Spheroids grown from mesothelioma cell lines acquire multicellular resistance to bortezomib. (a) M28 and REN monolayers and spheroids were treated with bortezomib 1, 5,10, 25 or 100 nM for 48 h. Apoptosis was measured in Hoechst-stained cells; cells with signs of nuclear condensation were considered apoptotic. Spheroids grown from both cell lines acquired a marked multicellular resistance to bortezomib even at the highest concentrations. (^*P<0.05 spheroids compared with monolayers, n=3). (b) REN monolayers and spheroids were treated with bortezomib (100 nM) or MG-132 (10 μM), a potent proteasome inhibitor, for 4 h before cells were lysed and analyzed for proteasomal activity. Monolayers and spheroids showed similar proteasomal activity at baseline and were both equally inhibited by bortezomib or MG-132 after 4 h. (^*P<0.05 compared with untreated control, n=3). (c) M28 and REN monolayers at standard confluence (70%), completely confluent monolayers (obtained by plating twice the number of cells to achieve at least 100% confluence) and spheroids were treated with bortezomib (25 or 100 nM). Completely confluent monolayers showed an apoptotic response comparable to monolayers and did not acquire multicellular resistance. (^*P<0.05 spheroids compared with monolayers and confluent monolayers, n=3)

Spheroids fail to upregulate Noxa after bortezomib but have a high expression of Bim. M28 and REN monolayers and spheroids were treated with bortezomib (25 or 100 nM) for 4 or 24 h. Whole cell lysates (50 μg) were resolved on SDS-PAGE and immunoblotted for a panel of pro-/anti-apoptotic Bcl-2 proteins. Both M28 and REN monolayers upregulated Noxa after bortezomib (25 nM) at 24 h. (Owing to the high degree of apoptosis, monolayers treated with 100 nM bortezomib at 24 h were not collected.) Spheroids, however, failed to upregulate Noxa, even after bortezomib 100 nM at 24 h. Interestingly, at baseline and during exposure to bortezomib, spheroids expressed more Bim protein than monolayers. (Representative of three experiments)

A Noxa peptide sensitizes spheroids to bortezomib-induced apoptosis. (a) Noxa was silenced in REN cells and, 24 h later, cells were plated as monolayers and treated with bortezomib (25 nM). After 4 h, cells were collected and lysed to verify the efficacy of Noxa siRNA (see immunoblot). No changes in other Bcl-2 proteins, Mcl-1 and Bim, were detected upon Noxa silencing, with or without bortezomib. After 24 h, Hoechst-stained cells were counted for the presence of apoptosis. Noxa siRNA significantly reduced the apoptosis induced by bortezomib. (^*P<0.05, n=3). (b) M28 and REN monolayers and spheroids were treated with bortezomib (25 nM), a cell-permeable R8-Noxa^BH3 peptide (50 μM) or a control R8 peptide (50 μM), either alone or in combination, for 24 h. R8-Noxa^BH3, but not the R8 peptide, eliminated the acquired multicellular resistance of spheroids to bortezomib without affecting monolayers. Interestingly, REN spheroids, but not M28, were sensitive to R8-Noxa^BH3 alone. (^*P<0.05 compared with untreated and R8 control; ⁺⁺P<0.05 spheroids versus monolayers, n=3)

ABT-737 eliminates multicellular resistance to bortezomib. M28 and REN monolayers and spheroids were treated with bortezomib (25 nM) and ABT-737 (1 μM), either alone or in combination, for 24 h. ABT-737 eliminated the acquired multicellular resistance of spheroids to bortezomib. Interestingly, when given alone, ABT-737 was able to induce apoptosis in spheroids but not in monolayers. (^*P<0.05 compared with bortezomib alone; ⁺⁺P<0.05 spheroids versus monolayers, n=3)

