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. 2022 Feb 20;12(4):e4321.
doi: 10.21769/BioProtoc.4321.

Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids

Zaineb Javed  1   2 , Beth L Worley  2 , Coryn Stump  2 , Sara S Shimko  2 , LaTaijah C Crawford  2 , Karthikeyan Mythreye  3 , Nadine Hempel  1   2
Affiliations

Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids

Zaineb Javed et al. Bio Protoc. .

Abstract

Three-dimensional (3D) cell culture models are widely used in tumor studies to more accurately reflect cell-cell interactions and tumor growth conditions in vivo. 3D anchorage-independent spheroids derived by culturing cells in ultra-low attachment (ULA) conditions is particularly relevant to ovarian cancer, as such cell clusters are often observed in malignant ascites of late-stage ovarian cancer patients. We and others have found that cells derived from anchorage-independent spheroids vary widely in gene expression profiles, proliferative state, and metabolism compared to cells maintained under attached culture conditions. This includes changes in mitochondrial function, which is most commonly assessed in cultured live cells by measuring oxygen consumption in extracellular flux assays. To measure mitochondrial function in anchorage-independent multicellular aggregates, we have adapted the Agilent Seahorse extracellular flux assay to optimize measurements of oxygen consumption and extracellular acidification of ovarian cancer cell spheroids generated by culture in ULA plates. This protocol includes: (i) Methods for culturing tumor cells as uniform anchorage-independent spheroids; (ii) Optimization for the transfer of spheroids to the Agilent Seahorse cell culture plates; (iii) Adaptations of the mitochondrial and glycolysis stress tests for spheroid extracellular flux analysis; and (iv) Suggestions for optimization of cell numbers, spheroid size, and normalization of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values. Using this method, we have found that ovarian cancer cells cultured as anchorage-independent spheroids display altered mitochondrial function compared to monolayer cultures attached to plastic dishes. This method allows for the assessment of mitochondrial function in a more relevant patho/physiological culture condition and can be adapted to evaluate mitochondrial function of various cell types that are able to aggregate into multicellular clusters in anchorage-independence. Graphic abstract: Workflow of the Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Keywords: Anchorage independence; Cancer metabolism; Extracellular flux assay; Live-cell metabolic assay; Ovarian cancer; Respiration; Seahorse XFp; Tumor spheroids.

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Conflict of interest statement

Competing interestsThe Agilent Seahorse XFp extracellular flux analyzer (S7802A) was awarded to Nadine Hempel through an equipment grant from Agilent.

Figures

Figure 1.
Figure 1.. Image of ES-2 cell spheroids pre and post extracellular flux assay run.
Spheroids remain intact after the completion of the assay. Scale bar = 1mm.
Figure 2.
Figure 2.. Optimization of spheroid number and ULA culture time.
A. Images of OVCAR3 spheroids after transfer into the XFp minicell culture plate wells. Scale bar=1 mm. B. Basal OCR measurements were obtained from the indicated number of spheroids per well, using the Seahorse XFp extracellular flux analyzer. Prior to the assay, spheroids were cultured in ULA plates for either 24 or 72 h (n=2, with mean OCR shown).
Figure 3.
Figure 3.. Loading the cartridge ports with test compounds.
A. Mitochondrial stress test. B. Glycolysis stress test.
Figure 4.
Figure 4.. Bioenergetics profiles of different cell lines and dependency on time in ULA culture conditions.
A. OCR measurements are shown over the course of the mitochondrial stress test assay (n=6). In the mitochondrial stress test, 1.0 µM Oligomycin and 1.0 µM FCCP were used. B. Basal OCR and ECAR values indicate that bioenergetic changes differ between cell lines, as cells are maintained for different times in anchorage-independent culture conditions. C. ECAR/OCR ratios provide information on the relative changes in oxidative phosphorylation in anchorage independence.
Figure 5.
Figure 5.. Changes in bioenergetics between attached and anchorage-independent conditions.
A. OCR and ECAR were measured during the mitochondrial stress test Assay (1.0 µM Oligomycin and 1.0 µM FCCP; n=9 OVCAR3; n=6 ES-2). B. Plots of basal OCR and ECAR measurements demonstrate the bioenergetic phenotypes of OVCAR3 and ES-2 cells cultured in attached and ULA conditions. C. Example of mitochondrial stress test analysis as described in “Data analysis” above (multiple unpaired t-tests).
See this image and copyright information in PMC

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