Application Of Near-infrared Fluorescence Imaging And Lugol’s Iodine Enhanced Micro-CT Imaging In The Evaluation Of Surgical Margin And Lymph Node Status Of Oral Squamous Cell Carcinoma

Oral squamous carcinoma cell was one of the most common malignancies in the head and neck.Despite the improvement of therapeutic method,the 5-year survival rate still less than60%.Locoregional recurrence was the main reason lead to failure treatment.Accurate assessment of OSCC surgical margin and regional lymph node status is an important measure to reduce regional recurrence.Postoperative pathological examination is the golden standard for evaluating the surgical margin and lymph node status of OSCC.However,due to the limitation of sampling,timeliness,workload and other factors,simple pathological examination cannot achieve the best.We need to find new methods to accurate assess the surgical margin and regional lymph node status of OSCC intraoperative/postoperative.With the development of technology,Near-infrared fluorescence imaging and Micro-CT imaging have been applied widely in the medical field.ICG^?-based NIR imaging could locate the primary tumor of lung cancer intraoperative and Micro-CT imaging could evaluate the surgical margin of in vitro specimens of breast cancer and lung cancer.In this study,we aimed to explore the feasibility of evaluating the surgical margin and regional lymph node status of OSCC intraoperative or postoperative by NIR imaging and Micro-CT imaging.One.Evaluation of OSCC surgical margin by ICG^?-based near infrared fluorescence molecular imaging and Lugol’s iodine enhanced Micro-CT imaging[Objective]Accurate assessment of OSCC surgical margin is an important measure to reduce its local recurrence.Due to the failure of traditional pathological examination to evaluate the whole OSCC tumor lesion,the missed diagnosis of positive surgical margin would lead to the local recurrence.This study aims to explore the feasibility of ICG^?-based near infrared fluorescence（NIR）molecular imaging and Lugol’s iodine enhanced Micro-CT imaging in evaluating the surgical margin of OSCC.[Methods]（1）ICG^?-based NIR imaging: Firstly,OSCC tumor implantation model in nude mice has been established to evaluated the feasibility of ICG^?-based NIR molecular imaging in detecting residual tumor tissue resulted by incomplete resection of primary tumor,and to observe the limited tumor volume which could be detected.Secondly,by conducting clinical trials,we have compared the boundary between fluorescence tumor margin and pathological margin.Finally,we further compare the difference of ICG^? uptake/bind between OSCC tumor cell and normal oral epithelium cell.（2）Lugol’s iodine enhanced Micro-CT imaging: On the basis of previous research of rabbit normal tongue tissues Lugol‘s iodine enhanced Micro-CT imaging,human OSCC samples were adopted and imaged via Lugol’s iodine enhanced Micro-CT.The optimal Lugol’s iodine concentration and staining time were determined before clinical application using tissue shrinkage,Micro-CT image quality,and effect on pathological diagnosis as assessment criteria.Next,6 OSCC specimens were used to verify the process feasibility of surgical margin imaging with the optimal parameters.Finally,the possible reason by which Lugol’s iodine could enhance Micro-CT imaging was validated in vitro.[Results]（1）ICG^?-based NIR imaging: by simulating the incomplete resection process of OSCC surgery,we found ICG^?-based NIR molecular imaging technology could successfully detect residual tumor lesions,and the minimum tumor volume that can be detected is up to 0.5 mm³.In the clinical trial,NIR molecular imaging of tumor lesion in vivo,in vitro,and surgical wound showed that this technique could assist the resection of primary tumor.After the operation,the tumor fluorescence boundary was compared with the actual pathological boundary,which showed that the fluorescence boundary of the tumor（181.25 mm²）was larger than the actual pathological boundary（95.09 mm²）（P<0.01）.Cytological experiments showed that OSCC tumor cells could absorb/bind more ICG^? than normal epithelial cells（HSC3 vs SCC9 vs Leu K-1: 0.01 mg/m L: 109.3±8.8 vs 74.3±3.9 vs 31.9±2.3;0.001 mg/m L:87.3±3.4 vs 60.1±0.7 vs 33.5±5.6）.（2）Lugol’s iodine enhanced Micro-CT imaging: Lugol’s iodine staining influenced specimen shrinkage,Micro-CT image quality,and pathological image quality in a concentration/time-dependent manner.After comprehensively considering these indicators,3%Lugol’s iodine staining for 48 h and for 12 h was found to be optimal for rabbit tongue tissues and OSCC samples,respectively.This method could provide a detailed 3D structure of OSCC samples compared with H&E sections.Moreover,tumor and normal tissues could be differentiated by their glycogen content,which has high affinity with Lugol’s iodine.[Conclusion]Both ICG^?-based NIR imaging technology and Lugol’s iodine enhanced Micro-CT imaging technology could effectively assist surgeons in the resection of primary OSCC lesions,which will reduce the occurrence of positive surgical margins and increase the long term survival.ICG^?