Biology:Multicellular tumor spheroids
Multicellular tumor spheroids (MCTS) are three-dimensional (3D) aggregates of cells that serve as widely used in-vitro models of tumors․ Unlike conventional two-dimensional (2D) cell cultures‚ MCTS enable cell-cell and cell-microenvironment interactions in 3D‚ and provide gradients of oxygen‚ nutrients‚ metabolites‚ and signaling molecules‚ as well as microenvironments similar to in-vivo tumors.[1][2]
The structure of MCTS is analogous to that of solid tumors with an outer layer of proliferating growth‚ a middle layer of quiescent cells‚ and a necrotic core in larger spheroids.[3] Cells in the outer proliferating layer are better supplied with oxygen and nutrients than the quiescent cells and can more quickly expel waste to the environment surrounding the spheroids‚ and are therefore capable of dividing more rapidly․ The cells in the quiescent layer live in hypoxic and nutrient-limited conditions‚ and are in a non-proliferative or low-proliferative state‚ while the cells in the necrotic core are severely hypoxic and are undergoing cell death (necrosis)․ This is due to limited diffusion of oxygen and nutrients‚ with a necrotic core developing in spheroids with larger diameters.[4] These traits make MCTS a useful model for tumor biology and response to therapy․
Preparation
MCTS are created using culture methods that prevent cells from adhering to flat surfaces‚ allowing cells to aggregate to form three-dimensional structures that more closely resemble the architecture and microenvironment found in solid living tumors․ The aggregation of cells through the cellular interactions of cell adhesion molecules (CAMs) leads to the formation of spheroids that compact into multicellular aggregates suspended in culture.[2]
Various experimental methods‚ such as the hanging drop method‚ spinner culture‚ pellet culture‚ and shaker based suspension culture systems‚ have been used for the production of MCTS․ These methods allow for spheroid formation of controlled size and reproducibility for research purposes.[2][5]
Applications
MCTS are also commonly used to model the drug resistance of tumors: their internal structure means that drugs can be considerably less effective in spheroids than when used in monolayers‚ since the drugs must diffuse through multiple layers of cells.[6]
Cancer treatment failure may also be due to the presence of a quiescent cell layer: since many chemotherapeutic agents are geared towards killing rapidly dividing cells‚ the proliferating outer layer of a MCTS may be killed‚ leaving the quiescent inner region and necrotic core relatively unaffected․ As such‚ MCTS may provide a more physiologically relevant model of drug response than 2D culture systems.[6]
Characterization
Imaging‚ molecular‚ and mechanical studies have characterized MCTS‚ providing insights on their architecture‚ cellular physiology‚ and physical properties.[1][2][7]
Imaging techniques
Fluorescence imaging is one of the most used methods to visualize crystalline cellular architecture and MCTS biological activity․ Fluorescent dyes and proteins can visualize proliferation‚ apoptosis‚ and hypoxia in MCTS․ Confocal microscopy is often used in conjunction with fluorescence imaging as it provides optical sectioning and 3D reconstruction of spheroids to visualize the internal cellular architecture.[2][7]
Molecular techniques
Gene expression analysis can be performed in MCTS using quantitative polymerase chain reaction (qPCR) and RNA sequencing to study changes in gene expression when growing cancer cells in three-dimensional structures in comparison to two-dimensional cultures.[2][8] QPCR-based methods are useful to quantify the expression of individual genes involved in tumor growth‚ metabolism and hypoxia. In contrast, RNA sequencing is better utilized to analyze thousands of genes at once.
