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Morphological and functional responses of three-dimensional-cultured colorectal cancer spheres to anticancer drugs
Yusuke Yoshimura, Seiichi Shinji, Yutaro Ogawa, Takeshi Yamada, Akihisa Matsuda, Kay Uehara, Yasuyuki Yokoyama, Goro Takahashi, Takuma Iwai, Toshimitsu Miyasaka, Shintaro Kanaka, Takanori Matsui, Koki Hayashi, Masakazu Fujiwara, Yuuki Shichi, Tomio Arai, Toshiyuki Ishiwata, Hiroshi Yoshida
Yusuke Yoshimura, Seiichi Shinji, Yutaro Ogawa, Takeshi Yamada, Akihisa Matsuda, Kay Uehara, Yasuyuki Yokoyama, Goro Takahashi, Takuma Iwai, Toshimitsu Miyasaka, Shintaro Kanaka, Takanori Matsui, Koki Hayashi, Hiroshi Yoshida, Department of Gastroenterological Surgery, Nippon Medical School, Bunkyo-ku 113-8603, Tokyo, Japan
Yusuke Yoshimura, Seiichi Shinji, Yutaro Ogawa, Masakazu Fujiwara, Yuuki Shichi, Toshiyuki Ishiwata, Division of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Itabashi-ku 173-0015, Tokyo, Japan
Tomio Arai, Department of Pathology, Tokyo Metropolitan Institute for Geriatrics and Gerontology, Itabashi-ku 173-0015, Tokyo, Japan
Author contributions: Yoshimura Y, Iwai T, and Shinji S conceptualized the study; Yoshimura Y, Ogawa Y, Shichi Y, and Fujiwara M were responsible for the investigation and the study’s methodology; Yamada T, Matsuda A, Uehara K, Ogawa Y, Takahashi G, Iwai T, Miyasaka T, Kanaka S, Matsui T, Hayashi K, Yoshimura Y, Arai T, and Yoshida H were responsible for the study’s resources; Yoshimura Y, Iwai T, and Shinji S were responsible for the writing - original draft; all authors were responsible for writing - review and editing; Iwai T and Shinji S supervised the study; Yamada T, Matsuda A, Uehara K, Ogawa Y, Takahashi G, Iwai T, Miyasaka T, Kanaka S, Hayashi K, Yokoyama Y, Arai T, and Yoshida H were responsible for the study validation.
Supported by the JSPS KAKENHI (Grants-in-Aid for Scientific Research C), No. 21K08744, No. 24K11881, No. 25K12018, No. 22K08882, and No. 22K08835.
Institutional review board statement: This study did not include any human or animal participants. Only established human cell lines obtained from a cell bank were used; therefore, institutional review board approval was not required.
Conflict-of-interest statement: The authors declare no conflicts of interest, including financial, personal, political, intellectual, or religious interests related to this work.
Data sharing statement: The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Corresponding author: Seiichi Shinji, MD, PhD, Associate Professor, Department of Gastroenterological Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku 113-8603, Tokyo, Japan.
s-shinji@nms.ac.jp
Received: November 3, 2025
Revised: November 28, 2025
Accepted: January 14, 2026
Published online: April 15, 2026
Processing time: 157 Days and 8.5 Hours
BACKGROUND
Three-dimensional (3D) culture systems closely recapitulate the tumor microenvironment, allowing the assessment of drug effects on cell-cell interactions and metabolic heterogeneity. Colorectal adenocarcinoma (CRAC) cells exhibit epithelial and mesenchymal features, reflecting tumor heterogeneity, which may influence drug sensitivity. However, the morphological alterations and functional states of the residual CRAC spheres after chemotherapy remain unclear. Understanding these morphological and metabolic adaptations may help identify cancer stem cell (CSC)-like and epithelial-mesenchymal transition (EMT)-related phenotypes that contribute to therapeutic resistance and recurrence.
AIM
To elucidate the morphological and functional responses of 3D-cultured CRAC spheres to anticancer drugs and to characterize the residual CSC/EMT phenotypes.
METHODS
Five CRAC cell lines were cultured as 3D spheres, treated with 5-fluorouracil, irinotecan, or oxaliplatin, and analyzed morphologically and molecularly.
RESULTS
All drugs significantly suppressed sphere growth, but the morphological and metabolic responses varied. Scanning electron microscopy revealed cell surface flattening or detachment in epithelial-type spheres, whereas mesenchymal-type SW480 spheres exhibited a bowl-shaped structure with depressed centers. ATP levels decreased in all lines, except for 5-fluorouracil-treated SW480 spheres, which retained ATP but showed reduced water-soluble tetrazolium 8 activity, suggesting metabolic dormancy. Caspase 3/7 activity markedly increased in irinotecan-treated HCT-15 cells. Quantitative reverse transcriptase PCR revealed the upregulation of CD44, ALDH1A1, and PROM1 in HCT-15 cells, and increased CD44/ALDH1A1 with reduced PROM1 in SW480 cells. CDH1, VIM, and ZEB1 levels increased in HCT-15 cells, whereas SW480 cells upregulated CDH1 and VIM but downregulated ZEB1, indicating partial EMT and mesenchymal-epithelial reverting transitions.
CONCLUSION
Anticancer drugs induce distinct morphological and molecular adaptations, and residual CRAC spheres acquire hybrid epithelial-mesenchymal phenotypes associated with chemoresistance.
Core Tip: This study analyzed the effects of 5-fluorouracil, irinotecan, and oxaliplatin on three-dimensionally cultured colorectal adenocarcinoma (CRAC) spheres. Using cell viability assays, apoptosis detection, and quantitative reverse transcriptase PCR, we found that the residual spheres displayed increased expression of cancer stem cell and epithelial-mesenchymal transition markers, with distinct profiles between epithelial (HCT-15) and mesenchymal (SW480) types. These results indicate that CRAC spheres that survive anticancer drug treatment acquire hybrid epithelial-mesenchymal phenotypes and stem cell-like features, providing insights into the mechanisms of drug tolerance and cancer recurrence in colorectal cancer.
