A549 cells formed multicellular spheroids within 48 hours and acquired resistance to bortezomib plus TNF-related apoptosis–inducing ligand (TRAIL). (A) Spheroids and microspheroids formed from A549 cells have short diffusion distances. Spheroids generated with 10,000 cells each show consistent discoid shape and size, shown from a side view (left panel) and from above (center panel), with a width of 750–1,000 μm and a thickness of 100 μm. Microspheroids generated from 250 cells each are spherical, with a diameter of 75–100 μm. The microspheroids are shown in the recesses of the gel used to form them (representative images shown). (B) A549 cells in monolayer undergo synergistic apoptosis to the combination of bortezomib plus TRAIL, whereas spheroids exhibit resistance. A549 monolayers (open bars) and spheroids (solid bars) were exposed to the proteasome inhibitor, bortezomib (100 nM), plus TRAIL (1 ng/ml) for 24 hours, and then examined for apoptosis after Hoechst 33342 nuclear staining. Neither monolayers nor spheroids responded significantly to TRAIL alone or to bortezomib alone. In cells grown as monolayers, the apoptotic response to the combination of bortezomib plus TRAIL was synergistic, because the response to the combination was statistically greater than the sum of the responses to the individual agents. Compared with monolayers, the spheroids were resistant (*different from the sum of the responses to TRAIL alone and to bortezomib alone; ^†different from monolayer; mean ± SD; n = 3; P < 0.01).

Apoptotic resistance of spheroids to bortezomib plus TRAIL is not likely due to the NF-κB pathway. (A) TRAIL fails to activate the NF-κB pathway in A549 cells. A549/NF-κB–luc monolayers were pretreated with nothing or with the NF-κB inhibitor, BAY 11-7082 (10 μM), for 1 hour, and then exposed to TRAIL (1 or 10 ng/ml) or TNF-α (50 ng/ml) for 5 hours. TNF-α induced a strong luciferase signal; the expected inhibition by BAY confirmed that the luciferase activity represented NF-κB signaling. TRAIL did not induce NF-κB signaling. (B) NF-κB is activated by TNF in monolayers (open bars) and spheroids (solid bars) equally, and is inhibited equally by bortezomib. A549/NF-κB–luc monolayers and spheroids were pretreated with nothing or with bortezomib (100 nM or 1,000 nM) for 1 hour, and then exposed to TRAIL (1 ng/ml) or TNF-α (50 ng/ml) for 5 hours. NF-κB was activated by TNF-α in both monolayers and spheroids, and was equally inhibited by bortezomib. NF-κB was not activated by TRAIL in either monolayers or spheroids (*different from baseline; ^†different from TNF-α stimulation; mean ± SD; n = 3; P < 0.01).

Balance of anti- and proapoptotic proteins and response to treatment is altered in spheroids, contributing to apoptotic resistance. (A) Expression of Bcl-2 increases and Mcl-1 decreases when A549 cells form spheroids. A panel of anti- and proapoptotic Bcl-2 family proteins were screened by immunoblot in A549 monolayers (M) and spheroids (S), before and after treatment with 100 nM bortezomib plus 1 ng/ml TRAIL for 2 and 4 hours. Bcl-2 was up-regulated after A549 spheroids formed spheroids, whereas Bcl-xL and Mcl-1 were down-regulated. At 4 hours after treatment, there was a significant up-regulation of Noxa in monolayers, but not in spheroids. (B) ABT-737, a BH3 mimetic that blocks Bcl-2, Bcl-xL, and Bcl-w, induces apoptosis in spheroids and eliminates apoptotic resistance. At baseline, the Bcl-2 family inhibitor, ABT-737, alone (10 μM) induced apoptosis in A549 spheroids without any effect on monolayers. After exposure to bortezomib plus TRAIL, 10 μM ABT-737 induced a significant increase in apoptosis of spheroids, so that the response to treatment was equal in monolayers (open bars) and spheroids (solid bars). The negative control enantiomer of ABT had no effect (*different from negative control for ABT; mean ± SD; n = 3; P < 0.01).

Bcl-2 family proteins contribute to the multicellular resistance of H1299 spheroids. (A) ABT-737 alone did not eliminate the multicellular resistance of spheroids. At baseline, the Bcl-2 family inhibitor, ABT-737, alone (10 μM) had no effect in H1299 monolayers (open bars) and spheroids (solid bars). After exposure to bortezomib plus TRAIL, 10 μM ABT-737 induced an increase in apoptosis of spheroids, but did not eliminate the multicellular resistance. The negative control enantiomer of ABT had no effect (*different from negative control for ABT; mean ± SD; n = 3; P < 0.01). (B) H1299 spheroids have higher expression level of Mcl-1 than A549 spheroids. By immunoblot, Mcl-1 was down-regulated in spheroids (S) compared with monolayers (M) in both A549 and H1299 cells. However, H1299 spheroids retained higher expression levels of Mcl-1 than A549 spheroids. (C) Mcl-1 small interfering RNA (siRNA) depletes the expression of Mcl-1 in H1299 monolayers and spheroids. Mcl-1 was depleted by siRNA, as measured at 48 hours after transfection, the time when apoptotic agents were added. (D) Depletion of Mcl-1 largely removes multicellular resistance in H1299 spheroids. Depletion of Mcl-1 by itself did not induce apoptosis in H1299 monolayers (open bars) and spheroids (solid bars), but significantly removed the multicellular resistance of H1299 spheroids to 100 nM bortezomib (Bz) plus 1 ng/ml TRAIL treatment for 24 hours. ABT-737 (10 μM) exposure plus Mcl-1 depletion induced death of monolayers and spheroids, and removed all multicellular resistance to bortezomib plus TRAIL (*different from spheroids treated with bortezomib plus TRAIL; mean ± SD; n = 3; P < 0.01).

