Multicellular spheroids are generated from mesothelioma cell lines. A, spheroids formed from >10,000 cells show baseline apoptosis. Spheroids generated over 24 h from different numbers of cells (M28, REN, and VAMT) were studied by immunoblot for baseline evidence of apoptosis, as determined by cleavage of poly(ADP-Ribose)polymerase (PARP). Spheroids formed from 25,000 cells per spheroid or greater showed evidence of cleaved PARP in REN and M28 cells, indicating the presence of apoptosis. (Tubulin is shown as a loading control; a representative blot is shown.) B, spheroids generated with 10,000 cells per spheroid showed consistent shape and size. Representative images of spheroids formed by 10,000 M28, REN, and VAMT cells at 24 h, viewed from above by inverted phase contrast microscopy of spheroids growing in wells and from the side (at left) by light microscopy of a paraffin-embedded section of an M28 spheroid. Spheroids were between 500 and 1000 μm in greatest width, and 100 μm in thickness. REN and VAMT spheroids were of a similar thickness, despite their smaller widths.

Multicellular spheroids acquire resistance to apoptosis. A, mesothelioma spheroids display resistance to various apoptotic stimuli. M28, REN, and VAMT monolayers and spheroids exposed to TRAIL (1 ng/ml) plus the proteasome inhibitor MG-132 (2.5 μm) or the histone deacetylase inhibitor trichostatin A (250 nm) for 24 h were studied for apoptosis using annexin V staining and confirmed by examination of condensed nuclei following Hoechst staining. Spheroids consistently showed resistance to the different apoptotic stimuli in all cell lines tested (n = 3, *, p ≤ 0.05 monolayer versus spheroids, mean ± S.D.). B, TRAIL diffuses uniformly within spheroids. After M28 spheroids were exposed to TRAIL (1 ng/ml) for 6 h, immunohistochemistry was performed to detect TRAIL within spheroids. TRAIL diffusion into the spheroids was complete within 6 h as demonstrated by the uniform staining of spheroids exposed to TRAIL, with no staining seen in unexposed spheroids (control) (representative images of four separate experiments). C, spheroids also display resistance to non-TRAIL combinations at 24 h. Combinations of non-TRAIL apoptotic agents that were found to induce apoptosis in mesothelioma cells in monolayers were then tested on monolayers and spheroids of M28, REN, and VAMT cells. Monolayer cells underwent apoptosis following MG-132 (2.5 μm) together with either sodium butyrate (10 mm) or trichostatin A (250 nm), whereas the same cells grown as spheroids were relatively resistant. Each agent, MG-132, trichostatin A, and sodium butyrate, had little effect alone, with apoptosis being <10% (not shown). Apoptosis was measured by quantification of nuclear condensation of Hoechst-stained cells, in this and remaining apoptosis studies (n = 3, *, p ≤ 0.01 monolayer versus spheroids, mean ± S.D.). D, spheroids are also resistant to TRAIL plus the sensitizing agent, anisomycin, at 6 h. After exposure to TRAIL (1 ng/ml) plus anisomycin (25 ng/ml) for 6 h, all three mesothelioma cell lines showed apoptosis, whereas spheroids displayed resistance. The same resistance was seen at 12 and 24 h (not shown) (n = 3, *, p ≤ 0.001 monolayer versus spheroids, mean ± S.D.).

