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The role of neutrophils in tumour biology is largely unresolved. Recently, independent studies indicated either neutrophil extracellular traps (NETs) or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, their individual or combined role in colonic adenocarcinoma is still unexplored. Colectomy tissue specimens and variable number of draining lymph nodes were obtained from ten patients with adenocarcinoma of the colon. NETs deposition and neutrophil presence as well as TF expression were examined by immunostaining. The effect of NETs on cancer cell growth was studied in in vitro co-cultures of Caco-2 cell line and acute myeloid leukemia primary cells. Proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry. TF-bearing NETs and neutrophil localization were prominent in tumour sections and the respective metastatic lymph nodes. Interestingly, neutrophil infiltration and NETs concentration were gradually reduced from the tumour mass to the distal margin. The in vitro-generated NETs impeded growth of cancer cell cultures by inducing apoptosis and/or inhibiting proliferation. These data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth.

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a ‘metastasis-like’ phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell’s migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Inflammation is a hallmark of colorectal cancer (CRC). Neutrophils are well-known mediators in tumor biology but their role in solid tumors, including CRC, was redefined by neutrophil extracellular traps (NETs). Given that it was recently demonstrated that platelet-derived polyP primes neutrophils to release NETs, we examined surgical specimens from CRC to investigate the presence of polyP, as a possible NET inducer. Biopsies with adenomas, hyperplastic polyps, inflammatory bowel disease and healthy colon tissues were used as controls. In all cases, the presence of polyP was apparent, with the main source of polyP being the mast cells. In all CRC and all adenomas with high-grade dysplasia, a substantial number of mast cells, more than 50%, co-expressed intracellularly polyP with CD68 surface antigen (CD68+), but this was not the case in the other examined disorders. PolyP-expressing mast cells were detected in close proximity with tumor cells and neutrophils, suggesting polyP expression by CD68+ mast cells among the stimuli which prime neutrophils to release NETs, in CRC. Moreover, the detection of CD68+ polyP-expressing mast cells could represent a potential prognostic marker in colorectal adenomas and/or carcinomas.

Uncoupling protein 1 （UCP1） is a proton transporter/channel residing on the inner mitochondrial membrane and is involved in cellular heat production. Using immunohistochemistry, we investigated the expression of UCP1 and UCP3 in a series of 98 patients with non-small cell lung cancer （NSCLC） treated with surgery. Expression patterns were correlated with histopathological variables, prognosis, and the expression of enzymes/proteins related to cell metabolism. Bronchial epithelium did not express UCPl or UCP3, while alveolar cells strongly expressed UCP1. In tumors, strong expression of UCP1 and UCP3 was recorded in 43/98 （43.8%） and 27/98 （27.6%） cases, respectively. UCP1 was significantly associated with squamous cell histology （P = 0.05）, whilst UCP3 was more frequently overexpressed in large cell carcinomas （P = 0.08）, and was inversely related to necrosis （P - 0.009）. In linear regression analysis, UCP1 was directly related to markers of glycolysis [hexokinase （HXKII） and phosphofructokinase （PFK1）] and anaerobic glucose metabolism [pyruvate dehydrogenase kinase （PDK1） and lactate dehydrogenase （LDH5）]. UCP3 was directly linked with a glucose transporter （GLUT2）, monocarboxylate transporter （MCT2）, glycolysis markers （PFK1 and aldolase）, and with the phosphorylation of pyruvate dehydrogenase （pPDH）. Kaplan-Meier survival analysis showed that UCP3 was significantly related to poor prognosis in squarnous cell carcinomas （P = 0.04）. UCP 1 and UCP3 are overexpressed in a large subgroup of non-small cell lung tumors and their expression coincides with increased glucose absorption, intensified glycolysis, and anaerobic glucose usage. Whether UCPs are targets for therapeutic interventions in lung cancer is a hypothesis that demands further investigation.

