Research Of Common Female Tumors Surgery Guided By The Second Near-infrared Window Fluorescence Imaging

Objective:Breast cancer,cervical cancer and endometrial cancer are the three major malignant tumors in women,seriously threatening female health and life.Surgery is the mainstay of treatment for patients with these tumors,but there are still some challenging problems in these operations.For example,up to 20% of patients after breast-conserving surgery require a second surgical resection due to residual tumor after the primary operation;the iatrogenic injury of the pelvic autonomic nerve during radical hysterectomy leads to postoperative voiding dysfunction(5-76%)in patients with cervical cancer;the accuracy of evaluation of depth of myometrial invasion of endometrial cancer during radical endometrial cancer remains to be improved.To improve the above surgical outcomes,it is necessary to develop a real-time intraoperative technique for visualization of residual tumor,small nerves,and tumor boundaries.The emergence of optical molecular imaging technology has brought new opportunities to solve the above difficulties,especially the second near-infrared window(NIR-Ⅱ,1,000–1,700 nm)fluorescence imaging technology has become an important research direction of surgical navigation with its unique advantages.Therefore,this research focuses on the above key challenges of common female tumors surgery,develops a new type of NIR-Ⅱ fluorescence imaging technique,and conducts basic and clinical application research to explore the clinical value of this technology in gynecological tumor surgery.This research is divided into three parts.The first part focused on preclinical research for development of multifunctional the NIR-Ⅱ fluorescent probe and new strategies to improve breast-conserving surgery outcomes.The second part is to carry out clinical trial for assessment the safety and feasibility of intraoperative NIR-Ⅱ fluorescence imaging with ICG in identifying pelvic autonomic nerves during surgery and to demonstrate the advantages of NIR-Ⅱ fluorescence imaging over NIR-I fluorescence imaging in identification of small nerves.The third part is to carry out clinical trial for assessment the safety and feasibility of intraoperative NIRⅡ fluorescence imaging with ICG in imaging of endometrial cancer and accuracy in determine the myometrial invasion depth.Methods:In the first part,the NIR-Ⅱ organic fluorescent molecule A1 was designed and synthesized with the structure of D1-π-A-D2-R.In order to facilitate biological application,the polyether coated nanoparticles A1 NPs were further prepared and characterized and cell experiments were carried out in vitro.Subsequently,the effect of A1 NPs-based NIRⅡ fluorescence imaging on intraoperative guidance of residual tumor and identification and resection of metastatic lymph nodes was demonstrated by constructing a mouse model of breast cancer in situ.Finally,the effect of A1 NPs-based new integrated strategy on improving breast-conserving surgery was evaluated.In the second part,a prospective open-label clinical study was carried out.10 patients with a cervical cancer diagnosed at stage IB1-ⅡA1 were enrolled in this study.All patients were intravenously injected with ICG at a dose of 5?mg/kg body weight 24 hours before the operation.During the operation,NIR-Ⅱ imaging/NIR-I imaging was performed in vivo to identify the pelvic nerves.After hysterectomy,the nerve tissue was isolated from the broken ends of uterine ligaments for frozen sections to observe the distribution of ICG.In addition,the adverse reactions within two weeks after ICG injections was observed and recorded.In the third part,a prospective open-label clinical study was carried out.8 patients diagnosed with endometrial cancer by preoperative hysteroscopic biopsy were included in this clinical trial.All patients received 5 mg/kg of indocyanine green intravenously 24 hours before surgery.During the operation,the surgeon observed the extent of the lesion and the depth of myometrial invasion after complete removal of the uterus and bilateral appendages,then to determine the extent of lymphatic dissection.Subsequently,NIRⅡ/NIR-I/visible light imaging was performed on the isolated uterus to observe the fluorescence imaging effect of the lesion,and the tissue was sampled,frozen sectioned,and the histopathological type,ICG distribution,and the fluorescence boundary and pathology of the lesion were observed under the microscope.Finally,using the depth of myometrial invasion determined by postoperative pathology as the gold standard,the accuracy of preoperative magnetic resonance imaging(MRI),intraoperative doctors’ naked eyes and NIR-Ⅱ fluorescence imaging in judging the depth of myometrial invasion was compared.Results:In the first part,a multifunctional NIR-Ⅱ fluorescent nanoparticle A1 NPs based on aggregation-induced emission(AIE)was successfully constructed.In vivo experiments indicate that NIR-Ⅱ fluorescence imaging of A1 NPs precisely detect microscopic residual tumor(2 mm in diameter)in the tumor bed and metastatic lymph nodes.More notably,a novel integrated strategy that achieves complete tumor eradication(no local recurrence and metastasis after surgery)is proposed.In the second part,superior hypogastric plexus(SHP),hypogastric nerve(HN),pelvic splanchnic nerves(PSN),and inferior hypogastric plexus and bladder branch(IHP-VF)of9 patients(90.0%)were clearly displayed intraoperative through NIR-Ⅱ fluorescence imaging.The mean fluorescence signal-to-background ratio(SBR)of them is 2.0±0.6,2.0±0.4,1.7±0.2,and 1.6±0.2,respectively.In addition,3 of 8 patients,NIR-Ⅱ imaging and NIR-I imaging was performed in turn to identify obturator nerve(ON),genitofemoral nerve(GN),and SHP nerves,it was found that NIR-Ⅱ imaging is better than NIR-I imaging in visualization small nerves less than 3mm,and the difference is statistically significant(P<0.05).ICG fluorescence was detected in frozen sections of a nerve specimen,and was mainly distributed in axons.One patient was found to have a transient liver injury during follow-up,and no ICG-related adverse reactions were observed in other patients.In the third part,the results showed that ICG-based NIR-Ⅱ fluorescence imaging could visualize endometrial cancer lesions with an average SBR of 4.0±2.4,but could not visualize benign uterine fibroids.At the microscopic level,ICG fluorescence was observed to have obvious co-localization effect with tumor tissue,while there was only weak ICG fluorescence signal in the surrounding normal myometrium.Linear regression analysis showed that the percentage of tumor area and fluorescence intensity was strongly correlated.Depth of myometrial invasion determined by postoperative pathology was taken as the gold standard,the analysis showed that the accuracy of the depth of myometrial invasion determined by NIR-Ⅱ fluorescence imaging reached 100%,which was higher than that of preoperative MR and intraoperative macroscopic observation.Conclusion:This study preliminarily proved: 1.NIR-Ⅱ fluorescence imaging based on A1 NPs could be used to accurately detect and eliminate residual tumors,and the new integrated strategy provides a new idea for reducing incomplete tumor resection in breast-conserving patients;2.The NIR-Ⅱ fluorescence imaging with ICG could display small pelvic autonomic nerves,and showed the dominant position of NIR-Ⅱ fluorescence imaging in displaying small nerves;3.The NIR-Ⅱ fluorescence imaging with ICG could be used in intraoperative visualization of endometrial cancer lesions and borders provides a theoretical basis for using this technique to accurately determine the depth of myometrial invasion.In conclusion,the above studies showed that NIR-Ⅱ fluorescence imaging technology based on novel A1 NPs or ICG can identify residual tumors,small nerves and tumor boundaries,and was expected to improve the outcome of common female tumors surgery.