Studying The Effect Of MDSC Recruited By HMGB1on The Peritoneal Implantation Of Colorectal Cancer After Surgery

Aims:Colorectal cancer has been the third incidence of cancer and the third cause of tumor death. Surgery remains the only treatment that has the possibility to completely cure colorectal tumor. However, the postoperative recurrence of colorectal tumor is a urgent issue needing to be addressed by clinical researchers. When surgery is used to treat cancer, itself is a trauma to body. Surgical trauma induces immune suppression through various mechanisms and immunosuppression is closely associated with tumor progression. The effect of surgical trauma in colorectal cancer therapy on the local recurrence of colorectal tumor in peritoneal cavity is remained to be known. This study was aimed at uncovering the contribution of surgical trauma to the local implantation in peritoneal cavity by a model of animal tumor surgery.Methods:Abdominal operation was made as the model of surgery trauma in colorectal tumor ectomy. Abdominal exudation was collected at24h after surgery, and detected by ELISA kit for HMGB1level. Exogenous HMGB1was injected into peritoneal cavity of mice one time per day for three days and then peritoneal lavage was collected. The ratio of MDSC to CD45positive cells and the number of MDSC in lavage were measure by flow cytometer. CT26cell was cultured and injected subcutaneously in mice which worked as tumor loading model. Two weeks later, implanted tumors were excised and surgical trauma in peritoneal cavity was made which mimiced operation for the abdominal tumor. HMGB1was blocked with HMGB1Box-A and MDSC was deleted with gemcitabine. The peritoneal lavage was collected and detected by flow cytometer for the percent of MDSC in CD45positive cells and the quantity of MDSC in the lavage. CT26cell was injected into different group mice including HMGB1blocking group, gemcitabine deleting group, mere surgery group and control group. Two weeks later, the tumor metastatic score was evaluated. Cells in the peritoneal lavage from HMGB1blocking group, gemcitabine deleting group, mere surgery group and control group were collected and detected by flow cytometer using DAF-FM and DCFH-DA probe for the NOS activity and ROS level respectively. CT26cell was injected into peritoneal cavity after surgery trauma followed by NOS inhibitor (L-NAME) and ROS scavenger (Tiron) injection at one time per day for three times. Two weeks later, the peritoneal metastatic score was evaluated.Results:The level of HMGB1in exudate from normal mice with surgery is4237.67ng/ml, and the concentration of HMGB1for tumor bearing mice with surgery is3300.67ng/ml. For the mice without surgery, the concentration is53.97ng/ml. Control mice had small percent of MDSC in CD45positive cells and small quantity of MDSC in lml lavage, corresponding to2%and2.4×10^3.100ng/ml HMGB1treated mice had10%MDSC in CD45positive cells and3.9×10^4MDSC in lml lavage and1000ng/ml HMGB1treated mice had30%MDSC in CD45positive cells and1×10^5MDSC in lml lavage. Both of them were higher than control group (p＜0.05). HMGB1Box-A treated mice had12%MDSC in CD45positive cells and0.75×10^6MDSC in lml lavage, while mere surgery mice had73%MDSC in CD45positive cells and6.0×10^6/ml lavage. Gemcitabine treated mice had28%MDSC in CD45positive cells and1.3×10^6/ml lavage. Tumor bearing mice without surgery had2.3%MDSC in CD45positive cells and2.0×10^3/ml lavage. The metastasis score showed that the total parietal peritoneal score for control group, HMGB1blocking group, gemcitabine deleting group and mere surgery group were2.0±1.0,5.0±1.0,7.3±3.0and10.4±3.5respectively, the difference in groups is significant (p=0.008). Total peritoneal score control group, HMGB1blocking group, gemcitabine deleting group and mere surgery group were6.7±1.5,11.0±1.0,13.5±3.8and18.8±3.5respectively. The difference in groups is significant (p=0.013). The result for NOS activity showed that MDSC in mere surgery group had higher fluorescence signal of DAF-FM DA compared with control group while both of gemcitabine and HMGB1Box-A groups had lower fluorescence signal compared with surgery group. For the ROS level, MDSC in surgery group, gemcitabine group and control group had similar fluorescence signal of DCFH-DA, while MDSC in HMGB1group had significant lower fluorescence signal than that in surgery group. However, MDSC in HMGB1group also had significant lower fluorescence signal than that in control group. When treated with NOS inhibitor and ROS scavenger, mice in surgery group, Tiron group and L-NAME group had total parietal peritoneal metastasis score corresponding to8.5±1.3,6.3±3.7and4.8±1.3respectively, and there was no significant difference in the groups (p=0.134). But, total peritoneal metastasis score for the three groups were16.3±1.0,12.3±3.7and9.7±1.5respectively, and there was significant difference in the groups (p=0.011). Meanwhile, the maximum diameter for the implanted tumor in three groups were0.7±0.1cm,0.5±0.1cm and0.5±0.1cm respectively, and there was significant difference in the groups (p=0.044).Conclusion:We used the model of animal tumor surgery to show that abdominal surgical trauma induce HMGB1which recruits large number of MDSC into peritoneal cavity. Blocking HMGB1could inhibit recruitment of MDSC and alleviate peritoneal implantation of colorectal cancer cell. The recruited MDSC produce NO to promote the form and growth of CT26implantation and ROS to favor the growth of CT26implantation.