Study On Antibody-Targeted Therapy For Human Gastric Cancer Stem Cells

PART ⅠSCREENING OF TARGETED ANTIBODY FOR HUMAN GASTRIC CANCER STEM CELLSThe incidence and death of gastric cancer ranked the second in our country. Gastric cancer has already been a terrible threat to human life and health. The prime cause of high mortality of gastric cancer is high proportion of metastasis and recurrence occurred after surgery, radiotherapy and chemotherapy. However, current clinical treatments have little effect on gastric cancer which is high metastasis and recurrence. Recently, it is found that Cancer stem cell(CSC) is the root cause of metastasis,recurrence and drug-resistance. Therefor, treatments of targeted CSC will be a new tragedy to reduce mortality. The study is aimed at screening out mAbs which can show powerful function to gastric stem cells so as to provide preferable precursor candidate of targeted CSC drugs.In the preliminary period, Immunology in mice with human gastric cancer cells which was adopted from human gastric cancer tissues. A 3120 hybridoma clones antibody library was established via hybridoma technique. Based on this library, we screened out 482 mAbs from 3120 mAbs by ELISA on SNU-5 cells and BGC-823 cells. For the sake of acquiring monoclonal antibody targeting human gastric CSC,144 mAbs were screened out from 482 mAbs by ELISA on SNU-5 Sphere cells. The study showed that 144 mAbs may target human gastric CSC. Sphere forming ability inhibition experiments showed that 64 mAbs can effectively reduce sphere forming rate. It turned out that 64 mAbs can not only target human gastric CSC, but also have function of gastric CSC’s metastasis, recurrence and drug-resistance.Sphere forming, ELISA on cells,immunofluorescence technique were used to screen out a functional antibody targeted gastric CSC. This study showed that positive rate of 10 mAbs in Sphere cells was increased.10 mAbs can co-express with CD90 and CD44 in gastric CSC(1.3-31.4 times). It confirmed that all 10 mAbs can target human gastric CSC. Therefor,we screened out 10 mAbs targetd human gastric CSC.PART IISTUDY ON TARGETED MONOCLONAL ANTIBODY 25G5 FOR HUMAN GASTRIC CANCER STEM CELLSStudies on 10 mAbs confirmed that mAb 25G5 expressed high positive rate and enriched in gastric cancer sphere cells(17.1%). The percentage of CD44+ and CD90+ cells population in sphere cells which were also mAb 25G5 positive was the highest among the 10 mAbs. Therefor,mAb 25G5 was screened out to be studied systematially.First, we cultured a lot number of 25G5 and purified 25G5 by Protein G affinity purification.It’s reported that drug-resistance invasive, self-renewal in vitro, and growth of gastric cancer xenograft in vivo is the characteristic of CSC. Cell immunofluorescence showed that antigen recognized by 25G5 co-expression with CD44 and CD90 on cell membrane. Then we applied the FACS to isolate 25G5+ cells for sphere culture, invasion assay, chemosensitivity and tumorigenicity test. The sorted 25G5+ cell presented stem cell-like features, including self-renewal [sphere formation rate(21.4±0.3)% vs(12.3±0.7)%],invasion ability[the number of invasive cells(244.5±13.5)vs(122.4±7.2)], drug-resistance [IC50(0.285μg/ml)vs(0.094μg/ml)] and the same as tumorigenic capacity.All these results demonstrated that 25G5+ cells were cancer stem cells featured with strong self-renewal, invasive and drug resistance.MAb 25G5 can target human gastric CSC.In vitro functional assay showed that mAb 25G5 significantly suppressed the sphere formation ability of SUN-5 cells, with the inhibitory rates being 46.4%(P<0.05). MAb 25G5 significantly suppressed the invasive ability of SNU-5 cells, with the inhibitory rates being 26.6～58.4%(P<0.05), the same as drug-resistance (IC500.341μg/ml～0.153μg/ml). All these experiments confirmed that 25G5 is a functional antibody which can effectively inhibit gastric CSC’s drug-resistance invasive, self-renewal.In summary, this study screened out 64 mAbs targeted human gastric cancer stem cells from 3120 mAbs.10 mAbs were established that can show functions of resistance to CSC. Then 25G5 was screened out to be studied systematically. The results showed that mAb 25G5 is an anti-gastric stem cell monoclonal antibody. At the same time,mAb 25G5 is a functional antiboty that can obviously inhibit gastric CSC’s drug-resistance invasive, self-renewal in vitro, and growth of gastric cancer xenograft in vivo. Antibody 25G5 is a preferable precursor candidate of targeted gastric CSC drugs.