| Objective:Lymph node metastasis is the main factor of poor prognosis and death in patients with prostate cancer.In the radical prostatectomy for patients with early prostate cancer.accurate staging depends on enough lymph nodes biopsy,which leads to early and correct postoperative adjuvant treatment.As a new method of intraoperative navigation,near-infrared fluorescence imaging has become more and more attractive,and the specific imaging and surgery targeting prostate specific membrane antigen(PSMA)has become a research hotspot.In this study,to observe the effectiveness and feasibility of fluorescence-assisted surgery in the identification of prostate and lymphoid tissue indocyanine green(ICG)was injected into the prostate to perform pelvic lymphography during the operation,more importantly,to evaluate its role in identifying cancerous lymph nodes.Secondly,a new prostate cancer specific fluorescent probe PSMA-12-IRDye800 CW,which can specifically bind to PSMA was developed to evaluate its preclinical efficacy and safety through cell experiments and animal model experiments.Methods:1.Sixty patients with prostate cancer were divided into two groups: the experimental group received fluorescence assisted robotic radical prostatectomy and lymph node dissection,and the control group received routine robot-assisted radical prostatectomy and lymph node dissection.ICG was injected into the prostate gland by transperineal route before operation,and the control group was treated routinely.The number of lymph node dissection was compared between both groups and the specificity of noncancerous and cancerous lymph node recognition were analyzed.At the same time,the safety and efficacy of the two operations were also compared.2.The specific fluorescent probe of PMSA-617 was prepared by optimizing the ligands of PSMA-12-IRDye800 CW used in clinical nuclear medicine.The effectiveness and specificity of the probe in PSMA expression positive(PSMA+)and PSMA negative(PSMA-)cell lines were observed by fluorescence microscope,its affinity to PSMA antigen was detected by fluorescence ELISA,and its cytotoxicity was evaluated by cell counting kit.The transplant prostate cancer xenografts nude mouse model prostate cancer was established,and the efficacy and specificity of PSMA-12-IRDye800 CW,in PSMA+and PSMA-cell tumor xenografts model in mice were observed by tail vein injection.The high dose probe was used to detect the tolerance,liver and kidney function and body weight of mice was evaluate in order to assess the toxicity of probe in vivo.Results:1.In the first part of the study,30 patients in the study group received transperineal prostate injection,and the fluorescence performed well in the study group which can be distinct from adjacent tissue.No adverse events were see related to ICG,which confirmed its safety.The fluorescence in pelvic lymphoid tissue was observed in all patients in the study group,which was characterized by tubules and lumps.The lymphoid tissues resected according to the fluorescence range were confirmed to be lymphatic vessels and lymph nodes by pathology.The total number of resected lymph nodes in all patients was 927,495 in the study group and 429 in the control group,of which 419 nodes developed fluorescence in the study group with high rate of 74.4%.A total of 40 cancerous lymph nodes were found in all patients,15 nodes in the control group(0.9%),and 25 in the experimental group(1.3%).The results showed that the fluorescent development of lymph nodes could well identify the lymphoid tissue,however,it had poor specificity for cancerous nodes as the fluorescence imaging could not distinguish between benign and malignanc,In terms of surgical effect,the total postoperative urine control rate of the two groups was 96.7%,no difference between the two groups was found;the incidence of lymphatic leakage in the study group was 6.7%(16.7%),and the biochemical recurrence rate was 3.3%(13.3%),both of which was lower than that in the control group,but the difference was not statistically significant.2.In the next part,the specific probe PSMA-12-IRDye800 CW was successfully synthesized,and its purity and physical properties were characterized with high stability.The results of fluorescence microscope showed that the probe could specifically bind to PMSA+LNCap cells and showed good fluorescence in a concentration-dependent manner,but not to PSMA-PC-3 cells.The results of ELISA further showed that the probe had a high affinity for PSMA+ cells with Km=13n M,but did not bind to PSMA-cells.In vivo experiments,it is confirmed that the probe has good specificity for PSMA+ tumor,and can actively concentrate to the tumor with high fluorescence intensity,and has a fast BK,which can be used for tumor-specific imaging.Finally,it was found that high concentration of the probe did not cause imaging on the liver and kidney function of mice,and had no effect on the general condition and weight change,so confirmed that is was a safe compound.Conclusion:Clinical trial conclusions:(1)the application of prostate regional 4-needle transperineal injection of ICG into the lobar glands of both sides of the prostate is safe and effective for intraoperative fluorescence imaging of prostate and lymphoid tissue,and can effectively avoid the disadvantage of leakage of fluorescence pollution in the existing methods.(2)ICG fluorescence-mediated prostate imaging can be used as an auxiliary method to identify the boundary between prostate and bladder neck,especially with prostatic surgery history.(3)the most important finding of this study is that ICG has high sensitivity to lymphoid tissue,which can be used as an auxiliary means to prevent omission in the process of lymph node dissection,and accurately seal the lymphatic vessels during operation to reduce the incidence of lymphatic leakage.Preclinical study of specific fluorescent probe we confirmed that PSMA-12-IRDye800 CW is a fluorescent probe with high affinity to prostate cancer cells,which can specifically transfer to future surgery.And the small molecular ligand probe is easy to synthesize and has high safety.So that a potential drug that can be used in clinical prostate surgery has the value of further transformation to clinical practice. |
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