Heparanase regulates secretion, composition, and function of tumor cell-derived exosomes

Abstract

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.

FIGURE 1.

Heparanase enhances the amount of exosomes secreted by tumor cells. A, CAG human myeloma cells were transfected with the cDNA for human heparanase (HPSE-high cells) or control vector (HPSE-low cells), and the amount of exosome protein accumulated over 42 h in the cell medium was quantified by BCA protein assay. In addition, exosome protein levels were measured following the addition of rHPSE to HPSE-low cells. *, p < 0.05 versus level of exosome protein in HPSE-low cells. B, characterization of exosomes. Micrographs from electron microscopy of negative stained particles (left panel, upper micrograph) or cryo-electron microscopy (lower panel) were of the size (30–120 nm) and shape consistent with their identity as exosomes (bar = 100 nm). Cryo-electron microscopy also demonstrated that the exosomes isolated from the HPSE-high and HPSE-low cells were similar in size (see Footnote 3), Right panel, Western blots of proteins (flotillin-1, clathrin heavy chain, and CD63) present in exosome preparations excluded by an iodixanol cushion. Each lane is loaded with material purified from an equivalent volume of conditioned medium. Thus, the much higher levels of the exosome markers seen in medium conditioned by CAG HPSE-high cells as compared with medium conditioned by HPSE-low cells indicate that HPSE-high cells are secreting substantially more exosomes. Pro-heparanase (65 kDa) was also detected in exosomes from HPSE-high cells. C, left panel, ARH-77 human lymphoblastoid cells also exhibited enhanced exosome secretion following transfection with the cDNA for heparanase. Right panel, exosome secretion by the human breast carcinoma cell line MDA-MB-231 was enhanced in response to the addition of 125 ng/ml recombinant heparanase. *, p < 0.05. D, left panel, decrease in exosome secretion following treatment of CAG HPSE-high with Hep III. *, p < 0.05. Right panel, a robust enhancement of exosome secretion is dependent on the active form of the heparanase enzyme. CAG cells transfected with a cDNA coding for mutated, enzymatically inactive forms of heparanase (M225 and M343) secrete low levels of exosomes as compared with cells transfected with the cDNA coding for the active form of heparanase (HPSE-high). *, p < 0.01 as compared with M225, M343 and HPSE-low cells. Error bars in panels A, C, and D indicate ± S.D.

FIGURE 2.

Exosomes from heparanase-high cells have altered composition and regulate the behavior of tumor and host cells. A, ELISA quantification of levels of syndecan-1 (SDC1), VEGF, and HGF present in exosomes isolated from conditioned medium of CAG cells expressing high (H) or low (L) levels of heparanase. Results from each ELISA assay are mean values from three different exosome preparations ± S.D. p < 0.01 for each of the proteins quantified. B, 100 μg of exosomes isolated from HPSE-high or HPSE-low cell conditioned medium were added to HPSE-low cells growing on fibronectin-coated wells. Following overnight incubation, the cells were stained with phalloidin and photographed, and the percentage of spread cells was determined. Bar = 200 μm. Results shown are representative panels from two different experiments using two different exosome preparations. The apparent reduction in cell numbers in photos of wells in which exosomes were added (left and middle panel) is due to the fact that exosomes also cause some cell aggregation. These aggregates remain suspended during the assay, therefore resulting in fewer cells attaching to the dish. C, 8 μg of exosomes purified from medium conditioned by HPSE-high or HPSE-low cells were added to endothelial cells, and the number of cells that invaded through Matrigel-coated chambers overnight was determined. Data are mean ± S.D. of three independent experiments. p < 0.01 for HPSE-high versus HPSE-low.