IL (interleukin)-6, an established growth factor for multiple myeloma cells, induces myeloma therapy resistance, but the resistance mechanisms remain unclear. The present study determines the role of IL-6 in re-establishing intracellular redox homoeostasis in the context of myeloma therapy. IL-6 treatment increased myeloma cell resistance to agents that induce oxidative stress, including IR (ionizing radiation) and Dex (dexamethasone). Relative to IR alone, myeloma cells treated with IL-6 plus IR demonstrated reduced annexin/propidium iodide staining, caspase 3 activation, PARP [poly(ADP-ribose) polymerase] cleavage and mitochondrial membrane depolarization with increased clonogenic survival. IL-6 combined with IR or Dex increased early intracellular pro-oxidant levels that were causally related to activation of NF-κB (nuclear factor κB) as determined by the ability of N-acetylcysteine to suppress both pro-oxidant levels and NF-κB activation. In myeloma cells, upon combination with hydrogen peroxide treatment, relative to TNF (tumour necrosis factor)-α, IL-6 induced an early perturbation in reduced glutathione level and increased NF-κB-dependent MnSOD (manganese superoxide dismutase) expression. Furthermore, knockdown of MnSOD suppressed the IL-6-induced myeloma cell resistance to radiation. MitoSOX Red staining showed that IL-6 treatment attenuated late mitochondrial oxidant production in irradiated myeloma cells. The present study provides evidence that increases in MnSOD expression mediate IL-6-induced resistance to Dex and radiation in myeloma cells. The results of the present study indicate that inhibition of antioxidant pathways could enhance myeloma cell responses to radiotherapy and/or chemotherapy.

The ␣5␤1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi-and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for ␣5␤1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and ␣5␤1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of ␤1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated ␣5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of ␣5␤1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.Adhesion molecules present on the endothelial cell (EC) 2 surface are vital regulators of vascular homeostasis and angiogenesis. Integrins are heterodimeric cell-adhesion receptors for extracellular matrix proteins, and are composed of noncovalently bound ␣ and ␤ subunits (1). During embryonic vascular development, as well as during tumor angiogenesis, the extracellular matrix protein fibronectin serves as an adhesive support and signals through ␣5␤1 integrin to regulate the spreading, migration, and contractility of ECs (1-3). Integrins do not have intrinsic enzymatic activity; their ability to transduce signals depends on recruitment of cytoplasmic linker and signaling proteins, and on the assembly of focal adhesions. Hence the processes that regulate integrin turnover at the cell surface may have implications for the regulation of angiogenesis in the context of therapies.The levels of ␣5␤1 integrin on the cell surface are maintained by recycling of endocytosed complexes back to the plasma membrane (PM). Internalization of the ␣5␤1 integrin complexes occurs via both clathrin-dependent and clathrin-independent endocytic pathways (4). Subsequently, these complexes traffic to early endosomes (EEs) and then to the perinuclear recycling compartment, from which they are recycled to the PM (5-7). This transport requires Rab11 and the activity of PKB/GSK3b (5). Binding of fibronectin to the ␣5␤1 integrin triggers endocytosis that does not involve integrin recycling; in this case the integrin complexes move from the EEs into the multivesicular endosomes for degradation (8). This phenomenon highlights the importance of ␣5␤1 integrin trafficking at the EE compartme...

BackgroundImmuno-oncology and cancer immunotherapies are areas of intense research. The numbers and locations of CD8+ tumor-infiltrating lymphocytes (TILs) are important measures of the immune response to cancer with prognostic, pharmacodynamic, and predictive potential. We describe the development, validation, and application of advanced image analysis methods to characterize multiple immunohistochemistry-derived CD8 parameters in clinical and nonclinical tumor tissues.MethodsCommercial resection tumors from nine cancer types, and paired screening/on-drug biopsies of non–small-cell lung carcinoma (NSCLC) patients enrolled in a phase 1/2 clinical trial investigating the PD-L1 antibody therapy durvalumab (NCT01693562), were immunostained for CD8. Additional NCT01693562 samples were immunostained with a CD8/PD-L1 dual immunohistochemistry assay. Whole-slide scanning was performed, tumor regions were annotated by a pathologist, and images were analyzed with customized algorithms using Definiens Developer XD software. Validation of image analysis data used cell-by-cell comparison to pathologist scoring across a range of CD8+ TIL densities of all nine cancers, relying primarily on 95% confidence in having at least moderate agreement regarding Lin concordance correlation coefficient (CCC = 0.88–0.99, CCC_lower = 0.65–0.96).ResultsWe found substantial variability in CD8+ TILs between individual patients and across the nine types of human cancer. Diffuse large B-cell lymphoma had several-fold more CD8+ TILs than some other cancers. TIL densities were significantly higher in the invasive margin versus tumor center for carcinomas of head and neck, kidney and pancreas, and NSCLC; the reverse was true only for prostate cancer. In paired patient biopsies, there were significantly increased CD8+ TILs 6 weeks after onset of durvalumab therapy (mean of 365 cells/mm2 over baseline; P = 0.009), consistent with immune activation. Image analysis accurately enumerated CD8+ TILs in PD-L1+ regions of lung tumors using the dual assay and also measured elongate CD8+ lymphocytes which constituted a fraction of overall TILs.ConclusionsValidated image analysis accurately enumerates CD8+ TILs, permitting comparisons of CD8 parameters among tumor regions, individual patients, and cancer types. It also enables the more complex digital solutions needed to better understand cancer immunity, like analysis of multiplex immunohistochemistry and spatial evaluation of the various components comprising the tumor microenvironment.Trial registrationClinicalTrials.gov identifier: NCT01693562.Study code: CD-ON-MEDI4736–1108.Interventional study (ongoing but not currently recruiting).Actual study start date: August 29, 2012.Primary completion date: June 23, 2017 (final data collection date for primary outcome measure).Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0326-x) contains supplementary material, which is available to authorized users.

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Senescence is a mechanism associated with aging that alters tissue regeneration by depleting the stem cell pool. Chronic obstructive pulmonary disease (COPD) displays hallmarks of senescence, including a diminished stem cell population. DNA damage from cigarette smoke (CS) induces senescence via the p16 pathway. This study evaluated the contribution of p16 to CS-associated lung pathologies. p16 expression was prominent in human COPD lungs compared with normal subjects. CS induces impaired pulmonary function, emphysema, and increased alveolar epithelial cell (AECII) senescence in wild-type mice, whereas CS-exposed p16 −/− mice exhibit normal pulmonary function, reduced emphysema, diminished AECII senescence, and increased pro-growth IGF1 signaling, suggesting that improved lung function in p16 −/− mice was due to increased alveolar progenitor cell proliferation. In conclusion, our study suggests that targeting senescence may facilitate alveolar regeneration in COPD emphysema by promoting IGF1 proliferative signaling.