AimsThe microtubule-associated protein 1 light chain 3 (LC3A) is an essential component of the autophagic vacuoles, forming a reliable marker of autophagic activity. In a previous study, the authors showed that LC3A immunohistochemistry renders three patterns of autophagic expression in breast carcinomas: diffuse cytoplasmic, perinuclear and ‘stone-like’ intracellular structures (SLS), each with a distinct prognostic relevance.MethodsTumour tissues from 155 patients with stage IIA–III colorectal adenocarcinomas, treated with surgery alone, were assessed immunohistochemically for LC3A. Median values were used as cut-off points to separate groups into low and high autophagic activity. Associations with prognosis and with lactate dehydrogenase-5 (LDH5) were sought.ResultsHigh SLS counts were associated with metastases and poor prognosis, while the prominence of the perinuclear pattern was linked to localised disease and good prognosis. The cytoplasmic pattern was irrelevant. Furthermore, patients with increased SLS numbers, but suppressed perinuclear expression, were associated with LDH5 overexpression and had an extremely poor prognosis (3-year survival 16.5%). The prognosis improved considerably when high SLS counts were accompanied by intense perinuclear expression (3-year survival 67%) and were optimal when SLS numbers dropped below median values, irrespective of perinuclear status (3-year survival 94–100%). Multivariate analysis showed that SLS and perinuclear patterns were independent predictors of death events.ConclusionsPerinuclear LC3A accumulation in colorectal tumour cells is a marker of good prognosis, presumably reflecting a basal autophagic activity. An abnormal or excessive autophagic response, as indicated by increased numbers of SLS, is linked to metastasis and poor prognosis.

The mechanism of Amifostine (WR-2721) mediated radioprotection is poorly understood. The effects of amifostine on human basal metabolism, mouse liver metabolism and on normal and tumor hepatic cells were studied. Indirect calorimetric canopy tests showed significant reductions in oxygen consumption and of carbon dioxide emission in cancer patients receiving amifostine. Glucose levels significantly decreased and lactate levels increased in patient venous blood. Although amifostine in vitro did not inhibit the activity of the prolyl-hydroxylase PHD2, experiments with mouse liver showed that on a short timescale WR-1065 induced expression of the Hypoxia Inducible Factor HIF1α, lactate dehydrogenase LDH5, glucose transporter GLUT2, phosphorylated pyruvate dehydrogenase pPDH and PDH-kinase. This effect was confirmed on normal mouse NCTC hepatocytes, but not on hepatoma cells. A sharp reduction of acetyl-CoA and ATP levels in NCTC cells indicated reduced mitochondrial usage of pyruvate. Transient changes of mitochondrial membrane potential and reactive oxygen species ROS production were evident. Amifostine selectively protects NCTC cells against radiation, whilst HepG2 neoplastic cells are sensitized. The radiation protection was correlates with HIF levels. These findings shed new light on the mechanism of amifostine cytoprotection and encourage clinical research with this agent for the treatment of primary and metastatic liver cancer.

Background The rheumatoid nodule is the most common extra-articular manifestation of rheumatoid arthritis. When present, it is readily identified in conventional hematoxylin and eosin sections. Case presentation We report a case with several rheumatoid nodules in a thyroid gland of a 33-year-old Greek woman with a 3-year history of rheumatoid arthritis treated with methotrexate, after having total thyroidectomy for hypothyroidism. Conclusion To the best of our knowledge, this is the first time that rheumatoid nodules have been encountered in the thyroid gland.

Objectives: We assessed the expression and the prognostic role of lactate dehydrogenase 5 (LDH5, the major LDH isoenzyme involved in anaerobic glycolysis) in patients with squamous cell head and neck cancer (SCHNC). Methods: LDH5 was assessed immunohistochemically in whole tissue sections from 141 patients with SCHNC. Of these, 102 were subjected to surgery with (90 patients) or without (12 patients) postoperative radiotherapy (group A), while 39 patients were treated with radical radiotherapy (group B). Results: Mixed nuclear/cytoplasmic LDH5 expression was detected in 72.5% of group A and 61.5% of group B patients. This was significantly related to T4-stage (p = 0.04) and hypoxia-inducible factor-1α (HIF-1α) expression (p = 0.002). In group A, high LDH5 was linked with poorer distant metastasis-free survival (p = 0.01) and disease-specific overall survival (OS; p = 0.009). In multivariate analysis, LDH5 (p = 0.002) and HIF-1α (p = 0.01) were independently linked with distant metastasis. LDH5 was also linked with death events (p = 0.005). In group B, high LDH5 expression was significantly associated with poorer local relapse-free survival (p = 0.009) and OS (p = 0.01). In multivariate analysis, only T stage was a significant predictor of death events (p = 0.04). Conclusions: LDH5 is highly expressed in SCHNC and is linked with local relapse, survival and distant metastasis, suggesting that LDH5 is a marker of radioresistance and a target for therapeutic interventions.