Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome

<p>Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.</p

ASS1 knockdown by siRNA increases the chemosensitivity of spheroids.

<p><b>(A)</b> Immunoblot analysis of M28, SARC, VAMT and ASS1-deficient MSTO spheroids, transfected with siRNA targeting ASS1 or control sequences show that ASS1 was efficiently knocked down in all the cells where it was expressed. <b>(B)</b> Apoptotic rates were measured for M28, SARC, VAMT and MSTO spheroids transfected with siRNA targeting ASS1 or control sequences and treated with bortezomib (BZ, 25 nM) or cis-platinum (200 μM) plus pemetrexed (10 μM)(C+P). In the 3 cell lines with baseline ASS1 expression, ASS1 siRNA increased the basal apoptotic rate and also increased the apoptotic response to chemotherapy. ASS1 siRNA had no effect in the ASS1-deficient MSTO spheroids. Apoptosis was quantified in a blinded fashion by assessing nuclear condensation in Hoechst-stained cells. (* <i>p</i> ≤ 0.05 n = 3).</p

ASS1 protein is upregulated in 3D in mesothelioma cells but not in normal mesothelial cells.

<p><b>(A)</b> ASS1 antibodies (BD clone 25, Sigma clone 2B10, and Millipore clone 2C10) were tested against ASS1-positive MSTO pIRES-ASS1 (as a positive control) and ASS1-negative MSTO pIRES-EV (as a negative control). Monoclonal antibody clone 2C10 provides the most accurate detection of ASS1 protein levels. <b>(B)</b> Immunoblot analysis of ASS1, ANXA4 and MVP in monolayers (m) and spheroids (s) grown from 6 mesothelioma cell lines (M28, SARC, VAMT, REN, JMN) and MSTO-211H (known to be ASS1-negative). MSTO pIRES-ASS1 cells were used as positive control for ASS1. ASS1 was the most consistently upregulated in 3D among the panel of cells tested. JMN and MSTO cells showed no ASS1 expression. <b>(C)</b> Immunoblot analysis of ASS1 in normal mesothelial cells grown as monolayers and spheroids. ASS1-negative MSTO pIRES-EV and ASS1-positive MSTO pIRES-ASS1 were used as controls for ASS1. Normal mesothelial cells did not upregulate ASS1 when grown in 3D.</p

ASS1 protein is expressed in mesothelioma samples and upregulated when compared to normal tissues.

<p><b>(A)</b> ASS1 stain intensity was measured in 88 mesothelioma samples by immunohistochemistry (each tumor in replicates of 3) in a semi-quantitative fashion as outlined in the Methods section. Only 8 samples out of 88 (10%) showed no ASS1 staining <b>(B)</b> ASS1 stain intensity was also measured in another 88 mesotheliomas with their paired 88 normal tissues. Analysis of matched pairs (tumor vs normal tissue control obtained from the same patients) of the 88 patients showed that ASS1 expression was significantly higher in mesothelioma (* <i>p</i> < 0.0001, n = 88 matched pairs).</p

More than 200 genes are differentially expressed in mesothelioma spheroids compared to monolayers.

<p>Venn diagrams showing the number of genes differentially expressed by spheroids of three mesothelioma cell lines M28, REN and VAMT, compared to respective monolayers. The diagram also shows the number of genes upregulated (138) and downregulated (71) in common by the three cell lines.</p

ASS1 gene expression is upregulated in 3D spheroids and mesothelioma patients.

<p>qRT-PCR results showing the Log2-transformed fold change in gene expression for ASS1, ANXA4 and MVP between M28, REN and VAMT spheroids and monolayers. Results were normalized to the gene expression levels of monolayers (<i>gray line</i>). Only ASS1 was upregulated in 3D in all three cell lines. (* <i>p</i> ≤ 0.05 n = 3).</p

Dkk-1 inhibits the high phosphate-induced osteogenic-like characteristics in rat mesenchymal stem cells.

<p>A) Rat mesenchymal cells treated with high phosphate and Dkk-1 were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Dkk-1 administration reduced nuclear translocation of β-catenin. Original magnification: 40x. B) BMP-2 mRNA expression in rat mesenchymal stem cells treated with high phosphate and Dkk-1 was determined by RT-PCR (a p<0.001 vs high phosphate treated cells). C) Calcium content and alkaline phosphatase activity (Units/mg protein) in rat mesenchymal stem cells treated with high phosphate and Dkk-1 (a p<0.001 vs high phosphate alone). Image is representative of three experiments.</p

TGF-β administration prevents osteogenic effects induced by high phosphate.

<p>A) High phosphate (P) increased the expression of BMP-2 while TGF-β or the combination of TGF-β plus high phosphate decreased significantly the expression of this osteogenic marker (a p<0.001 vs. all groups). Results are expressed as fold change vs. Control cells. B) High phosphate (P) decreased significantly SM22α and myocardin expression with respect to Control cells (b p<0.01 for SM22α and a p<0.001 for myocardin) and TGF-β group (c p<0.001). The combination of TGF- β and high phosphate (TGF-β + P) decreased the expression of SM22α and Myocardin although less than high phosphate alone (c p <0.001vs. TGF-β group). C) TGF-β alone did not change significantly the alkaline phosphatase activity. This activity increased after high phosphate treatment (a p<0.001 vs. all others groups). The combination of TGF-β and high phosphate for 14 days significantly decreased this activity when compared with high phosphate group. D) Calcium content was significantly increased after high phosphate treatment (a p<0.001 vs other groups). The combination of TGF-β and high phosphate prevented this increase of calcium induced by high phosphate alone.</p

Wnt/β-catenin pathway activation enhances the high phosphate-induced osteogenic-like characteristics in rat mesenchymal stem cells.

<p>A) Rat mesenchymal cells treated with high phosphate and CHIR98014 (0.4 µM) or lithium chloride (5 mM) were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Both Wnt activators (CHIR98014 and lithium chloride) increased nuclear translocation of β-catenin. Original magnification: 40x. B) BMP-2 protein and C) mRNA expression in rat mesenchymal stem cells treated with high phosphate and CHIR98014 or lithium chloride was determined by western blot and RT-PCR respectively (a p < 0.001 vs high phosphate alone). D) Calcium content and E) alkaline phosphatase activity in rat mesenchymal stem cells treated with high phosphate and CHIR98014or lithium chloride (a p<0.001 vs. high phosphate alone). Image is representative of three experiments.</p

TGF-β induces vascular smooth muscle cells differentiation of mesenchymal stem cells through nuclear translocation of Smad3.

<p>A) Rat mesenchymal cells treated with TGF-β for 14 days were stained for phospho-Smad3 immunofluorescence (red) and counterstained with DAPI (blue) to determine phospho-Smad3 subcellular localization. In TGF-β treated cells, positive phospho-Smad3 immunofluorescence was localized into the nucleus. Original magnification: 40x. B) Vascular smooth muscle actin (VSM-actin, green) was stained and the nuclei were counter-stained with DAPI showing cytoskeleton organization in Control cells and TGF-β treated cells. Original magnification: 20x.C) After 7 and 14 days, TGF-β induced the expression of vascular smooth muscle cells markers such as VSM-actin, SM22α, Myocardin and Myosin heavy chain with respect to control cells (a p<0.001 vs. control cells). Images are representative of three experiments.</p