Rapamycin inhibits mTOR and reduces the acquired apoptotic resistance in spheroids. A, PI3K/Akt/mTOR pathway is inhibited differently by rapamycin, PI-103, and LY294002. M28, REN, and VAMT cells grown in monolayers were exposed to nothing, rapamycin (5 nm), PI-103 (1 μm), or LY294002 (5 μm) for 4 h and then studied by immunoblot for expression of P-Akt (Ser-473) and P-S6K (Thr-389) as markers of Akt and mTOR activity, respectively. Rapamycin strongly inhibited mTOR activity in all tested cells but also produced a feedback Akt activation, PI-103 potently inhibited both Akt and mTOR, and LY294002 inhibited Akt and partially inhibited mTOR. B, PI3K/Akt/mTOR inhibitors reduce apoptotic resistance of spheroids, as measured by nuclear morphology. M28, REN, and VAMT cells grown as monolayers or spheroids were treated with TRAIL (1 ng/ml) plus anisomycin (25 ng/ml, T+A) with either no inhibitor, rapamycin (5 nm), PI-103 (1 μm), or LY294002 (5 μm) for 6 h. Apoptosis was measured by determination of characteristic nuclear morphology of Hoescht-stained cells. Inhibitors increased apoptosis in spheroids without a comparable effect in monolayers (n = 3, *, p < 0.05 compared with no inhibitor, mean ± S.D.). C, PI3K/Akt/mTOR inhibitors reduce apoptotic resistance of spheroids, as measured by PARP cleavage. For M28 cells studied under the same conditions as in Fig. 3B, apoptosis was assessed by PARP cleavage using immunoblot analysis. Inhibitors increased PARP cleavage induced by TRAIL plus anisomycin in spheroids while monolayers were not significantly affected. The pancaspase inhibitor z-VAD-fmk (50 μm) inhibited PARP cleavage confirming a caspase-dependent apoptosis. D, rapamycin reduces apoptotic resistance of spheroids to two different apoptotic regimens similarly. M28, REN, and VAMT spheroids were treated with TRAIL (1 ng/ml) plus anisomycin (25 ng/ml) for 6 h or with trichostatin A (250 nm) plus MG-132 (2.5μm) for 24 h in the presence of rapamycin (5 nm), PI-103 (1μm), or DMSO control. The experiments using TRAIL plus anisomycin are those shown in Fig. 3B, whereas the experiments using trichostatin plus MG-132 are shown here for the first time. Apoptotic resistance is defined here as the difference between the apoptosis of the monolayer and spheroid divided by that of the monolayer. Rapamycin reduced apoptotic resistance of spheroids to the two apoptotic combinations from 73 ± 14% to 44 ± 17%, or by ∼40%. PI-103, although targeting a broader range of kinases, was no more effective than rapamycin (n = 3).

In spheroids, the baseline activity of Akt/mTOR pathway is down-regulated but remains more activated than in non-malignant mesothelial cells. A, Akt/mTOR activity in spheroids is reduced compared with monolayers. Akt/mTOR pathway activity of M28, REN, and VAMT mesothelioma cells was reduced in the spheroids (s) as demonstrated by a lower expression of P-Akt (Ser-473), P-S6K (Thr-389), and P-4E-BP1 (Thr-37/46) compared with that in the monolayers (m). The total levels of Akt, S6K, and 4E-BP1 show no consistent change, and the tubulin levels confirm equal loading. B, Akt/mTOR activity in spheroids is greater than in normal mesothelial cells. Normal mesothelial cells (NMC) monolayers and both monolayers and spheroids from M28, REN, and VAMT cells were analyzed by immunoblot for P-Akt (Ser-473 or Thr-308) and P-S6K. In this experiment, cells were grown in reduced serum (2% fetal bovine serum) to reduce growth factor stimulation of the Akt/mTOR pathway. Although phosphorylation of Akt/mTOR pathway members in spheroids is lower than in the monolayer, spheroids nonetheless demonstrate a greater activation of the pathway than in their non-malignant counterparts. Below, the P-S6K panel has been overexposed to show more clearly the higher level of P-S6K in spheroids than in normal mesothelial cells. C, Akt and S6K phosphorylation is uniform throughout the spheroid. Staining of M28 spheroids with antibodies against P-Akt (Ser-473) and P-S6K (Thr-389) shows a uniform pattern of Akt and S6K phosphorylation throughout the spheroid. For the P-Akt, specificity is shown by a lack of staining with inclusion of a P-Akt Ser-473 blocking peptide (negative control). For the P-S6K staining, no primary antibody was used as a control and resulted in no staining (not shown). Two magnifications (20× and 40×) are shown.

