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Artesunate (ART), a water-soluble derivative traditionally used to treat malaria, has recently gained attention as a promising anticancer agent. Recently, studies have shown that ART can inhibit the migration and invasion of cancer cells. However, theses studies are always based on conventional two-dimensional (2D) culture models, which neglect the complex interactions between cells and the extracellular matrix (ECM) or in vivo microenvironment. To address these challenges, in this study, honeycomb-like gelatin methacryloyl (GelMA) hydrogel microspheres as a three-dimensional (3D) culture platform were introduced to mimic the native ECM microenvironment and assess the effects of ART on ovarian cancer cells. The results showed that these GelMA microspheres exhibited a uniform and highly porous structure, which supported cell adhesion, proliferation, and migration. Notably, 3D culture promoted DNA synthesis and induced G1-to-S phase transition, indicating enhanced proliferative capacity. Furthermore, ovarian cancer cells cultured in 3D exhibited stronger invasive and chemoresistant phenotypes. RNA sequencing revealed that after ART treatment, tumor-related proliferation and invasion genes of ovarian cancer cells were significantly up-regulated under 3D culture compared with 2D culture. This might be related to the up-regulation of TNF signaling pathway, NF-kappa B signaling pathway, and Pl3K-Akt signaling pathway. Correspondly, epithelial-mesenchymal transition (EMT) of ovarian cancer cells was markedly enhanced when cultured on GelMA hydrogel microspheres after ART treatment. These effects might be attributed to the altered microenvironment provided by the GelMA hydrogel microspheres, which could be responsible for the enhanced resistance of ovarian cancer cells to ART. These findings suggest that, compared to traditional 2D. These findings suggest that, compared to traditional 2D culture, the 3D extracellular matrix-based system better mimics the in vivo tumor architecture, thereby improving the sensitivity of anticancer drug screening and offering a more reliable platform for evaluating drug resistance and tumor cell heterogeneity.<\/jats:p>","DOI":"10.1007\/s10561-026-10226-2","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T06:20:37Z","timestamp":1787206837000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The evaluation of biological effects of artesunate on ovarian cancer using three-dimensional cell culture methods"],"prefix":"10.1007","volume":"27","author":[{"given":"Yijun","family":"Yuan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojing","family":"Luo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Longyu","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianwen","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinru","family":"Zou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Feng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Su","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongqin","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"issue":"1","key":"10226_CR1","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1186\/s12929-024-00994-y","volume":"31","author":"WH Abuwatfa","year":"2024","unstructured":"Abuwatfa WH, Pitt WG, Husseini GA (2024) Scaffold-based 3D cell culture models in cancer research. 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