Abstract 5117: Investigating the role of secreted plasminogen receptors in the establishment of the pre-metastatic breast cancer niche

Abstract Introduction: Breast cancer is the most common cancer in women, with 25% of patients presentingsecondary tumors at diagnosis. Metastasis, responsible for over 90% of cancer deaths, involves cancer cellsescaping the primary tumor to colonize distant organs. This process requires the preparation of target sites, known as premetastatic niches (PMNs). PMNs undergo structural remodeling facilitated by cancer cell-secreted factors, including the protease plasmin (Pm), which aids in cancer cell escape by degrading tissuebarriers. Cancer cells utilize plasminogen receptors (PgRs) to convert the blood protein plasminogen (Plg)into Pm. Our research has shown that conditioned media (CM) from breast cancer cells can activate Plg toPm, suggesting the presence of PgRs in these secretions. While cell surface PgRs are established in tumorprogression, the roles of secreted PgRs within the breast cancer secretome remains undefined. Additionally, the involvement of the Plg/Pm system in PMN modulation is unexplored. We have found that CM frommalignant BCCs has higher Pm-activating potential than from non-malignant BCCs. Protein purification ofCM identified several soluble PgR-active fractions, with phosphoglycerate kinase-1 (PGK-1) emerging asa key PgR candidate. Methods: CM was isolated from highly and weakly metastatic BCC lines (MDA MB 231 vs. MCF7 & 4T1vs. 67NR). Soluble proteins were separated by size- and charge-chromatography, and fractions wereassayed for PgR activity, measured through their ability to stimulate tPA-dependent Plg-to-Pm conversion.Active fractions were pooled and identified via mass spectrometry. Results: CM from MDA MB 231 and 4T1 cells exhibited higher PgR activity and distinct chromatographicprofiles compared to MCF7 and 67NR cells. Size-exclusion chromatography revealed multiple PgR activitypeaks. Mass spectrometry identified novel PgR candidates, including glycolytic enzymes and extracellularmatrix components, PGK-1, a glycolytic enzyme, was identified in a PgR activity peak, with recombinantPGK-1 showing significant in vitro Pm generation, confirming its PgR role. Conclusions: PGK-1 is secreted by breast cancer cells and has Pm-generating potential. Futureexperiments will validate PGK-1 and other proteins as PgRs and clarify their roles in metastasis. Citation Format: Gillian Okura. Investigating the role of secreted plasminogen receptors in the establishment of the pre-metastatic breast cancer niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5117.