Fibrin and Fibrinogen Play Complex Opposing Roles in Metastasis

Fibrin(ogen) has been identified as a major determinant of metastatic potential. However, it remains to be determined whether fibrinogen, fibrin polymer, or both is the molecular form of the protein conferring metastatic modifying activity. Here, metastatic potential was compared in C57BL/6-derived control mice (Fga^WT), mice with fibrinogen deficiency (Fga^-/-), and mutant mice carrying a form of fibrinogen locked in a nonpolymerizable state due to mutation of the native α chain thrombin cleavage site (Fga^EK). We first performed experimental metastasis assays using the C57BL/6-derived tumor cell lines B16-BL6 melanoma and Lewis lung carcinoma (LLC). Tumor cells were injected into the lateral tail vein, allowing for interrogation of the metastatic process after the tumor cells enter the circulation. Consistent with previous reports, Fga^-/- mice displayed significantly reduced metastatic potential relative to Fga^WT mice for both cell types. Fga^EK mice developed significantly fewer metastases than Fga^WT mice, but significantly more than Fga^-/- mice. We next performed spontaneous metastasis assays by injecting LLC cells into the dorsal subcutis, followed by surgical resection of the primary tumor 12 days later. Spontaneous pulmonary metastases were enumerated 2 weeks after tumor resection. Consistent with previous results, fibrinogen genotype had no impact on primary tumor growth. In this model, Fga^-/- mice had significantly fewer pulmonary metastases than Fib^WT mice. However, unlike what was observed with experimental metastasis, Fga^EK mice developed similar numbers of pulmonary metastases to Fga^WT mice. We hypothesized that nonpolymerizable fibrinogen in the tumor microenvironment promotes the intravasation of tumor cells, thereby abrogating any prometastatic potential conferred by fibrin polymerization once tumor cells have entered the vasculature. To test this hypothesis, we collected blood (700 μl ) from the right ventricle of GFP-expressing LLC-tumor bearing Fga^WT, Fga^EK, and Fga^-/- mice to culture viable circulating GFP-expressing tumor cells (CTCs). A significantly higher fraction of Fga^EK mice displayed detectable CTCs [12/22 (54.54%)] than Fga^WT [7/26 (26.92%)] or Fga^-/- [1/23 (4.34%)] mice. H&E staining of primary tumors revealed no significant genotype-dependent histological, morphological, or structural differences. Immunostaining of primary tumor tissue for cell proliferation markers (phospho-H3 & Ki67) and immune cell populations (i.e., macrophages, neutrophils and T cells) revealed no significant genotype-dependent differences. Immunofluorescent staining showed similar fibrin(ogen) deposition in Fga^WT and Fga^EK, and as expected no staining in Fib^-/- mice. Notably, analyses of primary tumor vasculature by anti-CD31 staining revealed a significant increase in both the number and caliber of vessels in tumors derived from Fga^EK mice relative to Fga^WT and Fga^-/- mice. Thus, nonpolymerized fibrinogen in the primary tumor microenvironment appears to increase metastatic potential by driving intravasation of tumor cells through a mechanism linked to enhanced tumor angiogenesis. Collectively, our study finds that fibrin(ogen) plays a multifaceted role in metastasis, promoting the ability of tumor cells to enter the vasculature from a primary tumor, while impeding the early survival of circulating tumor cells once in the vasculature.

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