Cell surface heparan sulfate released by heparanase promotes melanoma cell migration and angiogenesis

Abstract

Heparan sulfate (HS) proteoglycans are essential components of the cell-surface and extracellular matrix (ECM) which provide structural integrity and act as storage depots for growth factors and chemokines, through their HS side chains. Heparanase (HPSE) is the only mammalian endoglycosidase known that cleaves HS, thus contributing to matrix degradation and cell invasion. The enzyme acts as an endo-beta-D-glucuronidase resulting in HS fragments of discrete molecular weight size. Cell-surface HS is known to inhibit or stimulate tumorigenesis depending upon size and composition. We hypothesized that HPSE contributes to melanoma metastasis by generating bioactive HS from the cell-surface to facilitate biological activities of tumor cells as well as tumor microenvironment. We removed cell-surface HS from melanoma (B16B15b) by HPSE treatment and resulting fragments were isolated. Purified cell-surface HS stimulated in vitro B16B15b cell migration but not proliferation, and importantly, enhanced in vivo angiogenesis. Furthermore, melanoma cell-surface HS did not affect in vitro endothelioma cell (b.End3) migration. Our results provide direct evidence that, in addition to remodeling ECM and releasing growth factors and chemokines, HPSE contributes to aggressive phenotype of melanoma by releasing bioactive cell-surface HS fragments which can stimulate melanoma cell migration in vitro and angiogenesis in vivo.

Figure 1. Dose-dependent reduction of cell surface HSGAG with HPSE

A. Murine B16B15b melanoma cells were treated with 0-10,000 ng/ml HPSE. Cell surface HS level was detected by flowcytometry using mAb10E4. HPSE removes cell surface HSGAG in a dose-dependent manner, (green) 0 ng/ml HPSE, (blue) 100 ng/ml HPSE, (purple) 1,000 ng/ml HPSE, (light blue) 10,000 ng/ml HPSE. Appropriate controls were run to account for background staining, (black) no primary antibody control, (solid red) no primary and no secondary antibody control. B. HPSE-degraded cell surface HSGAG profile on silver stain. As expected, HPSE digestion generated HS fragments of about 10 kDa. Various concentrations of untreated HS was run to generate a standard curve for determination of concentration of HSGAG after densitometric analysis. Numbers on the right hand of figure refer to M.W. standards (kDa).

Figure 2

Endothelioma cell migration is not influenced by HPSE-degraded HS but VEGF stimulates wound healing. To study how exogenous addition of HS will influence endothelioma (b.End3) biological activity, we added HPSE-digested melanoma cell surface HS to serum-free endothelioma medium. HS treatment did not stimulate endothelioma cell migration while VEGF did (p< 0.05), as expected. HS, when added along with VEGF, did not augment VEGF response.

Figure 3

HPSE-degraded cell surface HS modulate melanoma cell migration. When HS (1 ng/ml) were added externally to melanoma cells in wound healing assays, there was increased migration compared to control (p< 0.05). Addition of VEGF (50 ng/ml) did not affect migration with or without HS.

Figure 4

HPSE-degraded cell surface HSGAG does not influence melanoma cell proliferation. Proliferation of melanoma cells were assayed by alamarBlue™, a non-toxic dye that monitors the reducing environment of the proliferating cell. Melanoma cell proliferation was monitored every 24 h for 72 h. Exogenous addition of VEGF (50 ng/ml) or melanoma cell surface HS (1 ng/ml) isolated by HPSE treatment did not influence cell proliferation.

Figure 5

HPSE-degraded cell surface HSGAG promotes angiogenesis in vivo. Blood vessel density was assessed by counting vessels within the tumor region in five different sections in each tumor. Tumor sections were photographed using Olympus DP70 camera, Olympus BX45 microscope and saved as JEPG format using DP Manager (Olympus). Tumor areas were measured by counting pixels on ImageJ software (NIH). Pixel counts were converted to mm² to present the number of vessels per unit area. Statistical analyses were done using SAS (Version 9.1.3) in an analysis of variance in a split-plot arrangement of treatments. A. Representative tumor sections from each treatment group (H & E). HPSE-treated cell surface HSGAG induced a significant increase in intratumor blood vessel (arrow) formation in animals compared to mock or HepIII treatment of HPSE-treated cell surface HSGAG (p<0.0001). Hep III treatment renders the HPSE-degraded fragments inactive, hence abolishes their biological activity. Presence of VEGF did not affect angiogenesis in all three groups (p=0.2-0.8). Notably, inside the tumors, absence of blood vessels, thereby lack of nutrition and oxygen led to areas of necrosis. B. Bar graph representation of mean blood vessel density with standard deviation plotted on a log scale. Bars with the same letter are not significantly different from each other.

Figure 5

HPSE-degraded cell surface HSGAG promotes angiogenesis in vivo. Blood vessel density was assessed by counting vessels within the tumor region in five different sections in each tumor. Tumor sections were photographed using Olympus DP70 camera, Olympus BX45 microscope and saved as JEPG format using DP Manager (Olympus). Tumor areas were measured by counting pixels on ImageJ software (NIH). Pixel counts were converted to mm² to present the number of vessels per unit area. Statistical analyses were done using SAS (Version 9.1.3) in an analysis of variance in a split-plot arrangement of treatments. A. Representative tumor sections from each treatment group (H & E). HPSE-treated cell surface HSGAG induced a significant increase in intratumor blood vessel (arrow) formation in animals compared to mock or HepIII treatment of HPSE-treated cell surface HSGAG (p<0.0001). Hep III treatment renders the HPSE-degraded fragments inactive, hence abolishes their biological activity. Presence of VEGF did not affect angiogenesis in all three groups (p=0.2-0.8). Notably, inside the tumors, absence of blood vessels, thereby lack of nutrition and oxygen led to areas of necrosis. B. Bar graph representation of mean blood vessel density with standard deviation plotted on a log scale. Bars with the same letter are not significantly different from each other.