Mechanical characterization
The mechanical characterization of tumor spheroids is important for understanding how their material properties influence cancer progression, including invasion, metastasis, and treatment response. Tumor spheroids exhibit complex viscoelastic behavior arising from cell–cell adhesion, cytoskeletal contractility, and extracellular interactions. These mechanical properties play a key role in collective cell migration, tissue shape remodeling, and mechanotransduction.[9][10][11] To quantify the mechanical properties of spheroids, researchers commonly employ bulk mechanical assays, including micropipette aspiration and compression testing. Micropipette aspiration applies a controlled pressure to induce deformation as the spheroid is aspirated into a pipette, allowing measurement of surface tension, viscosity, and elastic modulus through time-dependent creep response.[12] Compression-based methods, including parallel-plate compression and microtweezer-based assays, measure force–indentation relationships to estimate stiffness and distinguish between solid-like and liquid-like mechanical behavior.[13][14][15]
References
- ↑ 1.0 1.1 Madhavan, Mathangi; Jaiswal, Devina; Karlberg, Sarah; Duggan, Alexis; Almarshad, Hassan A.; Claffey, Kevin P.; Hoshino, Kazunori (2023-05-25). "Electron microscopy imaging and mechanical characterization of T47D multicellular tumor spheroids–Older spheroids reduce interstitial space and become stiffer" (in en). PLOS ONE 18 (5). doi:10.1371/journal.pone.0286291. ISSN 1932-6203. PMID 37228139. Bibcode: 2023PLoSO..1886291M.
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 Nath, Sritama; Devi, Gayathri R. (July 1, 2016). "Three-dimensional culture systems in cancer research: Focus on tumor spheroid model" (in en). Pharmacology & Therapeutics 163: 94–108. doi:10.1016/j.pharmthera.2016.03.013. PMID 27063403.
- ↑ Mehta, Geeta; Hsiao, Amy Y.; Ingram, Marylou; Luker, Gary D.; Takayama, Shuichi (2012-12-10). "Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy". Journal of Controlled Release. Drug Delivery and Cancer: Today's Challenges, Tomorrow's Directions. 164 (2): 192–204. doi:10.1016/j.jconrel.2012.04.045. ISSN 0168-3659. PMID 22613880.
- ↑ Friedrich, Juergen; Seidel, Claudia; Ebner, Reinhard; Kunz-Schughart, Leoni A. (February 12, 2009). "Spheroid-based drug screen: considerations and practical approach" (in en). Nature Protocols 4 (3): 309–324. doi:10.1038/nprot.2008.226. ISSN 1750-2799. PMID 19214182. https://www.nature.com/articles/nprot.2008.226.
- ↑ Lazzari, Gianpiero; Couvreur, Patrick; Mura, Simona (2017-08-30). "Multicellular tumor spheroids: a relevant 3D model for the in vitro preclinical investigation of polymer nanomedicines" (in en). Polymer Chemistry 8 (34): 4947–4969. doi:10.1039/C7PY00559H. ISSN 1759-9962. https://pubs.rsc.org/en/content/articlelanding/2017/py/c7py00559h.
- ↑ 6.0 6.1 Achilli, Toni-Marie; Meyer, Julia; Morgan, Jeffrey R. (July 12, 2012). "Advances in the formation, use and understanding of multi-cellular spheroids". Expert Opinion on Biological Therapy 12 (10): 1347–1360. doi:10.1517/14712598.2012.707181. ISSN 1744-7682. PMID 22784238.
- ↑ 7.0 7.1 Edmondson, Rasheena; Broglie, Jessica Jenkins; Yang, Liju (May 1, 2014). "Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors" (in en). Assay and Drug Development Technologies 12 (4): 207–218. doi:10.1089/adt.2014.573. PMID 24831787.
- ↑ Mullis, K.; Faloona, F.; Scharf, S.; Saiki, R.; Horn, G.; Erlich, H. (1986-01-01). "Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction" (in en). Cold Spring Harbor Symposia on Quantitative Biology 51: 263–273. doi:10.1101/SQB.1986.051.01.032. ISSN 0091-7451. PMID 3472723. http://symposium.cshlp.org/content/51/263.
- ↑ Charras, Guillaume; Yap, Alpha S. (April 2018). "Tensile Forces and Mechanotransduction at Cell–Cell Junctions". Current Biology 28 (8): R445–R457. doi:10.1016/j.cub.2018.02.003. ISSN 0960-9822. PMID 29689229. Bibcode: 2018CBio...28.R445C. https://linkinghub.elsevier.com/retrieve/pii/S096098221830157X.
- ↑ Lecuit, Thomas; Lenne, Pierre-François; Munro, Edwin (November 2011). "Force Generation, Transmission, and Integration during Cell and Tissue Morphogenesis" (in en). Annual Review of Cell and Developmental Biology 27 (27): 157–184. doi:10.1146/annurev-cellbio-100109-104027. ISSN 1081-0706. PMID 21740231. https://www.annualreviews.org/content/journals/10.1146/annurev-cellbio-100109-104027.
