Correlation between Spheroid Formation Ability and Reported Invasiveness of HEK-293, HT-29, MDA-MB-231, and HeLa Cancer Cell Lines in Commercial Well Plates
Abstract
Three-dimensional (3D) cell cultures, such as spheroids, are essential for replicating in vivo tumor environments, offering a more accurate model for cancer research and drug testing. Spheroids form through the self-aggregation of cells under specific conditions, enabling the study of cellular behavior, including invasiveness. In this study, we investigated the correlation between spheroid formation ability and the reported invasiveness of four widely used human cancer cell lines — Human Embryonic Kidney 293 (HEK-293), Human Colorectal Adenocarcinoma (HT-29), Human Breast Cancer (MDA-MB-231), and Human Cervical Cancer (HeLa) — using a commercial round bottom 96-well microplate and AggreWell™ microwells with varying cell seeding concentrations. Cells were cultured in highly viscous media and seeded at varying densities (1,000, 2,000, and 3,000 cells per well) to assess the effect of cell number on spheroid size over time. Microscopic analysis revealed distinct differences among the cell lines; HEK-293 and HT-29 cells formed compact, well-defined spheroids, with larger spheroids observed at higher seeding densities. In contrast, the more aggressive and invasive MDA-MB-231 and HeLa cells failed to form spheroids under these conditions. These findings demonstrate the intricate relationship between cancer cell aggressiveness, seeding density, and spheroid formation ability, which are critical factors in optimizing 3D culture-based drug development and cancer research
Keywords
3D cell culture Spheroid formation Cancer cell lines Tumor invasiveness Seeding density Drug testing models
Supporting Institution
Sao Paulo Research Foundation (FAPESP)
Thanks
Ismail Eş acknowledges the financial support of Sao Paulo Research Foundation (FAPESP), Brazil (Grant# 2015/14468-0 and 2018/23895-7)
References
- Ferlay, J., Colombet, M., Soerjomataram, I., Parkin, D.M., Piñeros, M., Znaor, A., & Bray, F. (2021). Cancer Statistics for the Year 2020: An Overview. International Journal of Cancer, 149, 778-789.
- DiMasi, J.A., Reichert, J.M., Feldman, L., & Malins, A. (2013). Clinical Approval Success Rates for Investigational Cancer Drugs. Clinical Pharmacology & Therapeutics, 94, 329-335.
- Wei, F., Wang, S., & Gou, X. (2021). A Review for Cell-based Screening Methods in Drug Discovery. Biophysics Reports, 7, 504.
- Kapałczyńska, M., Kolenda, T., Przybyła, W., Zajączkowska, M., Teresiak, A., Filas, V., Ibbs, M., Bliźniak, R., Łuczewski, Ł., & Lamperska, K. (2018). 2D and 3D Cell Cultures - A Comparison of Different Types of Cancer Cell Cultures. Archives of Medical Science, 14, 910-919.
- Fang, Y., & Eglen, R.M. (2017). Three-dimensional Cell Cultures in Drug Discovery and Development. SLAS Discovery: Advancing Life Sciences R&D, 22, 456-472.
- Dyson, P.J. (2019). The Rise of 3D Cellular Spheroids: Efficient Culture via Upward Growth from a Superamphiphobic Surface. National Science Review, 6, 1068-1069.
- Wang, X., Wang, Z., Zhai, W., Wang, F., Ge, Z., Yu, H., & Yang, W. (2021). Engineering Biological Tissues from the Bottom-up: Recent Advances and Future Prospects. Micromachines, 13, 75.
- Antoni, D., Burckel, H., Josset, E., & Noel, G. (2015). Three-dimensional Cell Culture: A Breakthrough in Vivo. International Journal of Molecular Sciences, 16, 5517-5527.
- Kronemberger, G.S., Matsui, R.A.M., de Castro, G.A.S., Granjeiro, J.M., & Baptista, L.S. (2020). Cartilage and Bone Tissue Engineering Using Adipose Stromal/Stem Cells Spheroids as Building Blocks. World Journal of Stem Cells, 12, 110.
- Ryu, N.E., Lee, S.H., & Park, H. (2019). Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells. Cells, 8, 1620.
- Roy, S.M., Garg, V., Barman, S., Ghosh, C., Maity, A.R., & Ghosh, S.K. (2021). Kinetics of Nanomedicine in Tumor Spheroid as an In Vitro Model System for Efficient Tumor-targeted Drug Delivery with Insights from Mathematical Models. Frontiers in Bioengineering and Biotechnology, 9, 785937.
