《Biomaterials Advances》：A scalable microbead-based cell culture platform presenting curvature cues for the enrichment of cancer stem cell-like phenotypes beyond 3D spheroid models
编辑推荐：
本研究提出一种基于3D微球的培养平台，能够均匀诱导癌细胞干细胞样表型，并大规模扩展恶性增强的细胞，其机制涉及曲率信号诱导的机械转导和细胞骨架重塑，优于传统2D和3D球体模型，为药物筛选和机制研究提供新工具。
Jemin Yeun|JeongYeon Kim|Minkyung Kim|Seonghyeon Park|Sung Hyun Yoon|Sang Yu Sun|Booseok Jeong|Sung Gap Im|Jieung Baek
韩国科学技术院（KAIST）化学与生物分子工程系，大田，34141，韩国
摘要
Cancer stem cells (CSCs) have been implicated as potential contributors to tumor recurrence, therapeutic resistance, and metastatic behavior. While traditional 3D spheroid models have advanced CSC research, their multilayered architecture introduces cellular heterogeneity and limits reproducibility. Here, we present a 3D microbead-based culture platform that enables spatially uniform induction of CSC-like phenotypes in cancer cells and scalable expansion of malignancy-enriched cells. By leveraging omnidirectional curvature cues provided from suspended microbeads, we achieved enhanced mechanotransductive stimulation across the entire ovarian cancer cell surface. To guide preferential adhesion to the beads and minimize nonspecific substrate attachment, we employed initiated chemical vapor deposition (iCVD) to coat the bottom surface with hydrophobic polymers, where poly(cyclohexyl methacrylate) (pCHMA) promoted microbead-specific adhesion effectively. Cancer cells cultured on this microbead-based system exhibited upregulation of the genes associated with tumor aggressiveness and invasive phenotypes exceeding those observed in conventional 2D monolayer and 3D spheroid models. Mechanistically, these effects were closely associated with curvature-induced RhoA signaling and cytoskeletal remodeling. Furthermore, this platform supported large-scale, high-throughput-compatible expansion of aggressive cancer cells, offering a robust tool for CSC-focused studies and drug screening. Our findings highlight the utility of curvature-mediated mechanobiology in engineering more physiologically relevant and scalable in vitro cancer models.
引言
Cancer is one of the leading causes of mortality worldwide, and its malignant traits pose major challenges to effective therapy [1]. A growing body of evidence has attributed the malignant phenotypes to a subpopulation of tumor cells with cancer stem cell (CSC)-like properties, which play a pivotal role in maintaining tumor heterogeneity and resistance to conventional treatments [2]. Thus, it has been considered that understanding the mechanisms underlying such CSC-like phenotypes and identifying drugs that can selectively target these aggressive subpopulations are essential steps in advancing cancer therapy.
Traditionally, patient-derived tumor cells, which were isolated directly from patients and sorted based on the CSC-specific markers, have been adapted to study CSC-related phenotypes for developing advanced cancer therapeutics [3]. However, this approach is hampered by seriously limited interpatient variability, low scalability, and complex handling procedures [4]. Moreover, such models commonly faced significant challenges for high-throughput drug screening and mechanistic studies due to their heterogeneity and ethical constraints. As a result, immortalized cancer cell lines have been increasingly utilized as alternatives, offering greater ease in cultivation and expansion [5]. Nevertheless, these cell lines often lack the metastatic potential observed in tumor cells in vivo, thereby limiting their translational relevance [2]. Recent efforts have thus focused on enhancing the CSC-like characteristics of cancer cell lines [6]. Such approaches have included co-culture with other cell types [7], exposure to geometrical cues [8], and the use of three-dimensional (3D) culture systems [9]. These advancements aimed to bridge the gap between conventional in vitro models and the complex behavior of metastatic cancer cells in vivo.
Among these strategies, 3D cancer cell culture has garnered particular attention for its improved ability to better mimic the tumor microenvironment compared to traditional two-dimensional (2D) monolayer cultures [10]. Various methods for spheroid generation have been suggested, such as culturing cells on hydrophilic ultra-low-attachment (ULA) surfaces [11], and our group previously developed a hydrophobic polymer thin film that induces spheroid formation via spontaneous interactions between cancer cells and surface-adsorbed proteins [12], [13]. This approach allowed for the formation of spheroids with uniform size and shape, and a far enriched population of CSC-like cells, all without the need for specialized equipment or complex procedures. However, despite these advances, challenges remain in achieving faithful CSC-like traits while maintaining spheroid uniformity and reproducibility for large-scale culture [15]. In particular, the intrinsic multilayered structure of spheroids induces cellular heterogeneity due to limited oxygen and nutrient diffusion, which can compromise cell viability, reproducibility, and scalability in downstream applications such as drug screening [16].
A promising strategy to overcome these challenges is to expose cancer cells to controlled physical and geometrical cues that can induce CSC-like phenotypes in a reproducible manner. Increasing evidence indicates that cellular mechanotransduction plays a critical role in regulating cancer cell plasticity, and that substrate geometry and mechanical properties strongly influence cancer cell behavior [17], [18]. Previous studies have demonstrated that convex and concave geometries can modulate cell shape, adhesion signaling, and downstream pathways, thereby promoting stem cell–like traits in specific subpopulations of cancer cells [19]. These findings highlight the importance of well-defined geometrical cues in priming CSC-associated phenotypes and provide a strong rationale for the use of microbead-based platforms to investigate geometry-driven CSC induction. Specifically, geometric cues at the tumor periphery were shown to prime a subpopulation of cancer cells toward a stem cell-like phenotype. This effect was mediated through orchestrated changes in cell shape, integrin α5β1 signaling, and activation of downstream pathways such as MAPK and STAT.