- ↑ Pegoraro, Adrian F.; Janmey, Paul; Weitz, David A. (2017-11-01). "Mechanical Properties of the Cytoskeleton and Cells" (in en). Cold Spring Harbor Perspectives in Biology 9 (11). doi:10.1101/cshperspect.a022038. ISSN 1943-0264. PMID 29092896. PMC 5666633. http://cshperspectives.cshlp.org/content/9/11/a022038.
- ↑ Guevorkian, Karine; Colbert, Marie-Josée; Durth, Mélanie; Dufour, Sylvie; Brochard-Wyart, Françoise (2010-05-24). "Aspiration of Biological Viscoelastic Drops" (in en). Physical Review Letters 104 (21). doi:10.1103/PhysRevLett.104.218101. ISSN 0031-9007. PMID 20867138. Bibcode: 2010PhRvL.104u8101G. https://link.aps.org/doi/10.1103/PhysRevLett.104.218101.
- ↑ Foty, Ramsey A.; Pfleger, Cathie M.; Forgacs, Gabor; Steinberg, Malcolm S. (1996-05-01). "Surface tensions of embryonic tissues predict their mutual envelopment behavior" (in en). Development 122 (5): 1611–1620. doi:10.1242/dev.122.5.1611. ISSN 0950-1991. PMID 8625847. https://journals.biologists.com/dev/article/122/5/1611/39045/Surface-tensions-of-embryonic-tissues-predict.
- ↑ Jaiswal, Devina; Cowley, Norah; Bian, Zichao; Zheng, Guoan; Claffey, Kevin P.; Hoshino, Kazunori (2017-11-22). "Stiffness analysis of 3D spheroids using microtweezers" (in en). PLOS ONE 12 (11). doi:10.1371/journal.pone.0188346. ISSN 1932-6203. PMID 29166651. Bibcode: 2017PLoSO..1288346J.
- ↑ Tomizawa, Yuji; Wali, Khadija H.; Surti, Manav; Suhail, Yasir; Kshitiz; Hoshino, Kazunori (March 2025). "Single-Cell Quantification of Viscoelastic Phase Transitions in 3D Tissues" (in en). Advanced Materials Technologies 10 (6). doi:10.1002/admt.202401302. ISSN 2365-709X. https://advanced.onlinelibrary.wiley.com/doi/10.1002/admt.202401302.
Further reading
- Lazzari, Gianpiero; Couvreur, Patrick; Mura, Simona (2017). "Multicellular tumor spheroids: a relevant 3D model for the in vitro preclinical investigation of polymer nanomedicines". Polymer Chemistry 8 (34): 4947–4969. doi:10.1039/C7PY00559H.
- Carver, Kyle; Ming, Xin; Juliano, Rudolph L. (1 January 2014). "Multicellular Tumor Spheroids as a Model for Assessing Delivery of Oligonucleotides in Three Dimensions". Molecular Therapy: Nucleic Acids 3 (3): e153. doi:10.1038/mtna.2014.5. PMID 24618852.
- Hirschhaeuser, Franziska; Menne, Heike; Dittfeld, Claudia; West, Jonathan; Mueller-Klieser, Wolfgang; Kunz-Schughart, Leoni A. (1 July 2010). "Multicellular tumor spheroids: An underestimated tool is catching up again". Journal of Biotechnology 148 (1): 3–15. doi:10.1016/j.jbiotec.2010.01.012. PMID 20097238.
- Yuhas, John M.; Li, Albert P.; Martinez, Andrew O.; Ladman, Aaron J. (1 October 1977). "A Simplified Method for Production and Growth of Multicellular Tumor Spheroids". Cancer Research 37 (10): 3639–3643. PMID 908012. https://cancerres.aacrjournals.org/content/37/10/3639.short.
- Kelm, Jens M.; Timmins, Nicholas E.; Brown, Catherine J.; Fussenegger, Martin; Nielsen, Lars K. (20 July 2003). "Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types". Biotechnology and Bioengineering 83 (2): 173–180. doi:10.1002/bit.10655. PMID 12768623. Bibcode: 2003BiotB..83..173K.
 |  |
