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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C & D

W17 - ECM Composition-Driven Enhancement of 3D Spheroid Formation and Invasion in Mesenchymal-Type TNBC Cells

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Do Hyeong Kim

College student
KAIST, United States

Last Author(s)

Jennifer H. Shin

Professor
KAIST
Daejeon, Republic of Korea

Co-Author(s)

Seongwoo Lee

Master Course Student
Korea Advanced Institute of Science and Technology
Daejeon, Taejon-jikhalsi, Republic of Korea

Introduction: : Triple-negative breast cancer (TNBC) is elucidated as a highly aggressive type characterized by poor prognosis and strong invasive capacity in the patient. The three-dimensional (3D) spheroid model of TNBC spheroid was suggested in recent studies to overcome the limitations of conventional 2D culture systems. However, the mesenchymal-type TNBC cell line (MDA-MB-231) is known for its difficulty in forming spheroids due to weak cell-cell interactions. In this study, a new method to enhance spheroid formation is suggested by complementing the ECM composition. We established a 3D model of the mesenchymal-type TNBC cell line, MDA-MB-231, using ultra-low attachment (ULA) plates and ECM supplementation with type I collagen (Col-I), a significant component of the interstitial stroma, to better mimic the native tumor microenvironment. This approach demonstrated that the ECM composition critically regulates the cohesion and invasiveness of TNBC spheroids. Given that type I collagen is a major structural protein in the stromal matrix, its supplementation mimics the tumor-associated interstitial ECM and facilitates biologically relevant cell–ECM interactions.

Materials and

Methods: : The MDA-MB-231 cells are seeded into ULA round-bottom 96-well plates. To create a combinatorial ECM environment, a mixture of 6 and 10 µg/mL Col-I was added to the culture medium. MDA-MB-231 breast cancer cells were used for 3D invasion assays and 2D spreading assays. Cellular response in the 3D invasion assay was observed through image-based morphological analysis on invasion depth and area by microscopy. ImageJ software (PIV Lab) was used for a 2D spreading assay to measure the spreading area and migration distance quantitatively.

Results, Conclusions, and Discussions:: The addition of Col-I promoted spheroid assembly by reinforcing intercellular cohesion. Circularity and solidity of spheroids measured on days 1,3,and7. Values are normalized, with 1.0 representing the ideal case (a perfect circle). Both metrics increased over time, indicating improved spheroid compaction and morphological uniformity. To evaluate the impact of ECM composition on spheroid behavior, we conducted both 2D spreading and 3D invasion assays following spheroid formation in ultra-low attachment plates supplemented with either 6 or 10µg/mL of Col-I. In the 2D spreading assay, spheroids were transferred onto collagen-coated surfaces, and cell migration was quantified after 24 hours. Spheroids formed under the 10µg/mL Col-I condition exhibited significantly greater spreading distances compared to those formed with 6 µg/mL, suggesting enhanced collective motility. In the 3D invasion assay, spheroids embedded in collagen matrices exhibited more profound and extensive invasive fronts at higher collagen concentrations. Live/Dead staining confirmed that overall cell viability (>98%) remained high across all collagen conditions. In this study, we successfully established a 3D spheroid model of the MDA-MB-231 cell by optimizing ECM composition using type I collagen. Collagen supplementation improved spheroid cohesion, effectively overcoming the limitation of weak cell–cell interactions in this cell line. Invasion and spreading assay results revealed that ECM composition plays a pivotal role in modulating both structural integrity and invasive behavior of TNBC spheroids. Spheroids cultured with a higher collagen concentration exhibited more pronounced invasive outgrowths compared to those formed under lower collagen conditions. These findings highlight the importance of ECM composition in regulating TNBC spheroid dynamics and provide a foundation for future mechanistic studies and drug screening using physiologically relevant 3D platforms. In further research, 3D invasion assays will be complemented with qRT-PCR analysis of EMT markers to quantitatively assess the mechanobiological responses of TNBC spheroids to different ECM environments. While the current model utilizes Col-I to mimic interstitial stroma, we plan to generate spheroids using type IV collagen and laminin to simulate basement membrane-like conditions. This will allow direct comparison of spheroid behavior under stromal versus vascular-like ECM contexts. Such analysis is expected to reveal how ECM composition influences TNBC invasiveness and epithelial–mesenchymal transition.

Acknowledgements and/or References (Optional): : Acknowledgement
This research was supported by the Boston-Korea Collaborative Research Program funded by the Ministry of Science and ICT and the Ministry of Health and Welfare of Korea (Project Number: RS-2024-00468873).

Reference
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