Date of Award
Master of Science
John Biggerstaff, Robert Mee
Although conventional therapies for metastatic cancers have made significant progress in recent years, they are relatively nonspecific and have many deleterious side-effects. Recently, novel therapies, including adoptive cellular immune therapies have had sporadic, but spectacular success in cancers such as malignant melanoma and renal cell carcinoma: tumors in which an immune response has been demonstrated. However, other physiological mechanisms, such as blood coagulation inhibit the immune response against cancers. Our previous work has shown that one of these coagulation proteins, soluble fibrin (sFn), inhibits unstimulated and activated lymphocyte adherence to tumor cells by blocking leukocyte integrin (CD11a/CD18) binding to tumor cell CD54, suggesting that sFn is an immunosuppressive agent in cancer. Since these receptors are also involved in lymphocyte/endothelial cell adherence and diapedesis (a necessary step in the immune response to cancer), it was hypothesized that sFn inhibits these functions, and that blockade of this inhibition using specific peptides would restore these immune responses. Fluorescently labeled lymphocytes and Interleukin-2 activated lymphocytes (LAK cells) were incubated with sFn (or its components; fibrinogen, Gly-Pro-Arg-Pro, or thrombin) in the presence or absence of specific blocking peptides. Lymphocyte and LAK cell adherence to endothelial cell monolayers was measured by perfusion at physiological shear rates in a microscope-mounted closed perfusion chamber, followed by image analysis using Image Pro Plus software. Diapedesis was measured by detection of fluorescence in 24-well microplates following immune cell incubation (18 h) with endothelial cell monolayers grown in transwells. SFn inhibited lymphocyte (54.1 + 11.3 %) and LAK cell (43.9 + 4.4 %) adherence to sFn pretreated endothelial cells, and intermediate values were obtained from sFn pre-treatment of only one cell type. Adherence was restored by peptide mediated blockade of sFn/CD54 binding, but not by CD11b blocking peptides. Diapedesis was also inhibited by sFn (lymphocyte 29.6 + 7.7 %; LAK 12.2 + 4.9 %) and restoration was observed using blocking peptides. These results confirm the stated hypotheses, and if physiologically relevant, suggest that sFn is an etiological agent in tumor growth and metastasis, and that blockade using fibrin specific peptides may enhance the effectiveness of adoptive immunotherapies.
Weidow, Brandy Lee, "The Role of Soluble Fibrin in Lymphocyte and LAK Cell Adherence to and Migration across Vascular Endothelial Cells: Implications for Immunotherapy and Cancer. " Master's Thesis, University of Tennessee, 2007.