The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions.

BackgroundWhile programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.MethodsBlood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+ and (CK)−CTCs (CD45−, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.ResultsCTCs were detected in 20/25 (80 %) patients, inclusive of CK+ CTCs (13/25, 52 %), CK−CTCs (14/25, 56 %), CK+ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+ CTCs; 4 of these patients had exclusively CK−/CD45−/PD-L1+ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45−CTC burden and low burden of apoptotic CTCs had worse overall survival.ConclusionsCTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+ and CK−CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2758-3) contains supplementary material, which is available to authorized users.

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Mesenchymal stem cells (MSCs) form part of tumor stroma, and are typically considered to be pro-tumorigenic, promoting continuing tumor growth and metastasis. Here, we describe a mechanism by which MSCs may be anti-tumorigenic, through inhibition of breast cancer cell migration and invasion, an important part of metastasis. MDA-MB-231 and T47D cells were co-cultured in a Transwell insert above MSCs or MSC conditioned media and their migration or invasion through Matrigel measured. The conditioned media was found to inhibit breast cancer cell movement. TIMP-1 and TIMP-2, inhibitors of matrix metalloproteinases (MMPs), were identified as candidates for this inhibition and enhanced secretion of MMPs was not sufficient to counter the anti-migratory effects of TIMP expression. Inhibition of TIMP activity showed that TIMP-1 and TIMP-2 together were largely responsible for the reduction of migration and invasion by MSCs. Therefore, MSCs may play anti-tumorigenic, anti-metastatic roles in tumor development, with the overall outcome dependent upon the balance of pro-and anti-tumorigenic molecules secreted.

Oncolytic adenoviral mutants have considerable activity in ovarian cancer. However, the mechanisms by which they induce cell death remain uncertain. dl922-947, which contains a 24 bp deletion in E1A CR2, is more potent than both E1A wild-type adenoviruses and the E1B-55K deletion mutant dl1520 (Onyx-015). We investigated the mode of death induced by three E1A CR2-deleted replicating adenoviruses in models of ovarian cancer and also the importance of E3 11.6 (adenovirus death protein) in determining this mode of death. Ovarian cancer cells were infected with dl922-947 (E3 11.6+) and dlCR2 (E3 11.6−). We also generated dlCR2 tSmac, which also encodes the gene for processed Smac/DIABLO. Classical apoptosis does not occur in adenoviral cell death and there is no role for mitochondria. Expression of Smac/DIABLO does not enhance cytotoxicity nor increase apoptotic features. A role for cathepsins and lysosomal membrane permeability was excluded. Autophagy is induced, but is not the mode of death and may act as a cell survival mechanism. There is no evidence of pure necrosis, while the presence of E3 11.6 does not modulate the mode or extent of cell death. Thus, E1A CR2-deleted oncolytic adenoviral cytotoxicity in ovarian cancer may define a novel mode of programmed cell death.