Near Infrared Fluorescence Visualization Of Key Molecules In Pancreatic Cancer Cell And Pancreatic Stellate Cell And Translational Therapy Of Pancreatic Cancer

BackgroundThe overall five-year survival rate of patients with pancreatic cancer(PC)is only 11%.Most patients are in advanced stage once they are diagnosed.Chemotherapy is an important treatment for advanced PC.However,PC patients have a very low response rate to the current standard chemotherapy regimen.One of the main reasons is that activated pancreatic stellate cells(aPSCs)in the tumor microenvironment(TME)of PC produce a large amount of dense extracellular matrix(ECM),which seriously reduces the intratumoral drug delivery and penetration efficiency.Studies have shown that direct elimination of aPSCs increases the risk of PC progression,invasion and metastasis.Therefore,restoring the physiological quiescent state of aPSCs to reprogram the TME of PC represents a safe and effective therapeutic strategy.During the development of PC,the vicious circle formed by bidirectional crosstalk between pancreatic cancer cells(PCCs)and PSCs makes it extremely challenging for aPSCs to revert to their physiological quiescent state.Due to good biocompatibility,biorecognition ability and chemical versatility,peptide amphiphiles(PAs)are expected to realize visualization of key molecules in PCCs and PSCs and targeted drug delivery.In recent years,there have been few studies on molecular imaging and translational therapy of PC via PAs.Based on the above clinical problems,this study will utilize a co-assembled PA drug delivery system(DDS)combined with fluorescence molecular imaging(FMI)to achieve near infrared(NIR)fluorescence visualization of key molecules in PCCs and PSCs and restoration of PSC quiescent state in the TME of PC,which will provide a new approach for stromal regulation and therapy for PC.ObjectiveThis study aims to synthesize a co-assembled PA-DDS CoA-A&B-γPGA,which can deliver metformin(MET)and all trans retinoic acid(ATRA)to both PCCs and PSCs,restore PSC quiescent state and reduce ECM production,thus enhancing the antitumor effect of gemcitabine(GEM)in PC.MethodsIn this study,Met-PepA and ATRA-PepB were prepared by solid-and liquid-phase synthesis,and PA co-assembly was implemented in combination with y-polyglutamic acid(y-PGA).The critical micelle concentration(CMC)of PAs was determined by light scattering method;The secondary structure of PAs was determined by circular dichroism(CD);The assembly morphology of PAs was observed by transmission electron microscope(TEM).In in vitro experiments,confocal laser scanning microscope(CLSM)imaging and flow cytometry were used to evaluate the targeted uptake ability of PCCs and PSCs to PAs;Western blot,immunofluorescence(IF),scanning electron microscopy(SEM)and three-dimensional(3D)cell culture were used to evaluate the ability of PAs to restore the quiescent state of PSCs.In in vivo experiments,PCC/PSC subcutaneous and orthotopic tumors were constructed to evaluate the tumor targeted accumulation of PAs,the restoration of PSC quiescent state,ECM modulation and combined GEM chemotherapy efficacy.Clinical tissue samples were obtained to assess the specificity and sensitivity of PAs for detecting patientderived PCs.ResultsCoA-A&B-yPGA constructed in this study co-assembled to form nanofibers with a diameter of about 7 nm and was verified to have good stability and biocompatibility.CoA-A&B-yPGA was able to target both PCCs and PSCs in vitro,and in vivo FMI validated its targeted accumulation in the AsPC-1/hPSC subcutaneous tumors at 24 h of intravenous injection,with the highest tumor-to-background ratio(TBR)of 3.88±0.28.In vitro and in vivo experiments validated CoA-A&B-γPGA significantly inhibited PCC paracrine-mediated PSC activation,restored the quiescent state of aPSCs and reduced ECM production.CoA-A&B-γPGA combined with GEM enhanced chemotherapy efficacy,inhibiting AsPC-1/hPSC orthotopic tumor growth by approximately 80%.Furthermore,CoA-A&B-yPGA showed high specificity and sensitivity for detecting PC in patient samples.ConclusionCo-assembled PA-DDS CoA-A&B-γPGA constructed in this study achieved the restoration of PSC quiescent state and suppression of ECM production in the THE of PC by Met and ATRA targeted delivery to both PCCs and PSCs.CoA-A&B-γPGA combined GEM treatment significantly enhanced the chemotherapeutic efficacy of PC,which provides a new idea for molecular imaging,drug delivery and precision therapy for PC.