High-Throughput Drug Screening of Multicellular Tumor Spheroids

Three-dimensional (3-D) culture models are increasingly being used for drug discovery in oncology, as they better represent the complexity of the in vivo microenvironment of solid tumors.1,2 By recapitulating cell-cell interactions, metabolic gradients and cell polarity, 3-D multicellular spheroids provide more physiologically relevant models than two-dimensional (2-D) cell culture. Despite these advantages, limitations of current technologies to enable simple and reliable assays have slowed the adoption of 3-D culture models for high-throughput screening (HTS).3,4

Automation-friendly spheroid microplates (Corning Incorporated, Corning, N.Y.), featuring the Corning Ultra-Low Attachment surface and clear, innovative round-well-bottom geometry, were used for generating, culturing and assaying 3-D multicellular spheroids. Further, through the use of the CellTiter-Glo 3D Cell Viability Assay (Promega, Madison, Wis.), the multicellular spheroids were assayed directly in the spheroid microplates, eliminating the need for a transfer step. The CellTiter-Glo 3D Cell Viability Assay, optimized for 3-D microtissue analysis, provides a ready-to-use, one-step reagent that measures ATP to quantify viable cells, making it well-suited for cell proliferation and cytotoxicity assays when screening 3-D multicellular spheroids.

Methods

Screening of multicellular tumor spheroids

BT-474 cells (ATCC HTB-20) cultured in growth medium (RPMI 1640, containing 10% fetal bovine serum [FBS] Corning) were plated in 40 μL per well volume using a Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific, Waltham, Mass.) in either 384-well flat, clear-bottom tissue-culture-treated microplates or 384-well spheroid microplates (Corning) at 5000 or 1000 cells/well to generate 2-D cell culture monolayers or 3-D multicellular tumor spheroids, respectively. Cells were cultured for 48 hours, and were then treated with chemotherapeutics or compounds from the Library of Pharmacologically Active Compounds (LOPAC1280, MilliporeSigma, St. Louis, Mo.). A CyBi-Well pipettor (Analytik Jena AG, Überlingen, Germany) was used for compound addition and mixing directly in the assay microplates. The final concentration of LOPAC1280 compound was 4 μM in medium with 0.1% DMSO. Dose-series of select compounds Iressa (Tocris Bioscience, Bristol, U.K.), rottlerin, methotrexate (MTX), idarubicin, ouabain and diphenyleneiodonium (DP) (all from Sigma-Aldrich) were applied at varying concentrations. Positive controls Iressa and daunorubicin, an antibiotic anticancer drug, were also included. Forty-eight hours post-treatment, cells were assessed for viability using CellTiter-Glo 3D Cell Viability Assay. Luminescent signal was read using an Infinite M100 PRO plate reader (Tecan Group Ltd., Männedorf, Switzerland) and normalized to vehicle control (growth medium containing 0.1% DMSO) from each culture tested. Hits were defined as compounds that elicited a change in the luminescent signal by at least three times the standard deviation of the vehicle control responses, averaged together for all four 384-well LOPAC1280 plates.

Results

2-D versus 3-D cell culture

BT-474 cells were cultured in either 384-well flat, clear-bottom tissue-culture-treated microplates or 384-well spheroid microplates. After 48 hours, the cells cultured in the spheroid microplate formed reproducible single spheroids in each well, while the cells cultured in the standard microplate formed a 2-D cell culture monolayer (Figure 1).

 Figure 1 – a) Forty-eight-hour 2-D and b) 3-D cultures of BT-474 cells in 384-well format microplates. Scale bar = 1000 μm.

HTS screening

To determine the differences between screening in 2-D versus 3-D, 48-hour cultures of BT-474 cells were exposed to compounds in LOPAC1280 for an additional 48 hours. Viability was assessed using CellTiter-Glo 3D Cell Viability Assay. Control compound Iressa displayed a comparable response between 2-D and 3-D cultures; however, 2-D cultures were more sensitive to control daunorubicin, as displayed by the larger reduction in luminescent signal observed in 2-D culture assay microplates (Figure 2).

 Figure 2 – LOPAC1280 screen results. Hits were defined as a change in RLU signal by at least 3σ of vehicle control response (medium), averaged together for all four 384-well LOPAC1280 microplates. Solid line indicates average normalized vehicle control response; dashed lines indicate 3σ threshold. LOPAC1280 compounds (LOPAC), control compounds Iressa (red) and daunorubicin (green) are shown.

Across the four LOPAC1280 microplates assessed, several toxic hits (compounds that displayed a significant decrease in relative light unit [RLU] signal) were observed. Further, unique to 3-D cell culture, proliferative hits (compounds that displayed a significant increase in RLU signal) were also identified (Table 1).

Table 1 – Hits identified from the LOPAC1280 screen

Dose-dependent toxicity

Select compounds were assessed in a dosedependent manner to evaluate toxicity potency differences between 2-D and 3-D cell culture of BT-474 cells (Figure 3). Dose-dependent responses for each compound were evaluated and compared (Table 2). Iressa, rottlerin and methotrexate (MTX) displayed comparable sensitivity between 2-D and 3-D cultures; however, idarubicin displayed a 20-fold left-shift in potency in 3-D compared to 2-D. Ouabain and diphenyleneiodonium (DP) showed a larger or more potent cytotoxic effect in 2-D.

Figure 3 – Dose response testing. 2-D and 3-D cultures of BT-474 cells were evaluated using chemotherapeutics and select hits from the LOPAC1280 screen, applied in a dose-dependent manner for 48 hours.
Table 2 – EC50 values (μM) in BT-474 cells

Conclusion

New technologies are emerging to assist researchers with the need to screen 3-D cell cultures in HTS formats. Spheroid microplate technology together with the CellTiter-Glo 3D Cell Viability Assay enable the generation, culture and assessment of 3-D multicellular tumor spheroids, all in the same microplate. The uniform and reproducible single spheroids that are formed in each well allow for robust assay performance in HTS.

References

  1. Kunz-Schughart, L.A.; Freyer, J.P. et al. The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J. Biomol. Screen. 2004, 9(4), 273–85.
  2. Pampaloni, F.; Raynaud, E.G. et al. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 2007, 8(10), 839–45.
  3. Vinci, M.; Gowan, S. et al. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biology 2012, 10, 29.
  4. Mehta, G.; Hsiao, A.Y. et al., Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J. Control Release 2012, 164(2), 192–204.

Ana Maria P. Pardo, Hannah J. Gitschier and David H. Randle are with Corning Incorporated, Life Sciences, 2 Alfred Rd., Kennebunk, Maine 04043, U.S.A.; tel.: 207-985-3111; e-mail: [email protected]; www.corning.com/lifesciences. Terry Riss is with Promega Corporation, Madison, Wis., U.S.A.

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