Abstract
Circulating soluble fibrin (sFn) is a marker for ongoing disseminated intravascular coagulation and may have prognostic significance, especially in metastatic cancers. Anti-coagulant therapies have been effective in reducing metastasis in several cancers, but with increased risk of bleeding. The authors have previously demonstrated that soluble fibrin (sFn), which is elevated in many cancer patients, enhances metastasis in an experimental model, and increases platelet/tumor cell adherence by cross-linking platelet aIIbb3 to tumor cell CD54 (a receptor for two of the leukocyte b2 integrins aLb2 and aMb2). sFn also binds to monocyte aMb2 (Mac1), and the peptide sequences of the fibrin(ogen) binding sites for aMb2 and CD54 have recently been identified. It was, therefore, hypothesized that sFn binding to these receptors would result in inhibition of monocyte/tumor cell adherence, and consequently cytotoxicity, which may be reversed by inclusion of blocking peptides. To test this, monocyte adherence to A375 melanoma cells was quantified at physiologically relevant shear rates (35–560 s−1) using a laminar flow perfusion chamber mounted on a Leica DMIRB inverted microscope equipped with a computer controlled digital imaging camera. Effector and target cells were untreated, or incubated with sFn (fibrinogen (Fg), 0.5 mg/ml; fibrin polymerization inhibitor Gly-Pro-Arg-Pro amide (GPRPa), 4 mM; and human thrombin (0.125 U/ml)) in the presence or absence of specific single or combined blocking peptides directed against the sFn binding sites on CD54 (P1) and aMb2 (P2), or the sFn g-chain binding sites for CD54 (P3) and aMb2 (P4). The effect of peptides on thrombin (0.125 U/ml) induced clotting of purified Fg (0.5 mg/ml) was assessed visually. The effect of sFn on monocyte cytotoxicity (effector: target 20:1) against green fluorescent protein (GFP) transfected A375 melanoma cells was measured by GFP release from lysed cells using a Perkin-Elmer Victor-3 96-well fluorescence plate reader. Pre-treatment of tumor cells with sFn significantly (P<0.01) increased monocyte adherence to 68.5 + 0.7% compared to the untreated control (32.9 +1.3%), whereas monocyte pre-treatment had no significant effect (P>0.05) on adherence. However, pre-incubation of both cells with sFn resulted in a significant (P<0.01) inhibition of adherence to 15.95 ± 1.0% (62% inhibition). sFn mediated inhibition of adherence was significantly reduced by pre-treatment of cells with a combination of P1 + P2 (to 22.3 ± 13.8% inhibition; P<0.01), and by pre-incubation of sFn with P3 + P4 (to 9.9 ± 3.6% inhibition; P<0.01). Single peptides blocked to an intermediate level, and controls performed appropriately. Furthermore, blocking peptides did not inhibit thrombin induced clotting of Fg. Pretreatment of both monocytes and A375 cells significantly inhibited specific cytotoxicity by 40% (P < 0.01 compared to untreated cytotoxicity − 28.6 ± 0.7%), and intermediate killing was observed when only one cell type was sFn treated. These results show that sFn incubation with both effector and target cells inhibits both cellular adherence and cytotoxicity by a mechanism involving monocyte aMb2 and tumor cell CD54. Adherence was restored by inclusion of sFn blocking peptides, which did not affect clotting. Clinically, these peptides may be effective therapeutically in reducing sFn mediated immunosuppression and may enhance the immune response to metastasizing cancer cells, without the risk of bleeding problems associated with other therapies.
Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing