Fibrin clot shields drive clonal selection of cancer cells: Procoagulant properties and proliferative survival of clot embedded cancer cells. a novel model of tumor microenvironment remodeling

Abstract Introduction: Cancer cells (CaCe) expressing Tissue Factor (TF) initiate thrombin generation and fibrin formation, contributing to cancer-associated hypercoagulability. The resulting fibrin clot shield (FCS) functions both as a protective barrier against therapeutic agents and as a scaffold facilitating CaCe migration. Our objective was to isolate and characterize cancer cells that migrate into the FCS and to determine involvement of a selective process. Materials and Methods: Highly procoagulant pancreatic CaCe (BxPC3) and invasive, highly procoagulant breast CaCe (MDA-MB-231) were cultured in RPMI-1640 medium supplemented with 10% human platelet-poor plasma (PPP) to induce FCS formation (Chi Mai Thromb Res 2024). Mechanical disruption of the clot enabled the isolation of CaCe embedded within the FCS, hereafter referred to as “clot cancer cells” (clot-CaCe). These clot-CaCe, as well as the remaining “paternal CaCe” (CaCe remaining adherent to the wells after clot removal), were subsequently cultured separately in RPMI-1640. BXPC3 and MDA-MB-231 CaCe cultured in conventional RPMI-1640 medium without plasma or coagulation activation to serve as the “control” condition. Cell viability and proliferation were assessed using the crystal violet assay. The procoagulant activity of the cancer cells was evaluated using the calibrated automated thrombogram (CAT® from Diagnostica Stago), as previously described (Tran et al Thromb Res 2024). Scanning electron microscopy (SEM) was used to analyze FCS structure and CaCe invasion within the fibrin network (method described in Tran et al Thromb Res 2024). Results: Clot-CaCe embedded in the fibrin network were successfully isolated and subsequently cultured. BxPC3 clot-CaCe showed higher proliferative capacity than their paternal adherent counterparts. Both BxPC3 and MDA-MB-231 clot-CaCe retained thrombin-generating capacity comparable to that of the parental CaCe. Moreover, both clot- and parental BxPC3 cells and MDA-MB-231 cells induced fibrin network formation when cultured in the presence of PPP. When clot-CaCe or parental-CaCe were exposed to normal PPP, they induced thrombin generation to a similar extent, with no significant difference compared to control CaCe. The SEM analysis showed that the clot-CaCe migrated into the FCS similarly to the paternal and control CaCe populations. However, fibrin networks generated by clot-CaCe exhibited a looser architecture, characterized by thicker fibers, larger pores, and fewer intersections compared to those formed by paternal adherent cells or control CaCe. Conclusion: We developed a novel methodology to study cancer cell behavior within fibrin networks formed through their inherent procoagulant activity. This study provides, for the first time, evidence that CaCe embedded within the fibrin clot network remain viable and preserve their proliferative and procoagulant potential to a similar degree as their progenitors. Moreover, clot-CaCe are capable of generating new fibrin networks. The resulting fibrin appears structurally softer. This observation supports the hypothesis that clot-embedded cancer cells may represent a distinct clone derived from the parental CaCe population. The newly formed fibrin network, in turn, serves as a scaffold supporting further CaCe migration and the formation of new colonies. Taken together, the data presented herein suggest that the fibrin clot network may promote selective survival and clonal expansion of cancer cells, pointing to a potentially novel mechanism of clonal selection and preservation within the hostile tumor microenvironment. The fibrin clot shield may serve as a protective barrier, shielding cancer cells from immune surveillance and the cytotoxic effects of targeted or cell-based anticancer therapies. Overall, this model provides a valuable tool to investigate cancer-associated hypercoagulability, thrombosis, disease recurrence, and therapeutic resistance. References: Tran et al. The procoagulant signature of cancer cells drives fibrin network formation in the tumor microenvironment and impacts its quality: Implications in cancer cell migration and the resistance to anticancer agents. Thromb Res. 2024;238:172–183. doi:10.1016/j.thromres.2024.04.015