Mapping Sentinel Lymph Node Metastasis By Dual-Probe Optical Imaging

Part ?The construction of lymphatic agent(Cy5.5-HA)and its application in imaging of sentinel lymph nodesObjective:To investigate the Cy5.5-HA as a lymphatic imaging agent and to evaluate its effect on the sentinel lymph node imaging and possible mechanisms,in order to provide accurate real-time intraoperative guidance for postoperative sentinel lymph node biopsyMethods:Cy5.5-HA was synthesized as a lymphatic imaging agent by conjugating hyaluronica acid(HA)with near-infrared fluorescent dye Cy5.5.The affinity of Cy5.5-HA to the lymphatic system was assessed using enzyme-linked immuno sorbent assay(ELISA)and immunofluorescence staining.Cy5.5-HA,which was synthesized with molecular weight of 5K,10K and 20K HA,was injected intradermally into the foot pad of the mouse,and the fluorescent imaging was performed every 10 minutes till 3 hours after injection.The migration of tracer in the lymphatic system and the residence in the sentinel lymph node were observed to optimize the best molecular weight.Cy5.5-HA and Cy5.5-PEG with the same molecular weight were injected into the foot pad of the mouse,and the migration in the lymphatic system and the residence in the sentinel lymph node of tracers was observed by fluorescent imaging.On day 5 of the model of local inflammation induced by turpentine,the same amount of Cy5.5-HA was injected on the inflammation side and the contralateral side,and the fluorescent imaging was performed every 10 minutes.Results:The binding of Cy5.5-HA to LYVE-1 was confirmed by ELISA and immunofluorescence staining.In the mouse model,the optimal imaging molecular weight of HA was 10 KDa,and the time window for sentinel lymph node imaging was 60 minutes to 180 minutes.Compared with control 10K Cy5.5-PEG in vivo imaging,Cy5.5-HA retented longger in sentinel lymph node indicating that Cy5.5-HA accumulation in the sentinel lymph nodes is associated with its specific binding,rather than simply related to the size of the molecular weight.Conclusions:Cy5.5-HA is an effective lymphatic imaging agent that can bind specifically to the sentinel lymph nodes in the lymphatic system.10K Cy5.5-HA expresses appropriate imaging time in mice.PART IIConstruction of tumor-specific probe(IRDye800-mAb)and its tumor tissue imagingObjective:The aim of this study is to construct tumor specific antibody probes and to assesse their sensitivity of imaging tumors for intraoperative differential diagnosis of lymph node metastases and partial replacement of intraoperative lymph node biopsy.Methods:Near infrared dyes IRDye800 were conjugated with anti-epidermal growth factor receptor(EGFR)antibody Cetuximab and human epidermal growth factor receptor 2(HER2)antibody trastuzumab(trastuzumab)separately,to synthesise IRDye800-Cetuximab and IRDye800-Trastuzumab two probes.After the UM-SCC-22B subcutaneous tumor model was established on the shoulder of BALB/c nude mice(n=6),fluorescent imaging was conducted 0,1,4,8,16.24.48 and 96 hous after IRDye800-Cetuximab(1 nmol)intravenous administration to optimize the best time to observe the tumor.After incubated with IRDye800-Cetuximab(5 umol/L,24 hours),different amounts UM-SCC-22B cells(IK,3K and 10K)were injected into BALB/c nude mice(n=6)subcutaneously and the mice were imaged immediately to figure out the minimum number of cells that can be distinguished by the probe.Different amounts UM-SCC-22B cells(1K,3K and 10K)were injected subcutaneously into BALB/c nude mice(n=4).24 hours after inoculation,IRDye800-Cetuximab(1 nmol)was injected intravenously,and 24 hours later,the mice were observed by fluorescent imaging to minimize cell count that can be imaged.Results:We successfully constructed near infrared tumor specific antibody probes(IRDye800-Cetuximab and IRDye800-Trastuzumab).The tumors can be imaged as early as 4 hours after intravenous probe injection and the contrast remained at high platform from 24 hours to 96 hours in the subcutaneous tumor model.To verify the sensitivity of the antibody probe,we figured out the sensitivity of IRdye800-Cetuximab in detecting UM-SCC-22B cells reached 1000 cell levels,approximately microns in size.Conclusions:We can conclude that the tumor specific antibody probe has a high sensitivity and a longer imaging time and therefore has a great potential for intraoperative imaging of metastatic lymph nodes.PART IIIMapping Sentinel Lymph Node Metastasis by Dual-probe Optical ImagingObjective:To investigate the synergistic effect of lymphatic imaging agent and tumor targeting agent to intraoperatively localize sentinel lymph nodes and the simultaneous detect tumor metastasis in sentinel lymph nodes in order to provide accurate real-time intraoperative guidance.Methods:Cv5.5-HA solution and IRDye800-Cetuximab solution were placed in near-infrared fluorescence imaging to distinguish their spectra.Fluc+ UM-SCC-22B cells and Fluc+SKOV-3 cells were injected into the foot pad of the mice to establish the sentinel lymph node metastasis model of head and neck cancer and ovarian cancer respectively.The tumor growth and lymph node metastasis were monitored by bioluminescence imaging.Dual tracer(IRDye800-Cetuximab,0.5 nmol in 100 uL,i.v.;Cy5.5-HA,0.2 nmol in 10 uL,locally)was given 6 weeks after the establishment of the cervical cancer lymph node metastasis model,and the imaging of the popliteal lymph nodes was performed.For the ovarian cancer lymph node metastasis model,dual tracer(IRDye800-Trastuzumab,0.5 nmol in 100 uL,i.v.;Cy5.5-HA,0.2 nmol in 10 uL,locally)was given 7 weeks after the establishment of the model,and optical imaging of the popliteal lymph nodes was performed.Pathological examination was made to confirm the imaging results.Results:The spectras of Cy5.5-HA and IRDye800-Cetuximab were well distinguished by near infrared imaging equipment.After a series of studies of IRDye800-labeled antibody and Cy5.5-labeled HA on tumor metastasis model mice,none metastatic lymph nodes,part of the metastatic lymph nodes and tumor tissue completely occupied lymph nodes were successfully separated,and results were confirmed by pathological results.Conclusions:Dual-tracer imaging with the combination of lymphatic mapping agents and tumor targeting agents can identify tumor metastases within SLNs,thus may provide accurate and real-time intra-operative guidance to spare the time spent waiting for a biopsy result.