Redefined complement C3c structures have significant increase in the plasma of ovarian cancer patients.

Abstract

Background: Ovarian cancer (OC) is the third among the most common gynecological cancers. Effective biomarkers are required for OC as in the case of other cancers. Therefore, here we explored whether plasma proteolytic products could serve as potential biomarkers.

Methods: We devised a platform that incorporates CyDye labeling, macroporous reversed-phase liquid chromatography, reducing/non-reducing SDS-PAGE, and fluorescence imaging. Paired preoperative and postoperative plasma samples from four patients were used to screen for possible proteolytic changes. For identified difference proteins, liquid chromatography-tandem mass spectrometry was used to analyze the protein digests using various proteases. Plasma samples from 33 healthy controls and 85 patients with OC were examined using enzyme-linked immunosorbent assay.

Results: Our analyses revealed that the circulating complement C3 derivative was present only in the diseased state. This 145-kDa species, under non-reducing conditions, could split into 72-, 39-, and 29-kDa fragments upon reduction, reminiscent of the C3c structure. While confirming the C3c identity, mass spectrometric analyses showed multiple C-terminal ends in the C3c α'1 fragment, which were utilized differently among patients with OC. Various ends were also observed in serum samples prepared using different complement activators, thus redefining C3c as a mixture of multiple molecular entities. ELISA assay targeting only canonical C3c demonstrated a strong correlation between increased plasma levels and the occurrence and progression of OC.

Conclusion: Our findings suggest that plasma proteolysis during complement deactivation is explicitly involved in ovarian tumorigenesis and the associated protein changes may aid in developing next-generation cancer biomarkers.