and response to melanoma therapy","volume":"5","author":"Robertson","year":"2024","journal-title":"Nat. Cancer"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1016\/j.molcel.2015.06.028","article-title":"Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis via Notch Signaling Inhibition of MITF","volume":"59","author":"Golan","year":"2015","journal-title":"Mol. Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1097\/CMR.0b013e32833d8d70","article-title":"Keratinocytes drive melanoma invasion in a reconstructed skin model","volume":"20","author":"Bergers","year":"2010","journal-title":"Melanoma Res."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Klicks, J., Ma\u00dflo, C., Kluth, A., Rudolf, R., and Hafner, M. (2019). A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells. BMC Cancer, 19.","DOI":"10.1186\/s12885-019-5606-4"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e719","DOI":"10.1038\/cddis.2013.249","article-title":"Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing","volume":"4","author":"Groeber","year":"2013","journal-title":"Cell Death Dis."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Michielon, E., L\u00f3pez Gonz\u00e1lez, M., Stolk, D.A., Stolwijk, J.G.C., Roffel, S., Waaijman, T., Lougheed, S.M., De Gruijl, T.D., and Gibbs, S. (2023). A Reconstructed Human Melanoma-in-Skin Model to Study Immune Modulatory and Angiogenic Mechanisms Facilitating Initial Melanoma Growth and Invasion. Cancers, 15.","DOI":"10.3390\/cancers15102849"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e3754","DOI":"10.7717\/peerj.3754","article-title":"Quantitative comparison of the spreading and invasion of radial growth phase and metastatic melanoma cells in a three-dimensional human skin equivalent model","volume":"5","author":"Haridas","year":"2017","journal-title":"PeerJ"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Nomdedeu-Sancho, G., Gorkun, A., Mahajan, N., Willson, K., Schaaf, C.R., Votanopoulos, K.I., Atala, A., and Soker, S. (2023). In Vitro Three-Dimensional (3D) Models for Melanoma Immunotherapy. Cancers, 15.","DOI":"10.3390\/cancers15245779"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1089\/ten.tea.2019.0319","article-title":"Bioprinted Skin Recapitulates Normal Collagen Remodeling in Full-Thickness Wounds","volume":"26","author":"Jorgensen","year":"2020","journal-title":"Tissue Eng. Part A"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1038\/nature00766","article-title":"Mutations of the BRAF gene in human cancer","volume":"417","author":"Davies","year":"2002","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1111\/1523-1747.ep12512500","article-title":"Some Aspects Of Melanin Biology: 1950\u20131975","volume":"67","author":"Jimbow","year":"1976","journal-title":"J. Investig. Dermatol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Marrapodi, R., and Bellei, B. (2024). The Keratinocyte in the Picture Cutaneous Melanoma Microenvironment. Cancers, 16.","DOI":"10.3390\/cancers16050913"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Dainese-Marque, O., Garcia, V., Andrieu-Abadie, N., and Riond, J. (2024). Contribution of Keratinocytes in Skin Cancer Initiation and Progression. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25168813"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e202212031","DOI":"10.1083\/jcb.202212031","article-title":"Melanoma cells repress Desmoglein 1 in keratinocytes to promote tumor cell migration","volume":"222","author":"Burks","year":"2023","journal-title":"J. Cell Biol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Loh, C.-Y., Chai, J., Tang, T., Wong, W., Sethi, G., Shanmugam, M., Chong, P., and Looi, C. (2019). The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and Challenges. Cells, 8.","DOI":"10.3390\/cells8101118"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Huang, F., Santinon, F., Flores Gonz\u00e1lez, R.E., and Del Rinc\u00f3n, S.V. (2021). Melanoma Plasticity: Promoter of Metastasis and Resistance to Therapy. Front. Oncol., 11.","DOI":"10.3389\/fonc.2021.756001"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/s41568-019-0154-4","article-title":"Phenotype plasticity as enabler of melanoma progression and therapy resistance","volume":"19","author":"Arozarena","year":"2019","journal-title":"Nat. Rev. Cancer"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5712","DOI":"10.1038\/ncomms6712","article-title":"Low MITF\/AXL ratio predicts early resistance to multiple targeted drugs in melanoma","volume":"5","author":"Krijgsman","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kawczak, P., and B\u0105czek, T. (2025). Pathway-Specific Therapeutic Modulation of Melanoma: Small-Molecule Inhibition of BRAF\u2013MEK and KIT Signaling in Contemporary Precision Oncology with a Special Focus on Vemurafenib, Trametinib, and Imatinib. J. Clin. Med., 14.","DOI":"10.3390\/jcm14227906"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4794","DOI":"10.1200\/JCO.23.01136","article-title":"Systemic Therapy for Melanoma: ASCO Guideline Update","volume":"41","author":"Seth","year":"2023","journal-title":"J. Clin. Oncol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1186\/s13046-018-0982-1","article-title":"TNF-alpha and metalloproteases as key players in melanoma cells aggressiveness","volume":"37","author":"Rossi","year":"2018","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3275","DOI":"10.1038\/s41388-018-0205-4","article-title":"AXL\/AKT axis mediated-resistance to BRAF inhibitor depends on PTEN status in melanoma","volume":"37","author":"Zuo","year":"2018","journal-title":"Oncogene"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1038\/s41698-021-00207-x","article-title":"PERK mediates resistance to BRAF inhibition in melanoma with impaired PTEN","volume":"5","author":"Qin","year":"2021","journal-title":"NPJ Precis. Oncol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"133","DOI":"10.5603\/NJO.2019.0025","article-title":"Mechanisms of melanoma resistance to treatment with BRAF and MEK inhibitors","volume":"69","author":"Bartnik","year":"2019","journal-title":"Nowotw. J. Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1503","DOI":"10.1007\/s11864-022-01006-7","article-title":"BRAF Inhibitor Resistance in Melanoma: Mechanisms and Alternative Therapeutic Strategies","volume":"23","author":"Zhong","year":"2022","journal-title":"Curr. Treat. Options Oncol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Valdez-Salazar, F., Jim\u00e9nez-Del Rio, L.A., Padilla-Guti\u00e9rrez, J.R., Valle, Y., Mu\u00f1oz-Valle, J.F., and Vald\u00e9s-Alvarado, E. (2024). Advances in Melanoma: From Genetic Insights to Therapeutic Innovations. Biomedicines, 12.","DOI":"10.3390\/biomedicines12081851"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Fontana, F., Sommariva, M., Anselmi, M., Bianchi, F., Limonta, P., and Gagliano, N. (2024). Differentiation States of Phenotypic Transition of Melanoma Cells Are Revealed by 3D Cell Cultures. Cells, 13.","DOI":"10.3390\/cells13020181"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"eadg6686","DOI":"10.1126\/sciadv.adg6686","article-title":"A human mucosal melanoma organoid platform for modeling tumor heterogeneity and exploring immunotherapy combination options","volume":"9","author":"Sun","year":"2023","journal-title":"Sci. Adv."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e2308525","DOI":"10.1002\/smll.202308525","article-title":"Mimicking Tumor Metastasis Using a Transwell-Integrated Organoids-On-a-Chip Platform","volume":"20","author":"Chen","year":"2024","journal-title":"Small"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1186\/s13046-025-03539-9","article-title":"High-throughput drug screening in advanced pre-clinical 3D melanoma models identifies potential first-line therapies for NRAS-mutated melanoma","volume":"44","author":"Angeli","year":"2025","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1007\/s00262-025-04269-9","article-title":"Modeling anti-tumor immune responses using patient-derived melanoma organoids","volume":"75","author":"Kaminska","year":"2025","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"104614","DOI":"10.1016\/j.ebiom.2023.104614","article-title":"Patient-derived melanoma organoid models facilitate the assessment of immunotherapies","volume":"92","author":"Ou","year":"2023","journal-title":"eBioMedicine"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2039","DOI":"10.1021\/mp200421k","article-title":"Fibroblasts Contribute to Melanoma Tumor Growth and Drug Resistance","volume":"8","author":"Flach","year":"2011","journal-title":"Mol. Pharm."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"10428","DOI":"10.1038\/s41598-017-10699-y","article-title":"Tumor cells and their crosstalk with endothelial cells in 3D spheroids","volume":"7","author":"Shoval","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"13191","DOI":"10.1038\/s41598-018-31502-6","article-title":"Tissue-engineered 3D melanoma model with blood and lymphatic capillaries for drug development","volume":"8","author":"Bourland","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1038\/nature08833","article-title":"RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth","volume":"464","author":"Hatzivassiliou","year":"2010","journal-title":"Nature"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/j.ccr.2012.01.017","article-title":"The RAF Inhibitor Paradox Revisited","volume":"21","author":"Cox","year":"2012","journal-title":"Cancer Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1023\/A:1020625423524","article-title":"Phase II Study of Marimastat (BB-2516) in Malignant Melanoma\u2014A Clinical and Tumor Biopsy Study of the National Cancer Institute of Canada Clinical Trials Group","volume":"20","author":"Quirt","year":"2002","journal-title":"Investig. New Drugs"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.ygyno.2005.12.020","article-title":"A phase III randomized trial of BAY 12-9566 (tanomastat) as maintenance therapy in patients with advanced ovarian cancer responsive to primary surgery and paclitaxel\/platinum containing chemotherapy: A National Cancer Institute of Canada Clinical Trials G","volume":"102","author":"Hirte","year":"2006","journal-title":"Gynecol. Oncol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1111\/ijd.14850","article-title":"The role of integrins in melanoma: A review","volume":"59","author":"Warda","year":"2020","journal-title":"Int. J. Dermatol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1182","DOI":"10.1007\/s10637-013-0001-5","article-title":"A phase Ib combination study of RO4929097, a gamma-secretase inhibitor, and temsirolimus in patients with advanced solid tumors","volume":"31","author":"Hirte","year":"2013","journal-title":"Investig. New Drugs"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Pagliuca, C., Di Leo, L., and De Zio, D. (2022). New Insights into the Phenotype Switching of Melanoma. Cancers, 14.","DOI":"10.3390\/cancers14246118"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Hossain, S.M., and Eccles, M.R. (2023). Phenotype Switching and the Melanoma Microenvironment; Impact on Immunotherapy and Drug Resistance. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24021601"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, T.L. (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform., 13.","DOI":"10.1186\/1471-2105-13-134"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"92102","DOI":"10.1172\/jci.insight.92102","article-title":"Transcriptional dissection of melanoma identifies a high-risk subtype underlying TP53 family genes and epigenome deregulation","volume":"2","author":"Badal","year":"2017","journal-title":"JCI Insight"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/12\/5314\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,14]],"date-time":"2026-06-14T04:10:57Z","timestamp":1781410257000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/12\/5314"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,12]]},"references-count":52,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["ijms27125314"],"URL":"https:\/\/doi.org\/10.3390\/ijms27125314","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,12]]}}}