-based NIR imaging technology has the advantage of real-time and accuracy while Lugol’s iodine enhanced icro-CT imaging technology has the advantage of three-dimensional imaging and high resolution.Two.Evaluation of OSCC regional lymph node status by ICG^?-based near infrared fluorescence molecular imaging and Lugol’s iodine enhanced Micro-CT imaging[Objective]Accurate assessment of regional lymph node status is essential for the treatment of oral squamous cell carcinoma（OSCC）patients.In this study,we aimed explore to the feasibility of ICG^?-based NIR molecular imaging and Lugol’s iodine enhanced Micro-CT imaging in evaluating the surgical margin of OSCC.[Method]（1）ICG^?-based NIR imaging: Firstly,we have performed SLN mapping in mice and New Zealand rabbit to stimulate the process of clinical trials.Secondly,29 patients were enrolled in the clinical trials with 13 patients receiving intravenous injection of ICG^? and 16 patients receiving peritumoral injection of ICG^?.During the surgery,the fluorescence-positive lymph nodes in vivo were sent to undergo frozen section after fluorescence intensity was recorded.After the cervical lymph nodes dissection,all lymph nodes were sorted by region,and the fluorescence intensity was recorded before the lymph nodes were sent for paraffin section.Finally,nude mice lymph node metastasis model were established and divided into two groups: blocked lymphatic vessels group and unblocked lymphatic vessels group.Twelve hours after 5 mg/m L ICG^? injected by tail vein,the fluorescence intensity of regional lymph nodes were detected.（2）Lugol’s iodine enhanced Micro-CT imaging: To explore the feasibility of Lugol’s iodine enhanced Micro-CT imaging in locating metastatic lesion in lymph nodes,nonmetastatic and metastatic lymph nodes from mice were used to mimic the imaging process.Then,the lymph nodes from OSCC patients were applied to verify the value of Lugol’s iodine enhanced Micro-CT imaging in revealing lymph nodes structure and locating metastatic lesions in lymph nodes.The glycogen content in nonmetastatic and metastatic lymph nodes was further detected by the use of a glycogen assay kit and PAS staining to explain the imaging differences between them.Finally,Ultrasound was adopted to accelerate the process of Lugol’s iodine staining.[Results]（1）ICG^?-based NIR imaging: In the animal simulation experiments of sentinel lymph node mapping,the relevant sentinel lymph nodes of different regions in mice and rabbit all could be detected.In the clinical trials,during the surgery,both intravenous or peritumoral injections with near-infrared（NIR）fluorescence imaging of ICG^? had their respective pros and cons in vivo,with the sensitivity and specificity being 62.5%/75% and 98.1%/89.1%respectively.After the surgery,both methods could reduce the pathological workload by preselecting the lymph nodes at-risk in the premise of accurate assessing the cervical lymph nodes stage.However,intravenous ICG^? administration was more valuable in determining all types of LN status according to the fluorescence intensity（area under the curve [AUC]: 0.91 vs.0.78,P<0.001）.In the experiments of exploring the source of ICG^? in regional lymph nodes,we found that part of ICG was drainage from the primary tumor and part of ICG^? was drainage from its own vessels.（2）Lugol’s iodine enhanced Micro-CT imaging: In nude mice,0.5% Lugol’s iodine staining for 4 h was the best parameter for normal lymph nodes.The metastatic foci in metastatic lymph nodes were also clearly outlined in this condition.For nonmetastatic lymph nodes from patients with OSCC,1% Lugol’s iodine staining for 12 h was the best parameter.However,due to the increased volume of metastatic lymph nodes,the image effect of 3% I2-IK staining for 12 h was superior to 1% Lugol’s iodine staining（TBR: 3% vs 1%,1.89 ± 0.10 vs 1.27 ±0.07,P < 0.001）.Compared with subsequent pathological sections,we found the CT intensity of metastatic foci in lymph nodes and muscle tissues were significantly higher than in nonmetastatic regions.Meanwhile,the glycogen content of metastatic foci in lymph nodes detected was also significantly higher than in nonmetastatic region.In addition,we found Lugol’s iodine staining process could be shortened to 2 hours after ultrasound vibration.[Conclusion]Both ICG^?-based NIR imaging technology and Lugol’s iodine enhanced Micro-CT imaging technology could effectively assist to evaluate the status of OSCC regional lymph nodes status accurately which conld avoide the regional recurrence.ICG^?-based NIR imaging technology could preselect the at-risk lymph node to improve the pertinence and accuracy of pathological examination and further improve the accuracy of selective lymph nodes dissection.Lugol’s iodine enhanced Micro-CT imaging technology could clearly display the regional lymph node structure and located the spatial location of tumor tissues,which could assist pathological examination and reduce the missed diagnosis of metastatic lymph nodes.