In this study, we present a 3D curvature-based platform for the spatially uniform induction of CSC-like phenotypes and large-scale expansion of malignancy-enriched cancer cells under physiologically relevant mechanical conditions. Compared to 2D monolayer curvature, such as convex and concave patterns that provide localized and unidirectional mechanical cues, we expected the 3D curvature to offer continuous and omnidirectional topographical stimulation across the entire cell surface with better mimicry of the 3D in vivo microenvironment. This approach involves the attachment of cancer cells on the surface of suspended microbeads, which provided uniform 3D curvature cues. Therefore, our system should be equipped with the capability to prevent the cells from adhering to the bottom flat substrates. Given that hydrophobicity of substrates influences critically cellular adhesion by regulating the adsorption profile of cell adhesive molecules (CAMs), we delicately guided the cellular adhesion preference between the bottom flat surface and the suspended curvature surfaces by coating the bottom surface with a series of polymers with varying hydrophobicity via initiated chemical vapor deposition (iCVD). Among these, we found that hydrophobic poly(cyclohexyl methacrylate) (pCHMA) thin film most effectively prevented nonspecific adhesion to the underlying culture dish while promoting preferential attachment of the cells to the suspended microbead surfaces (Fig. 1a). When the cancer cells and microbeads were co-seeded on conventionally-used tissue culture polystyrene (TCPS), they predominantly adhered to the TCPS surface rather than to the microbeads. In contrast, on the hydrophobic pCHMA-coated surface, cancer cells initially formed small spheroids and subsequently attached to the microbeads, promoting their spreading and adhesion (Fig. 1b, S1). To our knowledge, this is the first study to intentionally guide selective cell attachment onto 3D curved microstructures through substrate modification, thereby directing cellular growth specifically along the microbead surface. As a result of this curvature-guided growth, cancer cells cultured with the microbeads exhibited distinctly higher levels of CSC-associated traits compared to those grown on flat substrates [19]. Notably, in certain properties, these cells even surpassed the levels observed in 3D spheroid culture systems. We suggest that these phenotypic alterations are closely associated with cytoskeletal reorganization induced by the curved surface, and the subsequent RhoA-related cellular response. Furthermore, the microbead-based cell culture system is fully compatible with large-scale expansion of malignant cancer cells, demonstrating its potential for efficient CSC-enriched cell propagation. Altogether, this approach offers a promising tool for large-scale expansion of malignancy-enriched cells and more accurate screening of CSC-targeted therapeutics.
聚合物薄膜的合成与表征
To enable microbead-based culture, it is necessary to control the composition of the culture plate surface to prevent cell attachment to the plate surface itself, ensuring that cells attach exclusively to the surface of the microbeads. In that sense, the hydrophobicity of the plate surface to which cells adhere is a critical parameter in regulating cell adhesion, as it influences the adsorption profile of either CAMs or the cell membrane directly [22]. Considering all these aspects, a series of
讨论与结论
The large-scale expansion of cancer cells exhibiting CSC-like characteristics is a valuable strategy for dissecting key mechanisms underlying metastasis, recurrence, and therapeutic resistance, and for accelerating the development of treatments targeting highly malignant tumors. Here, we propose a scalable microbead-based culture platform that leverages a well-defined 3D curvature microenvironment to induce malignancy-associated phenotype in cancer cells without genetic engineering or chemical modifications.
通过化学气相沉积（iCVD）合成聚合物薄膜
The polymer films were coated on the tissue culture polystyrene (TCPS) following the reference protocol [80]. The temperature of the substrate was maintained at 28.5?°C for pHEMA, 29.5?°C for pEGDMA, 36?°C for pVBC, and 28?°C for pCHMA. For the deposition of pHEMA, HEMA (Sigma Aldrich) was heated at 55?°C and introduced into the iCVD chamber. The flow rate of the HEMA was 0.79 sccm, and the pressure of the chamber was maintained at 60 mTorr. UV light (λmax = 254?nm, VL-6-L.C, Vilber Lourmat) was used during the deposition process.
CRediT作者贡献声明
Jemin Yeun:撰写原始稿件、方法论设计、实验实施、数据分析、概念构建。
JeongYeon Kim:撰写原始稿件、方法论设计、数据分析、概念构建。
Minkyung Kim:方法论设计、数据分析、概念构建。
Seonghyeon Park:方法论设计、数据分析、概念构建。
Sung Hyun Yoon:撰写原始稿件、数据分析、概念构建。
Sang Yu Sun:数据分析、概念构建。
Booseok Jeong:数据分析。
伦理声明
This study doesn't include clinical experiments, animal experiments, or human subjects.
利益冲突声明
The authors declare that there are no known financial interests or personal relationships that could affect the objectivity of the research results.
致谢
This study was supported by the Nanomedical Devices Development Project of the National Research Foundation of Korea (NNFC) in 2025, as well as the Technology Development Program (RS-2024-00508684) funded by the Ministry of SMEs and Startups (MSS, Korea). It was also supported by grants from the National Research Foundation of Korea (NRF) provided by the Ministry of Science and Information Technology (MSIT) (numbers RS-2023-00213047 and RS-2024-00405818). In addition, this research was funded by the Ewha Womans University Research Grant in 2023.
