Karol Serwin

Innate Immunity Derived Factors as External Modulators of the CXCL12 - CXCR4 Axis and Their Role in Stem Cell Homing and Mobilization

The α-chemokine CXCL12 (stromal derived factor-1; SDF-1) and its corresponding GαI protein-coupled CXCR4 receptor axis play an important role in retention of hematopoietic stem progenitor cells (HSPCs) in bone marrow (BM) stem cell niches. CXCL12 has also been identified as a strong chemoattractant for HSPCs and implicated both in homing of HSPCs to BM after transplantation and in egress of these cells from BM into peripheral blood (PB). However, since CXCL12, as a peptide, is highly susceptible to degradation by proteolytic enzymes, its real biological availability in biological fluids may be somewhat limited. In this review, we will present data demonstrating that the CXCL12-CXCR4 axis is positively modulated by innate immunity-derived several external factors, ensuring that even low (near threshold) doses of CXCL12 still exert a robust chemotactic influence on HSPCs.

Clinical analysis of selected complement-derived molecules in human adipose tissue

BackgroundIt has been suggested that action of complement cascade [CC]-derived anaphylatoxins/molecules may represent a missing link between obesity and metabolic disorders. However, to date, the direct biochemical/immunomodulatory composition of the human AT environment remains poorly understood. In this study, we examined plasma and AT (subcutaneous and visceral/omental) levels of selected CC-derived anaphylatoxins/molecules, and adipsin as well as verified their associations with immune and stem cells chemoattractant - stromal-derived factor-1 (SDF-1).MethodsA total of 70 (35 subcutaneous and 35 omental) AT samples were obtained from patients undergoing elective surgery. Plasma and AT-derived interstitial fluid levels of C3a, C5a, C5b-9/membrane attack complex (MAC), complement factor D (adipsin) were measured using ELISA.ResultsAT levels of all examined substances were significantly lower than the corresponding levels in the plasma (in all cases P < 0.0000001). Moreover, in subcutaneous AT, robust C3a and adipsin concentrations were observed, whereas high levels of C5b-9/MAC were detected in the visceral depots. In addition, we established the correlations between analyzed molecular substances and body composition, BMI and/or the adiposity index of the examined patients.ConclusionsOur study demonstrated for the first time that significantly reduced levels of complement-derived molecules were present in human AT than in the peripheral blood, and that these factors are associated with the metabolic status of examined individuals. Moreover, in human AT, various associations between complement-derived molecules and SDF-1 levels exist.

Plasma and Adipose Tissue Levels of Selected Growth/Inhibitory Factors, Proteolytic Enzymes and Sphingosine-1-Phosphate in Humans

Recent studies have shown that adipose tissue (AT), while implicated in orchestrating the sophisticated process termed “immunometabolism,” may also serve as a potential niche for various bone marrow-derived (stem) cells. However, at present, the direct biochemical and immunomodulatory composition of the human AT environment has not been studied. Several substances that might play a crucial role in regulating stem cell migration and/or homing to AT, have been implicated, namely, hepatocyte/vascular endothelial growth factor (VEGF/HGF), leukemia inhibitory factor (LIF), and sphingosine-1-phosphate (SIP). Therefore, we examined and compared the AT concentrations of these substances between plasma, subcutaneous, and omental AT samples derived from 35 generally healthy subjects. VEGF, HGF, LIF, and metalloproteinases (MMP)-2 and MMP9 levels were measured using ELISA, and S1P concentrations were analyzed using reverse-phase high performance liquid chromatography. We found that AT levels of analyzed growth/inhibitory factors were generally comparable (VEGF and LIF) or even higher (HGF) than the corresponding levels in the peripheral blood, particularly in overweight/obese subjects. In subcutaneous AT, significantly lower VEGF and LIF concentrations were observed, and these were accompanied by higher MMP levels. No depot-specific differences in S1P concentrations were found in all examined groups. Moreover, we established several associations between analyzed molecular substances and body composition, BMI, or adiposity index of the examined patients. In conclusion, our study revealed that human AT possesses relatively high levels of selected growth/inhibitory factors and of chemoattractants involved in the regulation of stem cell trafficking, and these factors are associated with the metabolic status of an individual. Further studies are needed to clearly establish the role of these factors in the regulation of bone marrow-derived (stem) cell homeostasis and homing in human AT.

Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50–100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.

Abstract 1698: Novel evidence that blood plasma vitronectin is a major chemoattractant for cancer cells and its pro-migratory activity is suppressed/chaperoned after binding to fibrinogen

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background. One of the crucial problems with cancer therapy is migration of cancer cells that leave primary tumor and migrate to vital organs where they form metastases. Throughout the years several factors have been identified that direct metastatic process including growth factors, chemokines, bioactive lipids or extracellular nucleotides. To our surprise however, we noticed that highly diluted plasma (1%) possess remarkable chemokinetic activity against several cancer cell lines that highly exceeds those observed for optimal doses of chemokines (e.g. SDF-1) and growth factors (e.g. HGF/SF), that are considered as a main metastatic factors present in plasma. Aim of the study. Based on this observation our aim was to identify the “factor/s” responsible for this remarkable chemokinetic activity of normal diluted 1% plasma. Experimental strategies. We employed several human cancer cell lines and different dilutions of normal human, murine and bovine plasma and serum in Transwell migration assays, adhesion and cell signaling studies. We tested effect of heat inactivation, protease exposure, dialysis and molecular filtration on chemotactic activity of plasma. We also used gel filtration followed by hydrophobic interaction chromatography (HIC) to identified plasma fractions with the most potent chemotactic activity that were further analyze using MassSpec. Results. We found that remarkable chemokinetic activity of highly diluted 1% plasma against malignant cells, rapidly decreased at higher plasma concentration (>5%). Our initial characterization studies revealed that this “activity” is sensitive to proteolytic treatment, is not removed from plasma by dialysis and is temperature sensitive. The similar effect has been observed for diluted 1% serum however chemotactic responsiveness of serum was maintained with its higher concentrations. Based on this we hypothesized possible involvement of inhibitory effect of fibrinogen, which was subsequently confirmed in experiments where fibrinogen was removed from plasma or it was added to serum. Finally, gel filtration followed by HIC and MasSpec analysis allow us to identify several possible candidates that were further tested in in vitro experiments. These studies revealed that vitronectin (VTN) is a main factor responsible for observed chemotactic response and that its effect can be inhibited by fibrinogen. Conclusions. Our data indicate that VTN present in normal plasma is a main migration inducing factor responsible for metastasis of malignant cells and that VTN is more potent chemoattractant than already known pro-metastatic chemokines or growth factors. Pro-migratory effect of VTN is neutralized/chaperoned by fibrinogen what may explain preference in migration of tumor cells into lymphatic vessels and body cavities, where concentration of fibrinogen is low. Citation Format: Gabriela Schneider, Nichola C. Garbett, Ewa Bryndza, Agata Poniewierska-Baran, Michael L. Merchant, Karol Serwin, Zachariah P. Sellers, Barbara Dolegowska, Mariusz Z. Ratajczak. Novel evidence that blood plasma vitronectin is a major chemoattractant for cancer cells and its pro-migratory activity is suppressed/chaperoned after binding to fibrinogen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1698.