Fig 1.
A) Number of formed spheroids. Here, the number of single spheroids of roundish shape obtained per well for three tested methods used for the four glioblastoma cell lines and three different cell numbers is depicted. For BioFloat and magnetic bead technique all yields were equal to one and thus pooled. The 2D projected size is presented.(B, C). Graphs show the size of the formed spheroids as a function of time for the BioFloat (B) and magnetic bead (C) method. The circularity of the formed spheroids is shown as a function of time for the BioFloat (D) and magnetic bead (E) method. The relative height of spheroids for different techniques and all cell lines is presented (F). Error bars in B)-E) depict the standard error of the mean. Box plots in F) show the median (red line), the 25 and 75 percentile (blue box) and the maximal range (whiskers). Stars depict statistically significant results with p < 0.05. B) to E) For each cell line and cell number 16 spheroids were analyzed. F) nGBM10 Beads = 8, nGBM10 Biofloat = 8, nGBM4 Beads = 12, nGBM4 Biofloat = 9, nLN229 Beads = 11, nLN229 Biofloat = 8, nU138 Beads = 12, nU138 Biofloat = 8.
Fig 2.
Seahorse analyses showing a comparison between metabolic values in the different glioblastoma cell lines LN229, U138 and the primary cells GBM#4, GBM#10. Representative mitochondrial respiration measurement using the Seahorse XF Cell Mito Stress Test for U138, LN229, GBM#4 and GBM#10 spheroids generated from 30,000 cells. Oligomycin, FCCP, rotenone plus antimycin A are used as pharmacological tools to study mitochondrial respiration. Oligomycin inhibits ATP synthase, reducing oxygen consumption. Rotenone and antimycin A block the electron transport chain, preventing mitochondrial respiration and allowing measurement of non-mitochondrial oxygen consumption. Oligomycin reduces the oxygen consumption rate (OCR) through inhibition of ATP synthase. FCCP uncouples the proton gradient, maximizing oxygen consumption. Rotenone and antimycin A are electron transport chain inhibitors.
Fig 3.
(A) Graphic of the raw basal respiration data obtained from the Seahorse metabolic analysis. The data were normalized using different approaches and are shown in (B) for median normalization, (C) for median plus area normalization, and (D) for median plus cell number normalization. Error bars represent the standard error of the mean (SEM). Asterisks indicate statistically significant differences at p < 0.05. The number of samples used in each group was as follows: nGBM10 15k = 13, nGBM10 20k = 16, nGBM10 25k = 13, nGBM4 15k = 12, nGBM4 20k = 15, nGBM4 25k = 17, nLN229 15k = 13, nLN229 20k = 14, nLN229 25k = 20, nU138 15k = 16, nU138 20k = 15, nU138 25k = 17.
Fig 4.
Seahorse analyses showing the differences in the maximal respiration values of glioblastoma cell lines LN229, U138 and the primary cells GBM#4, GBM#10 after normalization of spheroids yielded from different cell seeding densities. (A) Graphic of the raw maximal respiration data obtained from the Seahorse metabolic analysis. The data were normalized using different approaches and are shown in (B) for median normalization, (C) for median plus area normalization, and (D) for median plus cell number normalization. Error bars represent the standard error of the mean (SEM). Asterisks indicate statistically significant differences at p < 0.05. The number of samples used in each group was as follows: nGBM10 15k = 13, nGBM10 20k = 16, nGBM10 25k = 13, nGBM4 15k = 12, nGBM4 20k = 15, nGBM4 25k = 17, nLN229 15k = 13, nLN229 20k = 14, nLN229 25k = 20, nU138 15k = 16, nU138 20k = 15, nU138 25k = 17.
Fig 5.
(A) t-SNE clustering of spheroids from the four cell lines, showing the distinct response of LN229 compared to the other GBM cell types. Each point represents a single spheroid. Each point corresponds to the data of a single spheroid. (B) Illustration of the energetic state of the four GBM spheroid types at baseline and after FCCP injection to achieve maximal respiration. Error bars represent the standard error of the mean (SEM). The following number of spheroids was used for each group: nGBM10k = 42, nGBM4 = 44, nLN229 = 47, nU138 = 48.
Fig 6.
Results of different normalization approaches and comparison to 2D.
(A) and (B) are graphs of the Pearson correlation coefficient of the raw or normalized OCR of basal (A) or maximal respiration (B) values with the cell number for 2D and 3D spheroids measurements. C) and D) are graphs of the relative variance of the raw or normalized OCR of basal (A) or maximal respiration (B) values for the 2D and 3D spheroids measurements.