Abstract

Previous studies established that the platelet/fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (NK) cells. However, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. It also remains to be determined if the link between fibrin(ogen) and innate tumor immunity is relevant to tumor growth in some contexts, or is strictly limited to early events important in metastasis.

To test the hypothesis that fibrin(ogen) promotes metastasis by distinct mechanisms linked to specific structure/function interactions with several cell types, we performed experimental metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. Expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)-inflammatory cell interactions mediated by αMβ2 in metastasis. To directly determine the importance of thrombin-mediated fibrin polymerization in metastasis, we analyzed metastatic potential in FibAEK mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a germ-line mutation in the Aα chain thrombin cleavage site. Metastatic potential in FibAEK mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymer formation in this process. However, the FibAEK mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains some significant prometastatic properties. In order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and FibγΔ5 mice, carrying a form of fibrinogen lacking the γ chain binding motif for the platelet integrin αIIbβ3. Surprisingly, this mutation had no impact on metastatic potential. Given the established importance of platelets in metastasis, these findings suggest that fibrin is capable of platelet stabilization through mechanism(s) independent of this αIIbβ3 binding motif.

Fibrin(ogen) appears to be important in metastasis across multiple tumor types, but its role in tumor growth is context dependent. Previous studies established that fibrin(ogen) promotes the growth of colonic adenocarcinoma in mice. To determine if fibrin(ogen) is mechanistically coupled to NK cell functions in this context, we depleted NK cells in cohorts of Fib-/- and control mice with an anti-NK1.1 antibody prior to subcutaneous inoculation of MC38 cells (a C57Bl/6-derived colonic adenocarcinoma). Depletion of NK cells had no impact on tumor growth in Fib+ mice. In mice with intact NK cells (i.e., inoculated with a nonspecific IgG), fibrinogen deficiency significantly limited tumor growth. However, depletion of NK cells resulted in a restoration of tumor growth in Fib-/- mice that was comparable to that observed in Fib+ mice.

Together, these studies suggest that both fibrin and fibrinogen play a significant role in metastasis. In addition to interactions with platelets, fibrin(ogen) appears to support metastasis through leukocyte interactions mediated by αMβ2. These studies also reveal an important context where fibrin(ogen) promotes tumor growth via mechanisms coupled to NK cell functions, opening the door to the possibility that targeting these interactions could provide a means to limit the progression of established tumors.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.