Apoptotic resistance of spheroids to bortezomib plus TRAIL is not due to the limited diffusion of reagents into the spheroids. (A) Bortezomib inhibits the proteasome activity equally in A549 monolayers (open bars) and spheroids (solid bars). Under baseline conditions, proteasome activity was lower in A549 cells in spheroids than in monolayers. After exposure to bortezomib (100 nM) for 4 hours, proteasome activity in both monolayer and spheroid was equally inhibited (*different from monolayer; ^†different from baseline; mean ± SD; n = 3; P < 0.01). (B) TRAIL induced similar Bid cleavage in monolayers and spheroids. A549 monolayers and spheroids were exposed to TRAIL (2.5, 5, or 10 ng/ml) with or without bortezomib (100 nM) for 16 hours. TRAIL-induced Bid cleavage was measured by immunoblot as a decrease in full-length Bid. TRAIL 2.5 and 10 ng/ml induced similar Bid cleavage in A549 monolayers and spheroids (see boxed areas). After combination therapy, Bid cleavage is enhanced in monolayers compared with spheroids, consistent with an enhanced Bid cleavage seen after apoptosis and widespread activation of caspases (2). (C) Microspheroids demonstrate apoptotic resistance similar to that of spheroids. A549 cells grown as monolayers (open bars), microspheroids (shaded bars), and spheroids (solid bars) were exposed to 100 nM bortezomib (Bz) plus 1 ng/ml TRAIL for 24 hours, and then studied for apoptosis after Hoechst 33342 nuclear staining. Despite their difference in size and shape, and the shorter diffusion distance in the microspheroids, microspheroids and spheroids showed similar apoptotic resistance to bortezomib and TRAIL (*different from monolayer; mean ± SD; n = 3; P < 0.01). (D) Apoptotic cells are distributed evenly throughout the spheroids. A549 multicellular spheroids were treated with bortezomib (100 nM) plus TRAIL (1 ng/ml) for 24 hours, and then prepared for immunohistochemistry. Nuclei of all cells are indicated by DAPI staining; apoptotic cells are those with cleaved caspase 3. After exposure to bortezomib plus TRAIL, apoptotic cells are located throughout the spheroids, not in any one particular region (negative control with no primary antibody showed no staining; positive control, thymus, showed characteristic staining for apoptotic cells).

Apoptotic resistance in spheroids is not due to the PI3K/Akt/ mTOR pathway. (A) The PI3K/Akt/mTOR pathway is inhibited by rapamycin and PI-103 in A549 monolayers and spheroids. A549 monolayers and spheroids were exposed to nothing, rapamycin (25 nM), or PI-103 (1 μM) for 4 hours, and then studied by immunoblot for expression of p-Akt (Ser473) and p-S6K (Thr389). Rapamycin inhibited mTOR activity, as shown by a decrease in the mTOR target, p-S6K, and PI-103 inhibited both PI3K and mTOR, as shown by a decrease in p-Akt and p-S6K. (B) PI3K/Akt/mTOR inhibitors do not reduce apoptotic resistance of spheroids to bortezomib plus TRAIL. A549 monolayers (open bars) and spheroids (solid bars) were treated with 100 nM bortezomib (Bz) plus 1 ng/ml TRAIL with either no inhibitor, 25 nM rapamycin, or 1 μM PI-103 for 24 hours. The inhibitors did not reduce the apoptotic resistance of spheroids to bortezomib plus TRAIL (*different from monolayer; mean ± SD; n = 3; P < 0.001).

H1299 cells form multicellular spheroids, acquire apoptotic resistance, and demonstrate an altered balance of Bcl-2 family proteins. (A) Spheroids and microspheroids formed from H1299 cells have short diffusion distances. H1299 spheroids generated with 10,000 cells each show a discoid shape and size similar to A549 spheroids, shown from a side view (left panel) and from above (center panel). Microspheroids generated from 250 cells each are spherical, with a diameter of 75–100 μm (representative images shown). (B) H1299 cells in monolayer undergo synergistic apoptosis to the combination of bortezomib plus TRAIL, whereas spheroids exhibit resistance. Bortezomib (100 nM) plus TRAIL (1 ng/ml) for 24 hours induced synergistic apoptosis in H1299 cells grown as monolayers. Compared with monolayers (open bars), the spheroids (solid bars) were resistant to this combinatorial treatment (*different from the sum of the responses to TRAIL alone and to bortezomib alone; ^†different from monolayer; mean ± SD; n = 3; P < 0.01). (C) H1299 microspheroids demonstrate apoptotic resistance similar to spheroids. H1299 cells grown as monolayers (open bars), microspheroids (shaded bars), and spheroids (solid bars) were exposed to 100 nM bortezomib (Bz) plus 1 ng/ml TRAIL for 24 hours, and then studied for apoptosis. Microspheroids and spheroids showed similar apoptotic resistance (*different from monolayer; mean ± SD; n = 3; P < 0.01). (D) Expression of Bcl-2 increases and Mcl-1 decreases when H1299 cells form spheroids. A panel of anti- and proapoptotic Bcl-2 family proteins were screened by immunoblot in H1299 monolayers (M) and spheroids (S), before and after treatment with 100 nM bortezomib plus 1 ng/ml TRAIL for 2 and 4 hours. Bcl-2 was up-regulated after H1299 spheroids formed spheroids, whereas Mcl-1 was down-regulated. At 4 hours after treatment, there was a significant up-regulation of Noxa in monolayers, but not in spheroids.