Tumor Spheroid Formation Assay

Cancer research is one of the major research fields in medicine today. Since the introduction of traditional therapeutics, considerable efforts have been made to optimize drug efficacy, minimize side-effects, and develop new cancer therapy methods [1]. In the past, the in vitro screening of anticancer drugs was mainly based on cytotoxicity assays using cancer cell lines grown as two-dimensional (2D) cultures. 2D assays have several strengths and contributed significantly to increase the knowledge of tumor biology but they also show important limitations [2, 3].

Conventional 2D cell cultures are not capable of mimicking the complexity and heterogeneity of naturally occurring human tumors with specific organization and architecture. That’s why numerous signals that govern different cellular processes are lost in 2D cell assays [4]. Xu and coworkers showed that only 50% of the acute hepatotoxic compounds under study were detected when screened on 2D monolayers cultures of hepatocytes [5]. Theses assays can lead to misleading and nonpredictive results that may partially account for the high attrition rate for cancer drugs entering early clinical trials in both in vivo and in vitro preclinical testing [1, 6]. 3D growth of immortalized established cell lines or primary cell cultures is seen as a more representative method to do in vitro drug screening as their extracellular matrix and network of cell-to-cell and cell-to-matrix interactions are similar to in vivo conditions and differ from the corresponding monolayer cultures [7].

Chemo- and radio-cytotoxicity are the most important areas where large spheroids are used. as the clinical response to chemical or physical treatments can also depend on parameters such as oxygen tension, compactness, apoptosis inhibition, damage repair and permeability [8, 9,10].

One of the 3D cell assay methods is the Liquid Overlay Technique (LOT). It makes use of a non-adhesive surface (e.g., super hydrophobicity) that prevents cellular adhesion. Therefore, the cells start to adhere to each other, forming aggregates and finally spheroids [11]. The LOT is one of the most explored methodologies due to its low costs, easy handling and its potential for being applied in high-throughput analysis [12].

In previous studies, the I.DOT successfully dispensed the lung carcinoma epithelial cell line A549 in a round bottom 384-well ULA plate that lead to tumor spheroid formation. This approach was able to show several advantages. One major aspect is the time saving. To dispense 20 µl of medium with cells in a full 384-well plate took around 3 minutes 25 seconds, whereas it took approximately 32 minutes with manual pipetting. Afterwards the dispensed cells showed a high cell viability, which is indicating that the automated dispensing has no effect on the cell viability. The subsequently formed spheroids even showed a more uniform size and shape compared to those with manual pipetting. All in all, usage of the I.DOT in creating tumor spheroids by LOT does not only save time, but also increases the quality of performing tumor spheroid assays.

Learn more about the I.DOT!


[1] Zanoni et al 2016 – §D tumor spheroid models for in vitro therapeutic screening

[2] Sams-Dodd, F. Target-based drug discovery: is something wrong? Drug Discov. Today 10, 139–147 (2005)

[3] Edwards, A. M. et al. Preclinical target validation using patient-derived cells. Nat. Rev. Drug Discov. 14, 149–150 (2015). [4] Costa, E. C., Gaspar, V. M., Marques, J. G., Coutinho, P., Correia, I. J., Evaluation of nanoparticle uptake in co-culture cancer models. PLoS One 2013, 8, e70072.

[4] Lee, G. Y., Kenny, P. A., Lee, E. H. & Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–365 (2007).

[5] Xu, J. J., Henstock, P. V., Dunn, M. C., Smith, A. R., Chabot, J. R., de Graaf, D., Cellular Imaging Predictions of Clinical Drug-Induced Liver Injury. Toxicol. Sci. 2008, 105, 97-105.

[6] Edmondson, R., Broglie, J. J., Adcock, A. F., Yang, L., Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. Assay Drug Dev. Technol.2014, 12, 207-218.

[7] Khaitan et al., 2006b

[8] Frankel, A., Buckman, R. & Kerbel, R. S. Abrogation of taxol-induced G2-M arrest and apoptosis in human ovarian cancer cells grown as multicellular tumor spheroids. Cancer Res. 57, 2388–2393 (1997).

[9] Dubessy, C., Merlin, J. M., Marchal, C. & Guillemin, F. Spheroids in radiobiology and photodynamic therapy. Crit. Rev. Oncol. Hematol. 36, 179–192 (2000).

[10] Kim, T. H., Mount, C. W., Gombotz, W. R. & Pun, S. H. The delivery of doxorubicin to 3-D multicellular spheroids and tumors in a murine xenograft model using tumor-penetrating triblock polymeric micelles. Biomaterials 31, 7386–7397 (2010).

[11] Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen24. LK. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.  Biotechnol Bioeng. 2003;83:173–80.

[12] Costa et al: Spheroids formation on non-adhesive surfaces by Liquid Overlay Technique: considerations and practical approaches


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