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Changes in cholesterol metabolism drive DAMP-dependent proliferation of EwS cell spheroids

To address possible differences in the metabolic rate of 3D- and 2D-cultured cells, we assessed their spare respiratory capacity (SRC), defined as the difference between basal and maximal respiratory capacity, which reflects the amount of extra ATP that can be generated by oxidative phosphorylation in the event of a sudden increase in energy demand. The SRC was calculated by measuring the oxygen consumption rate of the cells, using a Seahorse instrument, after sequential exposure to oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone/antimycin A. By inhibiting ATP synthase, oligomycin rapidly hyperpolarizes the mitochondrial membrane, thereby preventing protons from passing through the respiratory chain complexes. FCCP, which uncouples oxidative phosphorylation, reverses the hyperpolarized state caused by oligomycin by carrying protons across the mitochondrial inner membrane. Finally, rotenone/antimycin inhibit mitochondrial complexes I and III, completely halting mitochondrial respiration. Whereas resting state proliferation of 2D and 3D cells varied according to the cells examined (C1 cells proliferated more rapidly in 2D than in 3D culture but P2 cell spheroids proliferated faster than their monolayer counterparts), the SRC was three to fourfold greater in spheroids than in 2D cultures, irrespective of cell identity (Fig 3A). EwS spheroids may therefore dispose of more adequate means than monolayer cultures to adapt to stress.

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Figure 3. EwS spheroids have greater spare respiratory capacity (SRC) than 2D-cultured cells and changes in cholesterol metabolism drive damage-associated molecular pattern (DAMP)–dependent proliferation of EwS spheroids.

(A) SRC of P2 spheroids (3D) and adherent (2D) cells along with DAPI staining (4× magnification). Scale bar 1,000 μm. Right panel: bar graph showing quantification of the SRC (%), as calculated from the oxygen consumption rate of the indicated spheroids (SP) and adherent (Adht) cells in a Seahorse instrument (n ± 15 from representative values of two independent experiments). Oxygen consumption rate values were normalized for fluorescently identified cell counts (DAPI). Error bars represent + -SEM ****P < 0.0001 as assessed by ANOVA. Experimental setup (B, C, D, E, F, G, H, I). Cells were incubated with DAMPs and analyzed for cholesterol load and uptake. (C) Representative filipin staining (green) in basal conditions (resting state) of the indicated spheroids (SP) and adherent (Adht) cells. The bar represents 0.5 mm. (D) Total cholesterol content (free and esterified) was measured using a Cholesterol/Cholesterol Ester-Glo assay kit. The measurements are relative to those in adherent cells. Mean values ± SEM of two independent experiments in replicate are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired t test. (E) Representative image showing filipin MFI values and peak shifts in unstained, control, and DAMP-treated spheroids and adherent cells. (F) Flow cytometry quantification of filipin III MFI values after DAMP treatment for 2 d of 3D and 2D-cultured indicated cells relative to solvent-treated controls. Data represent mean values ± SEM of two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired t test. (C, D, G) Representative filipin staining (green) after 48 h of treatment with DAMPs (D) or solvent (C) of the indicated spheroids and adherent cells. DRAQ5 was used to label nuclear DNA (purple); the bar represents 0.5 mm. One representative image of at least two independent experiments is shown. (H) Total cholesterol load (free and esterified) was measured using a Cholesterol/Cholesterol Ester-Glo assay kit. Mean values ± SEM of two independent experiments in replicate are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired t test. (I) Total cholesterol quantification in P1 spheres treated for 48 h with DAMPs or solvent with or without simvastatin (5 μM). Mean values ± SEM of replicates in one experiment are shown. (J) Spheroids and adherent cells were incubated with fluorescently labeled cholesterol, treated with DAMPs or solvent for 30 h and labeled intracellular cholesterol load was determined via a plate reader. Mean values ± SEM of three independent experiments in replicate are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired t test. (K) Spheroids and adherent cells were incubated with cholesterol or mevalonic acid with or without simvastatin. Quantification of spheroid growth after 3 d of incubation in culture medium containing cholesterol (1 mM) or mevalonic acid (1 mM) with or without simvastatin (5 μM). Proliferation was assessed by MTT. Measurements are normalized to the solvent-treated control for each indicated cell line. Each experiment was performed at least three times. Mean values ± SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired t test. See also Fig S3.

