Fig 2: Anti-ENO1 mAb HuL227 reduces inflammation-enhanced chemotaxis and migration in androgen-independent prostate cancer PC-3 and DU145 cells. In CCL2-chemotaxis assay, PC-3 (A) or DU145 (B) were pretreated with TNFa for 4 hours and allowed to migrate toward chemoattractant CCL2 in the absence or presence with indicated concentrations of HuL227, 10 µg/mL human IgG1, and 10 mmol/L TXA for an additional 18 hours. Inhibition of migration (%) compared with the nontreatment was described. In migration assay, PC-3 (C) or DU145 (D) were treated with TGFß and allowed to migrate in the absence or presence with indicated concentrations of HuL227, 10 µg/mL hIgG1, and 10 mmol/L TXA for 18 hours. ##, P < 0.01; ###, P < 0.001 compared with untreated cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with cytokine-stimulated cells. E, Putative action mechanisms of HuL227 in prostate cancer. ENO1 was a molecular target commonly shared by prostate cancer cells, monocytes, osteoclasts, and endothelium cells in advanced prostate cancer TME. All these ENO1-expressing cells are crucial for the progressing of advanced prostate cancer to bone metastasis. HuL227 was hypothesized to inhibit the migration and invasion of prostate cancer cells from prostate to blood vessel and ultimately to bone. The induction effects of essential mediators, such as CCL2, TGFß, and TNFa, to drive this migration and invasion process could also be inhibited by HuL227. The angiogenesis crucial for tumor growth can be inhibited by HuL227 in prostate cancer as well.

Fig 3: Anti-ENO1 mAb HuL227 attenuates tumor growth and angiogenesis in a prostate cancer PC-3 subcutaneous xenograft model. Male nude mice were subcutaneously implanted with PC-3 for 3 days, randomized, and dosed with 30 mg/kg HuL227 or vehicle control twice a week. Tumor volumes (A) were measured twice a week. Data were shown as the mean volume ± SEM from n = 7 mice. At day 23, tumors were harvested for measurement of weight (B) and for immunofluorescence (IF) staining to detect the expression of tumor-infiltrating inflammatory monocyte marker CCR2 (C) and endothelial cell marker CD31 (E). DAPI staining (blue) was used to detect nuclei. Scale bar, 100 µm. D, CCR2 staining was quantified as the number of CCR2+ cells per 40X magnified field. F, CD31 staining was quantified as percentage of CD31+ area per region of interest. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with vehicle control.

Fig 4: Anti-ENO1 mAb HuL227 inhibits VEGF-A–induced plasmin activation and tube formation in HUVEC cells. After VEGF-A stimulation for 4 hours, surface ENO1 (green) was examined by IF staining (A). DAPI staining (blue) was used to detect nuclei. Scale bar, 10 µm. B, Plasmin activation was determined by fibrinolytic assay after VEGF-A stimulation for 4 hours in the absence or presence of indicated concentrations of control hIgG1, HuL227, and TXA. Inhibition of plasmin activity (%) compared with the VEGF-A stimulation only was described. C, HUVEC cells grown on matri-gel were treated with VEGF-A for 24 hours to examine the formation of capillary-like tubes which was quantified as manual counting of nodes. D, Inhibition of tube formation (%) compared with the VEGF-A stimulation only was described. Results were shown as mean ± SD. ##P < 0.01 compared with untreated cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with VEGF-A–treated cells.

Fig 5: Anti-ENO1 mAb HuL227 attenuates tumor growth and osteoclast activation in prostate cancer PC-3 intratibial xenograft model. Male NOD-SCID mice were intratibially implanted with PC-3 cells for 3 days, randomized, and dosed with 30 mg/kg anti-ENO1 mAb HuL227 or vehicle control twice a week. A, Dashed outlines in the presentative H&E staining indicated the boundary of PC-3 tumor growth in the bone lesion on day 28. Arrow heads indicated cartilages of the bone growth plate. B, MMP activity was measured on day 27 by using in vivo tracer of MMPs substrate to semiquantify tumor volume in bone. Data were shown as the mean fluorescence intensity (pmol) ± SEM from n = 5 mice. Representative images for each group were shown. C, Osteoclast activity (cathepsin K) was measured on day 15 and 22 by using in vivo tracer of cathepsin K substrate. Data were shown as the mean fluorescence intensity (pmol) ± SEM from n = 8 mice, except sham n = 1 for background. ###, P < 0.001; *, P < 0.05. Cat K, cathepsin K.