- Morán, M.C., Cirisano, F., & Ferrari, M. (2023). Spheroid Formation and Recovery Using Superhydrophobic Coating for Regenerative Purposes. Pharmaceutics, 15, 2226.
- Gaitán-Salvatella, I., López-Villegas, E.O., González-Alva, P., Susate-Olmos, F., & Álvarez-Pérez, M.A. (2021). Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering. Frontiers in Molecular Biosciences, 8, 672518.
- Rasouli, M., Safari, F., Kanani, M.H., & Ahvati, H. (2024). Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture. Springer.
- Anil-Inevi, M., Yaman, S., Yildiz, A.A., Mese, G., Yalcin-Ozuysal, O., Tekin, H.C., & Ozcivici, E. (2018). Biofabrication of In Situ Self Assembled 3D Cell Cultures in a Weightlessness Environment Generated Using Magnetic Levitation. Scientific Reports, 8, 7239.
- Minamikawa-Tachino, R., Ogura, K., Ito, A., & Nagayama, K. (2020). Time-lapse Imaging of HeLa Spheroids in Soft Agar Culture Provides Virtual Inner Proliferative Activity. PLoS One, 15, e0231774.
- Stepanenko, A.A., & Dmitrenko, V.V. (2015). HEK293 in Cell Biology and Cancer Research: Phenotype, Karyotype, Tumorigenicity, and Stress-induced Genome-phenotype Evolution. Gene, 569, 182-190.
- Gest, C., Joimel, U., Huang, L., Pritchard, L.L., Petit, A., Dulong, C., Buquet, C., Hu, C.Q., Mirshahi, P., & Laurent, M. (2013). Rac3 Induces a Molecular Pathway Triggering Breast Cancer Cell Aggressiveness: Differences in MDA-MB-231 and MCF-7 Breast Cancer Cell Lines. BMC Cancer, 13, 1-14.
- Sukjoi, W., Young, C., Acland, M., Siritutsoontorn, S., Roytrakul, S., Klingler-Hoffmann, M., Hoffmann, P., & Jitrapakdee, S. (2024). Proteomic Analysis of Holocarboxylase Synthetase Deficient-MDA-MB-231 Breast Cancer Cells Revealed the Biochemical Changes Associated with Cell Death, Impaired Growth Signaling, and Metabolism. Frontiers in Molecular Biosciences, 10, 1250423.
- Zhong, Z., Pannu, V., Rosenow, M., Stark, A., & Spetzler, D. (2018). KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 Through Microtubule and Heat Shock Proteins. Cancers, 10, 180.
- Vinci, M., Box, C., & Eccles, S.A. (2015). Three-dimensional (3D) Tumor Spheroid Invasion Assay. Journal of Visualized Experiments: JoVE, 52686.
- Bairoch, A. (2018). The Cellosaurus, a Cell-line Knowledge Resource. Journal of Biomolecular Techniques: JBT, 29, 25.
- Ellis, K., & Wood, R. (2023). The Comparative Invasiveness of Endometriotic Cell Lines to Breast and Endometrial Cancer Cell Lines. Biomolecules, 13, 1003.
- Lucey, B.P., Nelson-Rees, W.A., & Hutchins, G.M. (2009). Henrietta Lacks, HeLa Cells, and Cell Culture Contamination. Archives of Pathology & Laboratory Medicine, 133, 1463-1467.
- De Both, N.J., Vermey, M., Dinjens, W.N., & Bosman, F.T. (1999). A Comparative Evaluation of Various Invasion Assays Testing Colon Carcinoma Cell Lines. British Journal of Cancer, 81, 934-941.
- Alberti, L., Losi, L., Leyvraz, S., & Benhattar, J. (2015). Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells. PLoS One, 10, e0132977.