ABT-737 sensitization of spheroids to bortezomib is dependent on Bim and aided by ablation of Mcl-1. (a) Bim was ablated in M28 and REN cells and, 24 h later, cells were plated to generate monolayers and spheroids, exposed to bortezomib 25 nM and/or ABT-737 1 μM for 24 h and lysed and probed for Bim at 72 h after siRNA (see immunoblot insert), the time when cells were counted for the presence of apoptosis. The absence of Bim in spheroids completely abolished the apoptotic effect of ABT-737 when given alone and significantly reduced the effect when given with bortezomib. (^*P<0.05 compared with control siRNA, ⁺⁺P<0.05 spheroids versus monolayers, n=3). (b) Mcl-1 was ablated, as performed for Bim above (see immunoblot insert). The absence of Mcl-1 reduced the acquired multicellular resistance of spheroids to bortezomib. The absence of Mcl-1 also increased the apoptotic response to ABT-737 when given alone or in combination with bortezomib. (^*P<0.05 compared with control siRNA, ⁺⁺P<0.05 spheroids versus monolayers, n=3)

Spheroids are ‘primed for death'. BH3-profiling was performed on cells disaggregated from monolayers and spheroids grown from M28, REN, SARC and VAMT cells. The cells were gently permeabilized as described³⁹ and exposed to BH3 peptides (100 μM) for 90 min before JC1 was added for 30 min and depolarization measured as a percentage of the total. Cells from spheroids and monolayers were equally depolarized by Bim^BH3, the positive control, confirming a functioning apoptotic apparatus. However, cells disaggregated from spheroids were more sensitive to Bad^BH3, Hrk ^BH3 and ABT-737 than cells from monolayers, confirming that cells in 3D acquired apoptotic priming. Cells showed no response to an ABT-737 inactive enantiomer, used as negative control. (^*P<0.05 compared with monolayers, n=3)

Apoptotic priming of spheroids increases with elevated Bim expression. (a) Spheroids grown from M28, VAMT, REN and SARC cell lines were treated with R8-Noxa^BH3 (50 μM), ABT-737 (1 μM), bortezomib (25 nM) or their combination for 24 h. Whereas R8-Noxa^BH3 alone failed to induce apoptosis in M28 spheroids, ABT-737 alone induced apoptosis in the spheroids generated from all four cell lines. Nevertheless, when given with bortezomib, either R8-Noxa^BH3 or ABT-737 was effective in increasing apoptosis of spheroids. (^*P<0.05 compared with untreated spheroids, ^**P<0.05 compared with bortezomib alone, n=3). (b) Immunoblot analysis of Bim expression in M28, REN, SARC and VAMT monolayers and spheroids with densitometry shown for Bim, standardized for α-tubulin expression. Immunoblot and densitometry values are representative of three separate experiments. In all four cell lines, spheroids expressed increased Bim levels. Compared with monolayers, spheroids of M28 displayed a 1.9-fold increase in Bim levels whereas spheroids of REN, SARC and VAMT showed a 4.5-, 3.2- and 4.0-fold, increase, respectively

ABT-737 increases activity of bortezomib against mesothelioma cells in spheroids derived from tumors with high expression of Bim. (a) TFS generated from seven tumors were treated with bortezomib (50 nM), ABT-737 (2.5 μM) or their combination for 24 h. In the responsive tumors (n=5) (shown), ABT-737 increased apoptosis when given together with bortezomib. In unresponsive tumors (n=2) (not shown), ABT-737 with or without bortezomib had no effect (apoptosis <5%), (^*P<0.05 compared with no treatment, bortezomib alone or ABT-737 alone, n=5). (b) Confocal images representative of the responsive tumor fragment spheroids treated with bortezomib (50 nM) with or without ABT-737 (2.5 μM) for 24 h. Pan-cytokeratin (red) was used to identify mesothelioma cells within the spheroids and cleaved caspase-3 (green) to identify apoptosis. The merged color (yellow) identifies apoptotic mesothelioma cells. The mesothelioma cells are resistant to ABT-737 alone (not shown) and to bortezomib alone. The addition of ABT-737 to bortezomib, however, effectively increased apoptosis of the human mesothelioma cells. (c) Using a TMA generated with tissue from the original seven tumors tested for apoptosis along with normal pleural tissue as control, Bim expression was determined by immunohistochemistry and quantified using an imaging analysis system (ScanscopeXT, Aperio). Every sample was represented three times on the slide. Normal pleura and the two tumors not responsive to ABT-737 and bortezomib showed comparable, very low amounts of Bim. Conversely, the responsive tumors showed strong Bim staining. Intensity of staining was expressed as percentage compared with normal pleura. (^*P<0.05 compared with normal and non-responsive)