Fetuin-A triggers the secretion of a novel set of exosomes in detached tumor cells that mediate their adhesion and spreading

Abstract

Our goal in this study was to define the mechanisms by which fetuin-A mediates the adhesion of tumor cells. The data show that in the absence of fetuin-A, detached tumor cells secrete exosomes that contain most of the known exosomal associated proteins but lack the capacity to mediate cellular adhesion. In the presence of fetuin-A, the cells secrete exosomes, which contain, in addition to the other exosomal proteins, fetuin-A, plasminogen and histones. These exosomes mediate adhesion and cell spreading. Plasminogen is a participant in this novel adhesion mechanism. The data suggest that these exosomes play a role in tumor progression.

Figure 1

Cellular adhesion and spreading in the presence of fetuin-A and exosomes. In panel A, cells were allowed to adhere to microtiter wells in absence (SFM) or presence of fetuin-A (0.5 mg/ml), BSA Exo (20 µg/ml) and FetA exo (20 µg/ml). In B, cells in panel A, were fixed, stained and the absorbance determined.

Fig. 2

Secretion of GFP-CD-63 was monitored in live cells. The detached cells were either incubated with fetuin-A (0.5 mg/ml) without (control) or with BAPTA-AM on attached cells as described in Materials and Methods. The cells were photographed at the indicated time points.

Fig. 3

The secretion of exosomes as a function of intracelllar calcium ion concentration. In panel A, intracellular [Ca²⁺] increases determined by fura-2 AM as extracellular calcium and fetuin-A levels were adjusted were recorded as described in Materials and Methods. In panel B, exosomal secretion by detached cells incubated in the presence of BSA and fetuin-A and 2 mM elxtracellular calcium was determined by NanosightTM. Of note is the decreased secretion in the presence of BAPTA-AM.

Fig. 4

Proposed model for the fetuin-A mediated adhesion. Only FetA Exo whose secretion is mediated by fetuin-A and contain plasminogen and histones (open small spheres) mediate adhesion and preading. Exosomes secreted in the presence of BSA (control) represented by filled small spheres, do not mediate adhesion and cell spreading. Supplemental movie depicting exosomal secretion.