A YKL-40-neutralizing antibody blocks tumor angiogenesis and progression: a potential therapeutic agent in cancers

Abstract

Accumulating evidence has indicated that expression levels of YKL-40, a secreted glycoprotein, were elevated in multiple advanced human cancers. Recently, we have identified an angiogenic role of YKL-40 in cancer development. However, blockade of the function of YKL-40, which implicates therapeutic value, has not been explored yet. Our current study sought to establish a monoclonal anti-YKL-40 antibody as a neutralizing antibody for the purpose of blocking tumor angiogenesis and metastasis. A mouse monoclonal anti-YKL-40 antibody (mAY) exhibited specific binding with recombinant YKL-40 and with YKL-40 secreted from osteoblastoma cells MG-63 and brain tumor cells U87. In the functional analysis, we found that mAY inhibited tube formation of microvascular endothelial cells in Matrigel induced by conditioned medium of MG-63 and U87 cells, as well as recombinant YKL-40. mAY also abolished YKL-40-induced activation of the membrane receptor VEGF receptor 2 (Flk-1/KDR) and intracellular signaling mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (Erk) 1 and Erk 2. In addition, mAY enhanced cell death response of U87 line to γ-irradiation through decreased expression of pAKT and AKT and accordingly, abrogated angiogenesis induced by the conditioned medium of U87 cells in which YKL-40 levels were elevated by treatment with γ-irradiation. Furthermore, treatment of xenografted tumor mice with mAY restrained tumor growth, angiogenesis, and progression. Taken together, this study has shown the therapeutic use for the mAY in treatment of tumor angiogenesis and metastasis.

Figure 1. Anti-YKL-40 antibodies recognize both YKL-40 protein secreted from tumor cells and purified recombinant YKL-40

A. Recombinant YKL-40 was generated by a baculoviral system. His-tagged YKL-40 was purified through a Ni-NTA affinity-binding column followed by a desalting PD-10 column. Collected samples were analyzed by immunoblotting with an anti-6xhistidine antibody. B. YKL-40 levels in cell conditioned medium collected from MG-63 cells, U87 cells, and HMVECs as well as recombinant YKL-40 were detected by immunoblotting using rAY and mAY.

Figure 2. YKL-40 siRNA inhibits endothelial cell angiogenesis induced by YKL-40

A. Western blot. Conditioned media from MG-63 expressing siRNA1, siRNA2 and vector control were measured for YKL-40, and cell lysates were used for actin using immunoblotting followed by quantitative analysis. Intensity of YKL-40 and actin was analyzed with NIH ImageJ software and YKL-40 levels were normalized with actin expression. Subsequently, these levels were compared with the basal control arranged as one unit. B. Tube formation. DMEM serum-free medium of MG-63 cells expressing YKL-40 siRNA were collected after 24 hr and transferred to Matrigel. After 16-hr incubation, tubules were imaged and tube density was analyzed quantitatively. n=5. Control represents DMEM serum-free medium and MG-63 control indicates DMEM serum-free conditioned medium of MG-63 control cells. C. Cell migration. Same conditioned medium of MG-63 cells as described above was transferred to the bottom chamber of a transwell in order to test its effects on migration of HMVEC that were loaded onto the top chamber of the well. After 4-hr incubation, migrated cells were fixed and stained. The quantification was shown in the bottom panel. n=4. ^*P<0.05 compared with DMEM serum-free medium as controls, and ⁺P<0.05 compared with MG-63 control or siRNA1. Bars: 100 µm.

Figure 3. mAY blocks tube formation induced by cell conditioned medium containing YKL-40

A. mAY inhibits tube formation induced by both conditioned medium of MG-63 and U87 cells. MG-63 and U87 cells were pretreated with mAY or mIgG (10 µg/ml) for 24 hr and the conditioned medium was transferred to HMVECs for the tube formation. The data were quantified. n=3. B. mAY-inhibited tube formation is dose-dependent. Tube formation of HMVEC was determined in the presence of conditioned medium of U87 cells pretreated with mAY at 5, 10, 20 µg/ml or mIgG at 20 µg/ml for 24 hr. The data were quantified. ^*P<0.05 compared mIgG. n=4. C. mAY blocks tube formation induced by recombinant YKL-40. mAY or mIgG (10 µg/ml) was introduced to serum-free medium of HMVECs in the presence of recombinant YKL-40 from 50–250 ng/ml in Matrigel and quantification of tube formation was displayed. *P and ⁺P<0.05 compared with non-YKL-40-treated controls and corresponding mIgG treatments, respectively. n=4. Bars: 100 µm.

