Spheroid tumor

In cancer research, the multicellular tumor spheroid model is the most widely used. Spheroids are three-dimensional, often heterogeneous organoids containing gradients of cell proliferation, oxygen, and nutrients. Spheroids may be developed in suspension or in 3-D matrices. The former method has been adapted in various ways to include the use of spinner flasks (86), rotating cell... 

We and others have based invasion assays on preformed tumor spheroids generated either in hanging drops (88) or other nonadherent systems (61). These are then transferred to matrix monolayers or embedded in Matrigel and can be analyzed qualitatively or quantitatively, usually by microscopy and image analysis (see also Chapter 16). A96- or 1,536-well spheroid-based... 

Confrontation cultures between tumor spheroids and solid tissues 3-D from 3-D Insight into biological processes and involved molecules, mimic of in vivo microenvironment via invasion into embryoid bodies or explanted tissues Histological analysis and quantification necessary, low throughput (133, 134)... 

Green SK, Francia G, Isidore C et al (2004) Antiadhesive antibodies targeting E-cadherin sensitize multicellular tumor spheroids to chemotherapy in vitro. Mol Cancer Ther 3 149-159... 

Multicellular tumor spheroids an underestimated tool is catching up again. J Biotechnol 148 3-15... 

Wartenberg M, Donmez F, Ling FC et al (2001) Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells. FASEB J 15 995-1005... 

Ivascu A, Kubbies M (2006) Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen 11 922-932... 

Stein AM, Demuth T, Mobley D et al (2007) A mathematical model of glioblastoma tumor spheroid invasion in a three-dimensional in vitro experiment. Biophys J 92 356-365... 

Wartenberg M, Finkensieper A, Hescheler J et al (2006) Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis. Methods Mol Biol 331 313-328... 

Key words Migration, 3D, Tumor spheroids, Extracellular matrix, Drug response, Adhesion,... 

Fig. 1. Schematic overview of the tumor spheroid-based migration assay. Tumor spheroids (TS) are transferred from their cuiture vessei or piate into a 96-weii fiat-bottomed migration piate pre-coated with an extraceiiuiar matrix (ECM) protein of choice (in this case, geiatin). Digital images of the spheroids are then captured at t=0 and once every 24 h for a period of up to 72 h, exemplified here by CAL spheroids. Image analysis software is used to calculate the spheroid size and extent of migration. Scale bar=100 p.m.

Fig. 2. Representative images of CAL and CAL tumor spheroids after 48 h of migration in piates coated with different ECM proteins. CAL ceiis are iess migratory (images acquired using a x10 objective, scaie bar=100 p.m) than CAL (images acquired using a x4 objective, scaie bar=500 p.m) on aii substrates. The migrated ceii areas have been outiined manuaiiy for ciarity.

Human umbilical vein endothelial cells (HUVEC), used to exemplify tumor spheroid-based migration on a cell monolayer. 

Tumor Spheroid-Based Migration Assay 1. Appropriate culture medium for the specific tumor cell line(s) under test. 2. 96-well flat-bottomed tissue culture-treated plates. 

Initiate tumor spheroids using a method that results in the formation of compact, reproducibly sized spheroids in suspension, for example, ultralow attachment (11), agar-coated (13), poly-Hema-coated 96-well plates (12), or in hanging drops (14, 15) (see Note 2). 

For the tumor spheroid-based migration assay, prepare the EC... 

Tumor spheroids are an in vitro model that mimic micrometas-tases or tumor intervascular microregions thus they can recapitulate some features of in vivo tumor tissue (e.g., gradients of oxygen and proliferation (11, 18, 35)). Considering that in vivo the maximum distance that oxygen can diffuse from capillaries is 100 pm, the spheroid diameter must be above 300 pm in order to reliably include these features. 

Although all the methods mentioned above can generate reproducibly sized tumor spheroids, the one that we recommend is the method that uses round-bottom ultralow attachment 96-well plates (11). This method is the least time consuming (e.g., plates do not require any coating), reproducible (one centrally located spheroid/well is formed), and suitable for automated imaging. Thus it satisfies important requirements of cell-based HT assays. 

For the tumor spheroid-based migration assay on EC mono-layer, first aspirate the medium from the EC monolayer 96-well plate and then dispense 200 pL/well of fresh, warm EC culture medium. Then follow the procedure described in Subheading 3.4. 

Vinci M, Gowan S, BoxaU E et al (2012) Advances in establishment and analysis of 3D tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10 29. doi 10.1186/1741-7007-10-29... 

Kebn JM, Timmins NE, Brown CJ et al (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83 173-180... 

Offiier FA, Wirtz HC, Schiefer J et al (1992) Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayers. Damage to endothelium by oxygen-derived firee radicals. Am J Pathol 141 601-610... 

Francia G, Green SK, Bocci G, et al. Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids implications for multicellular resistance to alkylating agents. Mol Cancer Ther. 2005 4 1484-1494. 

Freyer, J.P., Schor, P.L., Jarrett, K.A., Neeman, M., Sillerud, L.O. (1991). Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids. Cancer Res. 51,3831-3837. 

Lieubeau-TeiUet, B., Rak, J., Jothy, S., Iliopoulos, O., Kaelin, W. and Kerbel, R. S. (1998). von Hippel-Lindau gene-mediated growth suppression and induction of differentiation in renal cell carcinoma cells grown as multicellular tumor spheroids. Cancer Res. 58,4957-4962. 

Rotmensch J, Whitlock JL, Culbertson S, Atcher RW Schwartz JL (1994) Comparison of sensitivities of cells to X-ray therapy, chemotherapy, and isotope therapy using a tumor spheroid model. Gynecologic Oncology 55 290-293. 

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