Study On Targeting IFN? To Regulate Tumor Microcirculation And Promote Chemotherapeutic Drug Delivery

Background:According to the latest global cancer statistics report of 2018,lung cancer remains the most common type of cancer in the world with morbidity and mortality.China is one of the countries with the highest incidence and mortality of lung cancer worldwide.Chemotherapy is still one of the most important method of lung cancer treatment.Cisplatin(Cpt)is the first-line drug used in the treatment of lung cancer.However,chemotherapy can only delay the development of the disease,only 40-50% remission rate.There are many factors that influence the responses of chemotherapy,including genetic mutations,gene amplification,or epigenetic changes that affect single-cell drug uptake,metabolism,or export.Insufficient penetration of chemotherapeutic drugs is a fundamental but rarely understood cause of chemotherapy failure.Factors including tumor vascular abnormalities,extracellular matrix composition,cell-cell adhesion,high interstitial fluid pressure could limit drug delivery.Tumor blood vessels is the first barrier for most anticancer drugs reaching the tumor site,which plays an important role in chemotherapeutic drugs delivery.Besides killing tumor cells,chemotherapeutic drugs can affect tumor endothelial cells,destroy blood vessels.However,the destruction of tumor vessels in turn will reduce the subsequent delivery of chemotherapeutic agents into the tumor tissue and weaken the effect of killing tumor cells,resulting in chemotherapy failure.The integrity of vascular structure guarantees the normal delivery of chemotherapy drugs.Complete vascular structure consists of endothelial cells,junctions,basement membrane,and pericytes.Conventional factors including Vascular endothelial growth factor(VEGF),Delta-like ligand 4(Dll4)and Angiopoietin 1(Ang1)mainly affect the proliferation,migration and stablization of endothelial cells.However,the metabolism of endothelial cells(ECs),which is crucial for the function of blood vessels but has been little understood.Endothelial cell metabolism depends mainly on glycolysis,which will changes along with the disease state.It was reported that inhibition of glycolytic activator 6-phosphofructo-2-kinase/ fructose-2,6-biphosphatase 3(PFKFB3)in endothelium tightened the vascular barrier and improved chemotherapy effect by promoting drug delivery.However,the mechanism of metabolic regulation of vascular endothelial cells is still unclear.Although chemotherapeutic agents might directly eliminate dividing endothelial cells by inhibiting enzymes involved in DNA replication or metabolism of microtubules,the large part of vascular damages may attributed to chemotherapy-induced inflammation.In the process of cyclopamine-induced tumor rejection,macrophages are activated to produce Interferon ?(IFN?),inhibiting angiogenesis.IFN? is a pleiotropic inflammatory factor that has long been praised as an important effector molecule against tumor immunity and can suppress tumor growth through various mechanisms.On the contrary,it has also been involved in promoting an outgrowth of tumor cells with immune evasive phenotype.An important mechanism of IFN? affecting tumors is modulating of tumor blood vessels.Our studies found that IFN? has a modulating effect on the tight junctions of brain endothelial cells under inflammatory conditions;IFN? reduced the expression of Dll4 signaling pathways in endothelial cells,and lowered the expression of adhesion protein N-cadherin on perivascuar cells,stripped them from the vessels,increased vascular endothelial permeability and might promote metastasis of tumor cells.But whether IFN? could affect the metabolism of endothelial cells has not been reported.Here,we assessed whether neutralizing IFN? can block cisplatin-induced vascular injury and improve delivery of chemotherapeutic drugs.Purpose:In this article,we mainly studied that neutralizing IFN? blocked cisplatininduced vascular damage and improved drug delivery.Furthermore,a metabolic mechanism was introduced to confirm that neutralizing IFN? promoted the delivery of chemotherapeutic drugs by reducing endothelial glycolysis,limiting the excessive production of lactic acid,blocking cisplatin-induced tumor vascular injury.This provides a new strategy for the treatment of lung cancer.Methods:1)Immunofluorescence(IF)was used to detect the effects of cisplatin alone or cisplatin combined with anti-IFN? on tumor blood vessels in LLC tumor tissues.2)Quantitative-PCR(Q-PCR)and Cytometric Bead Array(CBA)were used to detect the effects of cisplatin on the expression of vascular-related inflammatory factors in LLC tumor tissues.3)Seahorse was used to detect the effects of IFN? on glycolysis of endothelial cells(sEND.1).4)Flow cytometry was used to measure the effect of IFN? on endothelial(sEND.1)glucose uptake.5)Western blot was used to detect the effect of IFN? on signaling pathway and glycolysis-related proteins.6)The lactic acid detection kit was used to test the effect of IFN? on the production of lactate in endothelial cells(sEND.1).7)Immunofluorescence and western blot were used to detect the effect of lactic acid on the distribution of VE-cadherin and activity of lysosome.8)Permeability assay was used to examine the effect of lactic acid on endothelial cell(sEND.1)permeability.9)Detect the influence of different treatment(control,cisplatin alone,cisplatin combined with anti-IFN?,cisplatin combined with anti-IFN? and lactate)on drug delivery and chemotherapy effect.Results:Our study found that cisplatin damaged tumor blood vessels in LLC mouse model.By RT-PCR and CBA screening,we found IFN? was increased after two days of first injection of cisplatin in LLC tumor tissues.Neutralizing IFN? at two days after first injection of cisplatin blocked cisplatin-damaged tumor blood vessels.The further study found that IFN? increased endothelial(sEND.1)glycolysis and promoted excessive production of lactate through activating AKT signaling.Lactic acid promoted lysosomal pathway-dependent endocytosis of VE-cadherin and enhanced degradation of intracellular VE-cadherin,thereby destroying vascular integrity.Inhibition of lactic acid production reversed IFN?-induced VE-cadherin redistribution.Lactic acid abolished the effect of anti-IFN? on tumor vasculature in vivo.Finally,the study found that neutralizing IFN? promoted the delivery of chemotherapeutic drugs and enhanced the efficacy of chemotherapy.lactate damaged the promotion of neutralizing IFN? on tumor blood vessels in vivo.Therefore,our results indicate that restraining excessive production of lactate by neutralizing IFN? improves drug delivery and enhances chemotherapy effect in LLC model.ConclusionOur study found that cisplatin damaged tumor blood vessels by inducing IFN?,neutralizing IFN? at two days after cisplatin was first injected blocked cisplatin-damaged tumor blood vessels.IFN? promoted the production of lactic acid by increasing the glycolysis of endothelial cells.Lactic acid promoted endocytosis of VE-cadherin by activating lysosome,thus damaging vascular structure and function.Restraining excessive production of lactate by neutralizing IFN? improved drug delivery and promoted chemotherapy effect.