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Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis-compared to the high circulating tumor-cell numbers (CTCs)-appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid-size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light-scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC-association with in vivo spheroids. We propose that the mechanism involves a portable 'micro-niche' in spheroids that may support CSC-survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression. [ABSTRACT FROM AUTHOR]

Background: Serum concentration of fetuin A/α2HS-glycoprotein (AHSG) is a good indicator of liver cell function and 1-month mortality in patients with alcoholic liver cirrhosis and liver cancer. We intended to determine whether decreased serum AHSG levels are associated with long-term mortality and whether the follow-up of serum AHSG levels can add to the predictive value of the Child-Pugh (CP) and MELD scores. Methods: We determined serum AHSG concentrations in 89 patients by radial immunodiffusion. Samples were taken at the time of enrolment and in the 1st, 3rd, 6th, and the 12th month thereafter. Results: Forty-one patients died during the 1-year follow-up period, 37 of them had liver failure. Data of these patients were analysed further. Deceased patients had lower baseline AHSG levels than the 52 patients who survived (293 ± 77 vs. 490 ± 106 µg/ml, mean ± SD, p < 0.001). Of all laboratory parameters serum AHSG level, CP and ME D scores showed the greatest difference between deceased and survived patients. The cutoff AHSG level 365 µg/ml could differentiate between deceased and survived patients (AUC: 0.937 ± 0.025, p < 0.001, sensitivity: 0.865, specificity: 0.942) better than the MELD score of 20 (AUC: 0.739 ± 0.052, p < 0.001, sensitivity: 0.595, specificity: 0.729). Initial AHSG concentrations < 365 µg/ml were associated with high mortality rate (91.4%, relative risk: 9.874, 95% C.I.: 4.258—22.898, p < 0.001) compared to those with = 365 µg/ml (9.3%). Fourteen out of these 37 fatalities occurred during the first month of observation. During months 1—12 low AHSG oncentration proved to be a strong indicator of mortality (relative risk: 9.257, 95% C.I.: 3.945-21.724, p < 0.001). Multiple logistic regression analysis indicated that decrease of serum AHSG concentration was independent of all variables that differed between survived and deceased patients during univariate analysis. Mu ltivariate analysis showed that correlation of low serum AHSG levels with mortality was stronger than that with CP and MELD scores. Patients with AHSG < 365 µg/ml had significantly shortened survival both in groups with MELD < 20 and MELD ≥ 20 (p < 0.0001 and p = 0.0014, respectively). Conclusion: Serum AHSG concentration is a reliable and sensitive indicator of 1-year mortality in patients with alcoholic liver cirrhosis that compares well to the predictive value of CP score and may further improve that of MELD score. [ABSTRACT FROM AUTHOR]

Examines the three-dimensional structure of Asp189Ser trypsin mutant. Modeling of the crystal structure; Comparison of the structure of the mutant to those of wild-type trypsin and chymotrypsin; Accessible interface areas of trypsin and chymotrypsin/ogen dimers.