Synergistic Enhancement Of Erlotinib In The Treatment Of Pancreatic Cancer By CA4(combretastatin A4) Nanoparticles

Background:Pancreatic cancer(PC),which is caused by abnormal cell division and uncontrolled growth in pancreas,representing one of the most malignancies.Although with the development of medical treatment,the five-year survival rate of PC was less than 10% owing to the absence of early symptoms and the aggressiveness.PC has become the seventh leading cause of cancer in the world,its risk factors include gender,age,area,smoking,obesity,diabetes and chronic pancreatitis.Surgical resection is the optimal therapeutic choice for PC,which diagnosed at an earlier stage.Nevertheless,less than 20% of PC patients were suitable for this treatment due to at middle and late stage with high recurrence rate and poor prognosis.Chemotherapy,radiotherapy,biological targeting therapy and Chinese medicine treatment only be used as adjuvant therapy to improve their lives.The epidermal growth factor receptor(EGFR),a member of the tyrosine kinase family,is overexpressed in up to 90% PC and is considered to be a key therapeutic target for oncology.Erlotinib,a EGFR tyrosine kinase inhibitors,is approved by the Food and Drug administration(FDA)for PC,but single therapy has limited therapeutic effect.Combretastatin A4(CA4)is a typical vascular disrupting agents(VDAs),which can destroy the established tumor vascular system by targeting endothelial cells,selectively prevent tumor blood flow,resulting in extensive secondary necrosis due to ischemia,but the tumor can not be completely eradicated,so combined treatment is needed to play a role in a variety of mechanisms and inhibit tumor growth and its vascular system at the same time.Based on the Enhanced IVPermeability and Retention Effect(EPR)effect of nanoparticles in solid tumors,CA4nanoparticles(CA4-NPs)have stronger tumor enrichment,lower systemic toxic and side effects and longer circulation time in vivo.Objective:In this study,C57BL/6 female mice were used to construct subcutaneous PC model to explore the efficacy and safety of CA4-NPs combined with Erlotinib in the treatment of PC.In addition,these treatments effectiveness was further validated by constructing orthotopic PC model and to explore the effect of CA4-NPs and Erlotinib on intestinal flora of the orthotopic PC.Methods:C57BL/6 female mice were used to construct subcutaneous PC model.The treatment group was: PBS,Erlotinib,CA4-NPs and CA4-NPs+Erlotinib.The tumor weight,tumor volume and Ki-67 immunohistochemistry(IHC)were used to evaluate the efficacy of different groups on subcutaneous PC model.The toxicity of each treatment group was observed by monitoring the liver and kidney function.In addition,the possible mechanism of treatments was explored by CD31 IHC and Western Blotting.Finally,the orthotopic PC model was constructed to further verify the efficacy of CA4-NPs combined with Erlotinib,and further collection of faeces at the end of the experiment was used for 16 S r RNA sequencing analysis to explore the effect of CA4-NPs and Erlotinib on intestinal flora of the orthotopic PC.Results:1.Tumor inhibition results and toxicity analysis in the subcutaneous PC model were as follow: compared with the PBS group,CA4-NPs and Erlotinib inhibited tumor growth,while CA4-NPs+Erlotinib group had the smallest tumor weight and smallest volume.And the expression of Ki-67 was the lowest in the CA4-NPs+Erlotinib group.In addition,there was no impairment of liver and kidney function among these groups compared with the normal mice.2.The CD31 IHC results showed that compared with PBS,the expression of CD31 in the Erlotinib group decreased,that in the CA4-NPs group decreased significantly,and that in the combined treatment group was the lowest.The expression of EGFR/PI3K/AKT signaling pathway protein was monitored by Western Blotting,and the results showed that the expression of p-EGFR,p-PI3 K,and p-AKT significantly increased in the CA4-NPs group compared with the PBS group,that in the Erlotinib group downregulated,but the combination of CA4-NPs and Erlotinib group might downregulated more effectively.3.The murine orthotopic PC model was constructed to further verify the efficacy of CA4-NPs combined with Erlotinib.CA4-NPs and Erlotinib monotherapy inhibited tumor growth compared with the PBS group,while the combination of CA4-NPs and Erlotinib significantly inhibited the growth of the tumor,and the anti-tumor effect was the best.4.Compared with the PBS group,the total abundance of Bacteroidota and Firmicutes increased(77.3% vs 88.1% vs 86.9% vs 87.4%)and Proteobacteria decreased(6.5% vs 4.4% vs 5.8% vs 3.5%)after the treatment of CA4-NPs and Erlotinib alone or in combination,which tended to the normal intestinal flora.