To determine whether the increased expression of genes associated with inflammation and cholesterol metabolism might play a role in the observed DAMP-induced proliferation of 3D cultured EwS cells, spheroids of both primary EwS cultures and A673 cells were stimulated with several of the up-regulated mediators of inflammation, including CXCL10, CCL5, IL1-β, and CCL2. However, only minor changes in proliferation were observed, with variable responses among the cells tested (Fig S3A) suggesting that none of the tested mediators alone could explain the observed DAMP-induced proliferation. Based on the observation that DAMPs did not stimulate proliferation of adherent cells despite inducing the same and additional mediators of inflammation compared to spheroids, we did not pursue the investigation of the role of inflammatory mediators in DAMP-mediated promotion of 3D EwS cell growth.

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Figure S3. Effect of înflammatroy mediators, cholesterol precursors and cholesterol on tumor cell growth.

(A) Growth of spheroids, as measured by MTT 4 d after treatment with the indicated recombinant soluble mediators at the indicated concentrations. (B) Experimental setup for (C, D) Spheroids and adherent cells were incubated with cholesterol or mevalonic acid with and without MβCD. (C) Microscopy images showing P1 and P2 sphere formation after incubation with cholesterol (1 mM), mevalonic acid (1 mM), ± MβCD (5 mM) for 3 d. Scale bar = 50 μm. (D) Growth of spheroid-derived cells after treatment with cholesterol (1 mM) and mevalonic acid (1 mM) ± MβCD (5 mM). Proliferation was measured by MTT after 4 d of incubation. Data are normalized to solvent-treated controls for each indicated cell line. Each experiment was performed at least three times. Data represent mean values ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed unpaired t test.

Instead, we interrogated the effect of DAMPs on cholesterol uptake and synthesis based on the observations that numerous genes implicated in these two functions were up-regulated in EwS cells grown in 3D but not in 2D culture (Fig 3B). We first addressed possible changes in cellular cholesterol uptake and content after 3–5 d of stimulation with DAMPs using filipin as an indicator of cholesterol load. Filipin has been widely used for cytochemical localization of cholesterol in biological membranes, based on its ability to bind to unesterified 3β-hydroxy-sterols forming 25 nm complexes, but it is also applied to mark free cholesterol in cultured cells. Interaction with cholesterol alters the filipin absorption and fluorescence spectra, allowing visualization with a fluorescence microscope (20). 3D-cultured EwS cells displayed a two to fourfold greater baseline cholesterol load, as assessed by filipin fluorescence intensity and cholesterol quantification, than their 2D-cultured counterparts (Fig 3C and D). DAMPs further increased the intracellular cholesterol of 3D-cultured C1 cells, as measured by filipin fluorescence (Fig 3E–G), and cholesterol quantification revealed an up to threefold increase in cholesterol load (Fig 3H). In contrast, DAMPs either had no effect or even decreased the cholesterol load of 2D-cultured cells (Fig 3H). The baseline and the DAMP-induced total cholesterol load in spheroids were reduced by statins (Fig 3I) and by the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD) (Fig S3D). DAMPs increased cholesterol uptake in EwS spheroids but not in the corresponding adherent cells (Fig 3J).

To determine whether cholesterol has a growth stimulatory effect on EwS, spheroids from primary EwS and EwS cell lines were cultured in the presence of cholesterol or mevalonic acid (1 mM each) for 5 d and assessed for proliferation. Cholesterol and mevalonic acid stimulated EwS 3D cell growth and the effect was partially neutralized by 50 μM simvastatin (Fig 3K) and MBCD (Fig S3B–D). Together, these observations suggest that the increase in cholesterol load, resulting from augmentation in both uptake and synthesis, constitutes at least one mechanism by which DAMPs promote EwS cell growth in 3D culture.