Figure 4. mAY interrupts Flk-1/KDR and MAPK Erk signaling pathways induced by YKL-40

A. mAY blocks YKL-40-induced Flk-1/KDR expression. HMVECs pretreated with serum-free medium overnight were stimulated with 100 ng/ml recombinant protein YKL-40 from 0 to 40 hr or with 0–200 ng/ml YKL-40 for 24 hr. Conditioned medium from U87 cells in the presence or absence of 10 µg/ml mIgG or mAY was transferred to HMVECs for 24 hr as described in Figure 3A. Subsequently, all of the cell lysates were tested for Flk-1/KDR expression by immunoblotting. Flk-1/KDR data were normalized with actin levels and then compared with the basal serum-free level arranged as one unit. n=4, mean ± SE. B. mAY blocks YKL-40-induced tyrosine phosphorylation of Flk-1/KDR. HMVECs were pretreated with serum-free medium overnight in the presence of mAY or mIgG (10 µg/ml) and then directly stimulated with recombinant YKL-40 (100 ng/ml) for 10 min. n=2, mean ± SE. For treatment with cell conditioned medium, HMVECs were pretreated with serum-free medium overnight and then treated for 10 min with U87 cell conditioned medium that was exposed to mAY or mIgG (10 µg/ml) for 24 hr. Half of the cell lysates were immunoprecipitated with an antibody against phosphorylated tyrosine protein (pY20) followed by immunoblotting using an antibody against Flk-1/KDR. The remaining lysates were used directly for immunoblotting against total Flk-1/KDR. IgG of the anti-pY20 antibody was also tested as loading controls. pFlk-1/KDR was normalized with total Flk-1/KDR. n=3, mean ± SE. C. mAY blocks YKL-40-induced MAPK Erk 1 and Erk 2 activation. The same conditions of HMVECs as described in B were collected and immunoblotted with antibodies against pErk 1, Erk 2, and total Erk followed by quantification. pErk 1 and pErk 2 levels were normalized with total Erk 1 and Erk 2. SF indicates serum-free basal DMEM and Con represents DMEM serum-free conditioned medium alone. n=2, mean ± SE.

Figure 5. mAY enhances tumor cell death responses to γ-irradiation and decreases endothelial cell angiogenesis

A. γ–irradiation induced YKL-40 expression. U87 cells were treated with 0, 3, 5, or 10 Gy of γ-irradiation and 48 hr later, cell conditioned medium was subjected to testing YKL-40 levels and cell lysates were examined for actin levels. YKL-40 levels were quantified by normalization with actin. The basal level of YKL-40 without γ-irradiation was set as one unit. n=2, mean ± SE. B. mAY decreases cell viability in response to γ-irradiation. In the upper panel, U87 cells treated with 10 Gy γ-irradiation as described above in the presence of mAY or mIgG (10 µg/ml) were determined for cell viability in cultured condition. Attached and detached cells were quantified under a microscope. In the bottom panel, 96 hr following treatment with 10 Gy γ-irradiation above, U87 cells were assayed for cell death as described in the Method. Red fluorescence indicates dead cells and green fluorescence represents live cells. *P<0.05 compared with the control or mIgG-treated group. n=5. Bars: 50 µm. C. mAY decreases pAKT and AKT levels. U87 cells treated with γ-irradiation for 48 hr as described in B were tested for pAKT (ser473), AKT, PI3K, and actin. pAKT and AKT levels were normalized with actin and subsequently, these levels were compared with their basal expressions without γ-irradiation arranged as one unit. n=2, mean ± SE. D. mAY blocks γ-irradiation-induced angiogenesis. Conditioned medium of U87 cells as described in C was transferred to HMVECs for testing tubules in Matrigel. n=4. *P<0.05 compared with cells without γ-irradiation and ⁺P<0.05 compared with control or mIgG.

Figure 6. mAY inhibits tumor growth, angiogenesis and progression

A. U87 cells were injected subcutaneously into SCID/Beige mice and mAY or mIgG (5 mg/kg body weight) was injected subcutaneously twice a week from week 3. Tumor volume was evaluated, n=5. *P<0.05 compared with corresponding mIgG. B. Tumor samples were collected and subjected to IHC of CD31 staining. Brown color indicates positive staining for blood vessels. Bar: 100 µm. Vessel density was quantified using ImageJ software. *P<0.05 compared with corresponding mIgG. C. Liver was removed from mice and processed for H & E staining. Dark blue cells are tumor cells. Bar: 100 µm.