Kuyu Plakalarında Dört Kanser Hücre Hattının Sferoid Oluşumu ve İnvazivlik Özelliklerinin Karşılaştırılması
Abstract
Üç boyutlu (3D) hücre kültürleri, özellikle sferoidler, in vivo tümör ortamlarını taklit etmek için büyük önem taşır ve kanser araştırmaları ile ilaç testleri için daha doğru bir model sunar. Sferoidler, belirli koşullar altında hücrelerin kendi kendine bir araya gelmesiyle oluşur ve bu sayede hücresel davranışlar, istilacılık da dahil olmak üzere incelenebilir. Bu çalışma, İnsan Embriyonik Böbrek 293 (HEK-293), İnsan Kolorektal Adenokarsinom (HT-29), İnsan Meme Kanseri (MDA-MB-231) ve İnsan Serviks Kanseri (HeLa) olmak üzere dört farklı hücre hattının sferoid oluşturma yeteneğini ticari bir yuvarlak tabanlı 96 kuyucuklu mikroplak ve AggreWell™ mikrokuyucuklar kullanarak değerlendirmektedir. Hücreler, yüksek viskoziteli ortamda kültürlenmiş ve kuyucuk başına farklı hücre yoğunluklarında (1.000, 2.000 ve 3.000 hücre) ekilerek hücre sayısının zaman içinde sferoid boyutu üzerindeki etkisi incelenmiştir. Mikroskopik analizler, hücre hatları arasında belirgin farklılıklar olduğunu ortaya koymuştur; HEK-293 ve HT-29 hücreleri, daha yüksek ekim yoğunluklarında daha büyük olmak üzere, kompakt ve iyi tanımlanmış sferoidler oluşturmuştur. Buna karşılık, daha agresif ve istilacı özelliklere sahip olan MDA-231 ve HeLa hücreleri bu koşullar altında sferoid oluşturamamıştır. Bu bulgular, kanser hücrelerinin agresifliği, ekim yoğunluğu ve sferoid oluşturma yeteneği arasındaki karmaşık ilişkiyi ortaya koymakta olup, 3D kültür temelli ilaç geliştirme ve kanser araştırmalarının optimizasyonu için kritik faktörlerdir.
Keywords
3D hücre kültürü Sferoid oluşumu Kanser hücre hatları Tümör invazivliği Hücre ekim yoğunluğu İlaç testi modelleri
Supporting Institution
Sao Paulo Eyaleti Araştırma Fonu (FAPESP)
Thanks
Bu çalışma Sao Paulo Eyaleti Araştırma Fonu tarafından University College London'da yapılan 2018/23895-7 numaralı proje için desteklenmiştir.
References
- Ferlay, J., Colombet, M., Soerjomataram, I., Parkin, D.M., Piñeros, M., Znaor, A., & Bray, F. (2021). Cancer Statistics for the Year 2020: An Overview. International Journal of Cancer, 149, 778-789.
- DiMasi, J.A., Reichert, J.M., Feldman, L., & Malins, A. (2013). Clinical Approval Success Rates for Investigational Cancer Drugs. Clinical Pharmacology & Therapeutics, 94, 329-335.
- Wei, F., Wang, S., & Gou, X. (2021). A Review for Cell-based Screening Methods in Drug Discovery. Biophysics Reports, 7, 504.
- Kapałczyńska, M., Kolenda, T., Przybyła, W., Zajączkowska, M., Teresiak, A., Filas, V., Ibbs, M., Bliźniak, R., Łuczewski, Ł., & Lamperska, K. (2018). 2D and 3D Cell Cultures - A Comparison of Different Types of Cancer Cell Cultures. Archives of Medical Science, 14, 910-919.
- Fang, Y., & Eglen, R.M. (2017). Three-dimensional Cell Cultures in Drug Discovery and Development. SLAS Discovery: Advancing Life Sciences R&D, 22, 456-472.
- Dyson, P.J. (2019). The Rise of 3D Cellular Spheroids: Efficient Culture via Upward Growth from a Superamphiphobic Surface. National Science Review, 6, 1068-1069.
- Wang, X., Wang, Z., Zhai, W., Wang, F., Ge, Z., Yu, H., & Yang, W. (2021). Engineering Biological Tissues from the Bottom-up: Recent Advances and Future Prospects. Micromachines, 13, 75.
- Antoni, D., Burckel, H., Josset, E., & Noel, G. (2015). Three-dimensional Cell Culture: A Breakthrough in Vivo. International Journal of Molecular Sciences, 16, 5517-5527.
- Kronemberger, G.S., Matsui, R.A.M., de Castro, G.A.S., Granjeiro, J.M., & Baptista, L.S. (2020). Cartilage and Bone Tissue Engineering Using Adipose Stromal/Stem Cells Spheroids as Building Blocks. World Journal of Stem Cells, 12, 110.
- Ryu, N.E., Lee, S.H., & Park, H. (2019). Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells. Cells, 8, 1620.
