{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T01:53:35Z","timestamp":1774317215252,"version":"3.50.1"},"reference-count":37,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T00:00:00Z","timestamp":1761091200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000925","name":"NHMRC Ideas Grant","doi-asserted-by":"publisher","award":["APP1181179"],"award-info":[{"award-number":["APP1181179"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001171","name":"Cancer Institute NSW Translational Program Grant","doi-asserted-by":"publisher","award":["2020\/TPG2081"],"award-info":[{"award-number":["2020\/TPG2081"]}],"id":[{"id":"10.13039\/501100001171","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001777","name":"University of Wollongong","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001777","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Bill Gole MND postdoctoral Fellowship"},{"name":"FightMND"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background\/Objectives: Cutaneous squamous cell carcinoma (cSCC) is a very common skin malignancy of the head and neck area, with a propensity to spread to local lymph nodes. Epithelial-to-mesenchymal transition (EMT) and cancer-associated fibroblasts (CAFs) play a well-documented role in the progression of the disease. In this study, we developed and characterised multicellular tumour spheroids (MCTS) composed of patient-derived metastatic cSCC cell lines\u2014each exhibiting distinct phenotypes\u2014combined with either dermal- or lymph node-derived fibroblasts. We aimed to investigate how these cellular combinations influence MCTS formation, spatial architecture, and invasive behaviour. We hypothesised that the interplay between different cSCC and fibroblast cell combinations would differentially influence spheroid formation and invasion. Methods: Using live-cell microscopy we assessed the spatial architectures specific to each cell line-fibroblast combination and evaluated the expression of EMT and CAF markers. Furthermore, we utilised MCTS in invasion models to investigate associations between the mode of invasion and the EMT phenotype of the cancer cell line. Results: We show that metastatic cSCC\/fibroblast MCTS self-organise into distinct spatial architectures. They also invade through collagen in a manner influenced by fibroblasts but dominated by the EMT status of the originating cancer cells. Conclusions: These findings highlight the physiological relevance and utility of MCTS as models for investigating tumour\u2013stroma interactions and invasion dynamics in metastatic cSCC.<\/jats:p>","DOI":"10.3390\/cancers17213399","type":"journal-article","created":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T02:03:48Z","timestamp":1761185028000},"page":"3399","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Spatial Organisation and Invasive Behaviour of Metastatic Cutaneous Squamous Cell Carcinoma-Derived Multicellular Spheroids Reflect Tumour Cell Phenotype"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5665-9811","authenticated-orcid":false,"given":"Benjamin","family":"Genenger","sequence":"first","affiliation":[{"name":"School of Science and Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1016-1710","authenticated-orcid":false,"given":"Jessica","family":"Conley","sequence":"additional","affiliation":[{"name":"School of Science and Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5066-3291","authenticated-orcid":false,"given":"Chelsea","family":"Penney","sequence":"additional","affiliation":[{"name":"School of Science and Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1764-3809","authenticated-orcid":false,"given":"Luke","family":"McAlary","sequence":"additional","affiliation":[{"name":"School of Science and Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1153-4609","authenticated-orcid":false,"given":"Jay R.","family":"Perry","sequence":"additional","affiliation":[{"name":"School of Science and Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia"},{"name":"Graduate School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4547-6611","authenticated-orcid":false,"given":"Bruce","family":"Ashford","sequence":"additional","affiliation":[{"name":"Graduate School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia"},{"name":"Illawarra Shoalhaven Local Health District, NSW Health, Wollongong, NSW 2500, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5570-9645","authenticated-orcid":false,"given":"Marie","family":"Ranson","sequence":"additional","affiliation":[{"name":"School of Science and Molecular Horizons, Faculty of Science, Medicine and Health, University of Wollongong, Wollongong, NSW 2522, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1111\/ajd.13830","article-title":"Defining the incidence of cutaneous squamous cell carcinoma in coastal NSW Australia","volume":"63","author":"Wilson","year":"2022","journal-title":"Australas. 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