Identification Of GEBP11 Targeting To Vasculature Of Gastric Cancer And Its Inhibition Of Angiogenesis

【Background】Angiogenesis and vasculature play important roles in the growth and metastasis of solid tumors. The progress, character and density of microvasculature are related to growth, invasion, metastatic and prognosis of tumor. Microvascular diagnosis and anti-vascular therapy have become an intensive studies field in cancer research. However, the means to detect tumor vasculature and anti-vascular drugs are not satisfactory. This could be explained by the deficiency of molecules targeting to tumor vasculature. It was found that tumor vessels had characteristics different from normal vessels, which would provide molecules targeting possibility for diagnosis and therapeutics for these heterogeneity molecules in tumor vasculatures. Now MVD count is the popular used methods to estimate the vasculatures in tumor, which, however, cannot evaluate the functions of them. Molecular imaging technology developed in the last several years provides the possibility to do it. And the radiation therapeutics targeting the receptors in tumor vasculatures hopes to promote anti-vascular therapy. These two means rely on efficient targeting molecules. In our previous works, we have found a peptide GEBP11 targeting to the vascular endothelial cells in gastric cancer by panning phage library.【Objectives】1. To identify the ability of GEBP11 to target the vasculature of gastric cancer in vivo.. 2. To make the isotope probes of GEBP11 and perform SPECT imaging and targeting therapy of gastric cancer in vivo.. 3. To identify the effects of GEBP11 on angiogenesis in gastric cancer and approach the possible mechanism.【Methods】1. The blue phage plaque formation assay and competitive binding assay in vivo. were performed to investigate the homing specificity of the IN11 phage which displayed the peptide GEBP11. 2. Immunofluorescent staining was used to study the binding specificity of synthesized peptide GEBP11. 3. Immunocytochemical or immunofluorescent staining was performed to identify the location and internalization of GEBP11 on Co-HUVECs. 4. Immunohistofluorescent staining was used to identify the binding specificity of GEBP11 in gastric cancer tissues. 5. GEBP11 was labeled with ⁹⁹Tc^mO₄^- using direct labeling method, and was labeled with ¹³¹I using NBS labeling method. Then the labeled peptides were validated for radiochemical yield, specific activity, and in vitro. stability. 6. The bioactivity of ⁹⁹Tc^m-GEBP11 or ¹³¹I-GEBP11 was validated by cell receptor autoradiography in cultured HUVECs. 7. Receptor binding assay was performed in vitro. to analyze quantitatively the binding specificity and affinity of GEBP11 to HUVEC, the receptor intensity on HUVEC, and the difference of affinity of GEBP11 binding to Co-HUVEC and HUVEC. 8. The radioactivity of all organs of nude mice injected with ¹³¹I-GEBP11 was determined and the radioactivity of all organs per g (%ID/g) was calculated to analyze the specific distribution of ¹³¹I-GEBP11. 9. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with ⁹⁹Tc^m-GEBP11 was obtained 0.5-24 h after being injected to identify the targeting ability of labeled peptide to tumor tissues. 10. MTT assay on HUVECs and antitumor assay in nude mice bearing tumor xenografts of human gastric cancer were performed to evaluate the effects of ¹³¹I-GEBP11 on HUVECs and xenografts. 11. Immunohistochemical staining was used for MVD counting in tumor. 12. H.E. staining, blood cells analysis and blood biochemical indicator analysis were performed to investigate the injury of liver and bone marrow. 13. Tube formation assay in matrigel, CAM angiogenesis assay and angionenesis induced by matrigel in mice were performed to identify the effects of GEBP11 on angiogenesis. 14. The cellular mechanisms of angiogenesis inhibition of GEBP11 were clarified by proliferation assay, cellular cycle and apoptosis analysis, invasion and migration assay and adherency assay. 15. The differential expression genes in Co-HUVECs treated by GEBP11 or not were screened by microarray.【Results】1. The binding ability of GEBP11 targeting to gastric cancer vasculature in vivo. and its binding characteristicsThe titer of IN11 phage recovered from tumor tissue was higher than that of control phage or IN11 phage but from control tissue. GEBP11 peptide inhibited homing effect of the IN11 phage to tumor tissue in nude mice bearing human gastric cancer xenografts, and the inhibition intensity was related to the concentrate of GEBP11. By immunohistofluorescent staining, GEBP11 was co-located with anti-Fâ…§antibody in tumor tissue. However, URP or GEBP11 in control tissues was not co-located with anti-Fâ…§antibody.Immunocytochemistry or immunofluorescence microscopy results showed that GEBP11 was stained on the membrane and perinuclear cytoplasm of Co-HUVECs, but not in HUVECs, GES cells, SGC7901 cells and AGS cells. We also found that GEBP11 peptide was abundant on membrane of ECs at 4℃, and they would be internalized into cytoplasm at 37℃. And the immunohistofluorescence microscopy results suggested that GEBP11 was co-located on the human gastric cancer tissues with anti-CD31 antibody, while URP or GEBP11 on the chronic gastritis tissues was not co-located with anti-CD31 antibody.2. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with ⁹⁹Tc^m-GEBP11By direct labeling GEBP11 with ⁹⁹Tc^mO₄^-, we acquired high radiolabeling efficiency at 90-98%, as well as high specific activity beyond 100 Ci/mmol. in vitro. stability test indicated that ⁹⁹Tc^m-GEBP11 is fine in vitro. stability. By using NBS labeling method, we acquired high radiolabeling efficiency at 90% or more, as well as high specific activity beyond 10 Ci/mmol with ¹³¹I-GEBP11 being fine stability either. Receptor autoradiography showed that there were more obvious silver particles on Co-HUVECs than on HUVECs, which indicated that ⁹⁹Tc^m-GEBP11 or ¹³¹I-GEBP11 has fine biological activity. Receptor binding assay in vitro. showed that the binding of GEBP11 by Co-HUVECs was higher than that of HUVECs. The binding constant of ⁹⁹Tc^m-GEBP11 was calculated by Scatchard analysis. The Kd value of Co-HUVECs and HUVECs were determined to be 1.972 nM, 2.489 nM. The number of binding sites for the labeled peptide (receptor density) was estimated as 5.7×105 per Co-HUVEC and 3.3×105 per HUVEC, respectively.Biodistribution data of ¹³¹I-GEBP11 in nude mice bearing tumour xenografts of human gastric carcinoma showed that the radiotracer exhibited a quick decrease in radioactivity over time in blood and primary organs. Highest activity concentration was observed in kidneys. After 2 h p.i., the tumor radioactivity was higher than that in most of other organs; After 48h p.i., tumor accumulation in the tumour xenografts was 15 times higher than that in the intestine. The tumor/non-tumor ratios steadily increased over time, which showed that ¹³¹I-GEBP11 had the specifical targeting ability to tumor tissues in vivo.. Gamma camera images of nude mice bearing tumor xenografts of human gastric carcinoma injected with ⁹⁹Tc^m-GEBP11 showed the tumors could be visualized as early as 12 h and the activity was higher than that of heart until 24 h. The most clearly visualized imaging appeared at 18-24 h. Compared to ⁹⁹Tc^m-GEBP11, the radioactivity of tumor in nude mice injected with ⁹⁹Tc^m-URP was constantly lower than that of heart.3. ¹³¹I-GEBP11 inhibited the growth of xenografts in nude miceThe cell kill assay in vitro. showed that ¹³¹I-GEBP11 could inhibit the growth of ECs at a radioactivity dependent manner. Na¹³¹I had no inhibition on ECs. GEBP11 began to inhibit the ECs's growth when the concentrate of GEBP11 reached 10μg/ml. Antitumor assay in vivo. showed that the tumor control rate of eADM was highest at 64.9%, and that of ¹³¹I-GEBP11 was second at 56.3%. Compared with eADM and ¹³¹I-GEBP11, Na¹³¹I and GEBP11 had no inhibition effects on tumor. By life span analysis, ¹³¹I-GEBP11, eADM and GEBP11 groups could prolong the life span. PLT decrease and liver injure occurred in therapy groups with ¹³¹I-GEBP11, which was lower than that in eADM groups. Immunohistochemical staining showed that MVD counts in tumor in ¹³¹I-GEBP11 and GEBP11 groups were lower than that in the NS group.4. GEBP11 was identified to have ability to inhibit angiogenesisBy tube formation assay in matrigel, CAM angiogenesis assay and angionenesis induced by matrigel in mice, GEBP11 was identified to have ability in inhibiting angiogenesis. Proliferation assay, cellular cycle and apoptosis analysis, invasion and migration assay and adherency assay showed that GEBP11 could inhibit the proliferation of Co-HUVECs and HUVECs, induce the apoptosis of ECs, but not alter the cell cycle of ECs. Additionally, GEBP11 appeared to inhibit the ECM degradation, migration and adhere of ECs.The total RNA of HUVECs, Co-HUVECs and GEBP11-treated Co-HUVECs were extracted. The quality of extracted RNA was evaluated by agarose electrophoresis and analysis of Lab-on-chip. After microarray hybridization and data normalization, more than 30000 gene spots were detected. There were 1202 down-regulated genes and 2104 up-regulated genes in Co-HUVECs treated by GEBP11. And there were 579 down-regulated genes and 194 up-regulated genes in Co-HUVECs vs. HUVECs.【Conclusions】1. GEBP11 peptide has the ability in targeting to gastric cancer vasculature in vivo., and its binding to the receptor on EC membrane promotes its internalization ability into cytoplasm.2. The SPECT imaging of ⁹⁹Tc^m-GEBP11 can show the tumor mass clearly and ¹³¹I-GEBP11 has anti-tumor effect with weak toxicant and secondary effect, which is the foundation for them to develop nuclide probe or radiotherapeutics drugs targeting to tumor vasculature.3. GEBP11 can inhibit angiogenesis, which comes true probably through its inhibition effects on the proliferation, invasion, migration and adherence of ECs. GEBP11 induces the changed expression of many genes, which provides important clew for approaching the molecular mechanism of GEBP11 inhibiting angiogenesis.