S6K ablation inhibits the acquired apoptotic resistance of spheroids and reproduces the effect of rapamycin. Upper panel, the knockdown of S6K (S6K-kd) reduced the resistance of M28 spheroids to TRAIL plus anisomycin (see the asterisk). The effect of S6K-kd was the same as the effect of rapamycin on control cells. Indeed, when combined, rapamycin did not add to the effect of S6K-kd, suggesting that they acted identically. Conversely, monolayers were not significantly affected either by S6K siRNA or by rapamycin (n = 3, *, p ≤ 0.001 scramble versus S6K duplexes, mean ± S.D.). Lower panel, S6K was effectively ablated by siRNA, as measured 48 h after transfection, the time when agents are added.

FLIPs and Bcl-2 levels increase in some spheroids but do not explain mTOR-dependent apoptotic resistance. A, spheroids of M28 and VAMT exhibit increased FLIPs and Bcl-2 protein levels. A panel of pro-/anti-apoptotic proteins was screened by immunoblot in M28, REN, and VAMT monolayers and spheroids. Compared with monolayers, spheroids formed by two of the three cell lines displayed increased levels of FLIPs and Bcl-2 proteins. The levels of other proteins did not show a consistent change that would account for acquired resistance in spheroids. B, mTOR inhibition does not modulate FLIPs and Bcl-2 levels. Neither pharmacological nor molecular inhibition of mTOR signaling modified FLIPs and Bcl-2 protein levels as evidenced by immunoblot analysis of monolayers and spheroids formed from control or S6K-kd M28 cells, treated with or without rapamycin (5 nm) for 4 h.

Rapamycin increases caspase-8 cleavage in spheroids by actions distal to Bid, presumably at the mitochondria. A, rapamycin increases TRAIL-induced caspase-8 cleavage in spheroids. Spheroids but not monolayers grown from M28 cells displayed increased caspase-8 cleavage when rapamycin (5 nm) was added to TRAIL plus anisomycin, as shown by the appearance of p26 and p41/43 caspase-8 fragments in the immunoblot performed after 6 h. The increase in caspase-8 cleavage was associated with increases in apoptosis, as shown by PARP cleavage. B, rapamycin increases apoptosis in spheroids by actions requiring Bid. Bid was knocked down by siRNA to isolate the death receptor from the mitochondrial apoptotic pathways. Upper panel, in control M28 cells, as expected, monolayer cells were sensitive to TRAIL plus anisomycin-induced apoptosis, whereas spheroid cells were resistant but rendered more sensitive after rapamycin. In cells without Bid, however, monolayers and spheroids failed to undergo apoptosis after TRAIL plus anisomycin, and rapamycin did not increase the apoptotic rate in the spheroids (n = 3, *, p ≤ 0.001 no inhibitor versus rapamycin, mean ± S.D.). Lower panel, Bid was effectively ablated by siRNA, as measured 48 h after transfection, the time when agents are added. C, rapamycin increases caspase-8 cleavage in spheroids by actions distal to Bid. Bid was knocked down by siRNA to permit identification of the site of action of rapamycin. Bid-kd prevented the increase in caspase-8 cleavage induced by rapamycin in control spheroids treated with TRAIL plus anisomycin. Because Bid is necessary for the rapamycin-induced increase in caspase-8 cleavage, mTOR is acting, not at the death receptor pathway, but distal to Bid. Caspase-8 cleavage paralleled apoptosis as shown by PARP cleavage. Addition of z-VAD-fmk (50 μm) inhibited both caspase-8 cleavage and apoptosis.

mTOR/S6K contributes to acquired multicellular apoptotic resistance at the level of the mitochondria. Scheme of the apoptotic signaling circuitry activated by TRAIL plus mitochondria sensitizers or stressors. Extrinsic (death receptor) and intrinsic (mitochondrial) apoptotic pathways, linked by the BH3-molecule Bid, can activate an effective apoptosis in mesothelioma cells (47). Truncated Bid (tBid), generated by caspase-8 activated at the death-inducing signaling complex (DISC) by TRAIL, signals to the mitochondria. When the mitochondrial threshold for apoptosis is lowered by concurrent DNA damaging or other sensitizers such as anisomycin, the mitochondria can respond to tBid and activate downstream caspases and activate caspase-8 by feedback. In our study, Bid was necessary for the ability of rapamycin to enhance caspase-8 cleavage and apoptosis indicating that mTOR/S6K acted distal to Bid. (Schematic diagram modified from Refs. 23 and 47).