Construction And Visualization Of Molecular Imaging Probes For Targeting Neurometastatic In Pancreatic Cancer

Background:One of the most destructive tumors in the digestive system,Pancreatic Cancer(PNI),is linked to local recurrence,distant metastasis,and a unfavourable prognosis..However,rare attempt was made to indentified the PNI intraoperative,which may result in the failure to recognize the negative margin of the infiltrated nerve intraoperatively.Growth associated protein-43(GAP-43)expression is significantly high in pancreatic cancer,particularly in PNI.To facilitate precise R0 excision of the tumor,we planned to develop a fluorescent probe for intraoperative imaging of the PNI using GAP-43 as the target and indocyanine green(ICG)as the carrier.Objective:We created a molecular fluorescence probe actively targeting PNI lesions with GAP-43 as the active target and incyanine green(ICG)as the carrier in order to map and visualize the PNI lesions in pancreatic cancer operatively and guide the accurate R0 resection of PNI lesions in real time.How:(1)Key target level:Public databases and clinical tissue chip samples werewere applied to demonstrate the high expression of GAP-43 in pancreatic cancer,which is associated with poor prognosis and PNI.(2)Probe level:The GAP-43Ra-ICG-PEG active targeting molecular fluorescence probe’s fluorescence imaging capacity,safety,and stability were assessed by in vitro and in vivo investigations using GAP-43 as an active target;(3)Cell level:Nerve cell(PC12)and tumor cell(Miapaca2)transwell co-culture model was used to simulate PNI in vitro and validate the targeting of the probe in vitro;.(4)Animal level and tissue level:The mouse sciatic nerve invasion model was established to verify the active targeting of the probe in vivo.Simulated surgery combined with clinical surgical navigation system to evaluate the potential clinical application of the probe.To demonstrate that the nerve,rather the tumor,was the probe’s active target,the sciatic nerve damage model was developed.The active targeting of the probe in vivo was verified by immunofluorescence and immunohistochemistry,and the toxicity of the probe was verified by pathology.Result:(1)Key target level:Both public databases and pancreatic cancer tissues have confirmed that GAP-43 is highly expressed in pancreatic cancer,especially in PNI..(2)Probe level:GAP-43 Ra-ICG-PEG,a fluorescent nanoprobe with active targeting of PNI in pancreatic cancer,was synthesized successfully.It was circular-like and had an average Zeta potential of-12.17mV.It had similar excitation and emission spectra to ICG,and could maintain stability in solution.(3)Cell level:Compared with non-cocultured nerve cells,co-cultured nerve cells(PC12)had significantly higher fluorescence signal(P<0,001)..(4)Animal and tissue level:In the sciatic nerve invasion experiment of pancreatic cancer,the fluorescence signal of probe GAP-43Ra-PEG-ICG at PNI of animal was 3.21 times than that of ICG-NP group(P<0.001),and significantly higher than that of contralateral normal nerve group.R0 excision was achieved in only 60%of mice with the naked eye,whereas,100%R0 accuracy was achieved under probe-based fluorescence navigation.The injury model showed that the fluorescence signal of the injured nerve was 2.29 times that of the contralateral nerve after injection of the probe(P<0.001).Conclusion:GAP-43 was highly expressed in pancreatic cancer tissues,and PNI was significantly correlated with GAP-43 expression levels.We successfully constructed an active targeting near infrared fluorescence(NIRF)probe GAP-43 Ra-ICG-PEG.In vitro PNI model,GAP-43Ra-ICG-PEG specifically bound GAP-43 positive nerve cells.The visualization of PNI lesions of pancreatic cancer was effectively realized in the preclinical model(in vivo),providing a new technique and method for NIRF-guided precision resection of pancreatic cancer,especially R0 resection of PNI lesions of pancreatic cancer..