- Roy, S.M., Garg, V., Barman, S., Ghosh, C., Maity, A.R., & Ghosh, S.K. (2021). Kinetics of Nanomedicine in Tumor Spheroid as an In Vitro Model System for Efficient Tumor-targeted Drug Delivery with Insights from Mathematical Models. Frontiers in Bioengineering and Biotechnology, 9, 785937.
- Morán, M.C., Cirisano, F., & Ferrari, M. (2023). Spheroid Formation and Recovery Using Superhydrophobic Coating for Regenerative Purposes. Pharmaceutics, 15, 2226.
- Gaitán-Salvatella, I., López-Villegas, E.O., González-Alva, P., Susate-Olmos, F., & Álvarez-Pérez, M.A. (2021). Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering. Frontiers in Molecular Biosciences, 8, 672518.
- Rasouli, M., Safari, F., Kanani, M.H., & Ahvati, H. (2024). Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture. Springer.
- Anil-Inevi, M., Yaman, S., Yildiz, A.A., Mese, G., Yalcin-Ozuysal, O., Tekin, H.C., & Ozcivici, E. (2018). Biofabrication of In Situ Self Assembled 3D Cell Cultures in a Weightlessness Environment Generated Using Magnetic Levitation. Scientific Reports, 8, 7239.
- Minamikawa-Tachino, R., Ogura, K., Ito, A., & Nagayama, K. (2020). Time-lapse Imaging of HeLa Spheroids in Soft Agar Culture Provides Virtual Inner Proliferative Activity. PLoS One, 15, e0231774.
- Stepanenko, A.A., & Dmitrenko, V.V. (2015). HEK293 in Cell Biology and Cancer Research: Phenotype, Karyotype, Tumorigenicity, and Stress-induced Genome-phenotype Evolution. Gene, 569, 182-190.
- Gest, C., Joimel, U., Huang, L., Pritchard, L.L., Petit, A., Dulong, C., Buquet, C., Hu, C.Q., Mirshahi, P., & Laurent, M. (2013). Rac3 Induces a Molecular Pathway Triggering Breast Cancer Cell Aggressiveness: Differences in MDA-MB-231 and MCF-7 Breast Cancer Cell Lines. BMC Cancer, 13, 1-14.
- Sukjoi, W., Young, C., Acland, M., Siritutsoontorn, S., Roytrakul, S., Klingler-Hoffmann, M., Hoffmann, P., & Jitrapakdee, S. (2024). Proteomic Analysis of Holocarboxylase Synthetase Deficient-MDA-MB-231 Breast Cancer Cells Revealed the Biochemical Changes Associated with Cell Death, Impaired Growth Signaling, and Metabolism. Frontiers in Molecular Biosciences, 10, 1250423.
- Zhong, Z., Pannu, V., Rosenow, M., Stark, A., & Spetzler, D. (2018). KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 Through Microtubule and Heat Shock Proteins. Cancers, 10, 180.
- Vinci, M., Box, C., & Eccles, S.A. (2015). Three-dimensional (3D) Tumor Spheroid Invasion Assay. Journal of Visualized Experiments: JoVE, 52686.
- Bairoch, A. (2018). The Cellosaurus, a Cell-line Knowledge Resource. Journal of Biomolecular Techniques: JBT, 29, 25.
- Ellis, K., & Wood, R. (2023). The Comparative Invasiveness of Endometriotic Cell Lines to Breast and Endometrial Cancer Cell Lines. Biomolecules, 13, 1003.
- Lucey, B.P., Nelson-Rees, W.A., & Hutchins, G.M. (2009). Henrietta Lacks, HeLa Cells, and Cell Culture Contamination. Archives of Pathology & Laboratory Medicine, 133, 1463-1467.
- De Both, N.J., Vermey, M., Dinjens, W.N., & Bosman, F.T. (1999). A Comparative Evaluation of Various Invasion Assays Testing Colon Carcinoma Cell Lines. British Journal of Cancer, 81, 934-941.
- Alberti, L., Losi, L., Leyvraz, S., & Benhattar, J. (2015). Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells. PLoS One, 10, e0132977.
Details
| Primary Language | English |
|---|---|
| Subjects | Plant Tissue and Cell Culture, Cancer Biology, Biofabrication, Tissue Engineering |
| Journal Section | Research Articles |
| Authors | |
| Early Pub Date | June 16, 2025 |
| Publication Date | |
| Submission Date | February 2, 2025 |
| Acceptance Date | May 5, 2025 |
| Published in Issue | Year 2025 Volume: 37 Issue: 2 |
