Metabolomics Investigation On The Biological Effects Of A Tumor Vessel Targeting Protein TTF-pHLIP

Tumor vasculature is crucial to supply oxygen and nutrients for cell survival and almost all tumors share sustained angiogenesis capability.Compared to traditional targeted therapy,direct target to the tumor vasculature can be applied in almost all kinds of tumors and the therapeutic effects could take place in several hours.In addition,the drug resistance problems caused by target mutation may be avoided since the markers expressed on the endothelial cells are stable.Tumor vessel target therapy aims to deprive tumor cells of oxygen and nutrients by blocking blood flow,preventing waste from being excreted,and ultimately killing tumor cells.tTF-pHLIP is a coagulation protein consisting of truncated tissue factor(tTF)and low pH targeting peptide(pHLIP),which can target to tumor vessel and induce blood clotting.Although tTF-pHLIP has shown superior anti-tumor effects in previous studies,it still lacks in-depth research on its drug efficacy and safety.Therefore,the comprehensive study of its biological effect is of great significance to the understanding of its clinical effect and its rational application as well as the development of vascular targeted therapyConsidering that tTF-pHLIP targets to the vessels in the acidic tumor microenvironment,BALB/c mice,C57 mice and C57 melanoma mice were selected as animal models.Systematic and in-depth investigation on the toxic mechanism of tTF-pHLIP with different compositions,different dose exposures and its biological effects were conducted by NMR,GC and LC-MS based metabolomics and qRT-PCR methods.Firstly,BALB/c mice were used as experimental subjects to investigate the effects of high dose exposure of tTF,tTF/pHLIP(tTF and pHLIP mixture)and tTF-pHLIP on the metabolic profile of mice.The results showed that the effects of different exposures on the overall metabolism of mice were ranked from large to small:tTF-pHLIP(30?g)>tTF-pHLIP(20 ?g)>tTF/pHLIP(20 ?g)>tTF(20 ?g).Increased consumption of glucose metabolism,amino acid metabolism and energy metabolism,and elevated levels of serum albumin and globulin suggest that tTF-pHLIP may activate the body's immune response.The increase of choline,phosphorylcholine and ethanolamine related to cell membrane synthesis and the increase of serum cholinesterase level indicate that tTF-pHLIP may affect the synthesis or degradation of cell membrane.Finally,high dose(30?g)tTF-pHLIP also resulted in strong fatty acid metabolism and oxidative stress in mice.Taken together,high dose exposure of tTF-pHLIP may cause metabolic disorders in the body due to inserting into the cell membrane randomly.Next,the effect of therapeutic dose(2.5 ?g)of tTF-pHLIP exposure on the metabolic levels of C57 mice was investigated.Unlike tTF-pHLIP high-dose exposure,the therapeutic dose of tTF-pHLIP only had a slight effect on liver and kidney metabolism in mice.The energy metabolism,glucose metabolism and amino acid metabolism of the mice changed,and no significant changes in fatty acid metabolism were observed,indicating that the therapeutic dose of tTF-pHLIP was less toxic to the body.Finally,B16F10 melanoma mice were selected as the study subjects.The mice in the healthy control group were given different doses of tTF-pHLIP for treatment or no treatment.The metabolic profiles of plasma and organ tissues were analyzed.comparative analysis.The results showed that tumor progression resulted in significant changes in central carbon metabolism,tricarboxylic acid cycle,de novo fatty acid synthesis,pentose phosphate pathway and glutamine-related pathways in mice.The metabolic activities of glucose and glutamine,which are closely related to tumor growth,are abnormally changed in mice,and the body consumes nutrients such as visceral sugars and amino acids to increase the body's fatty acid synthesis to meet the needs of tumor growth.However,tTF-pHLIP treatment can significantly inhibit tumor growth,and at the same time relieve the tumor-induced TCA cycle and abnormal fatty acid metabolism,but cannot restore the metabolic changes of internal organs.The treatment of tTF-pHLIP also causes oxidative stress in the body.However,increase the number of treatments may result in rebalance of metabolic profiles of organs.In summary,this thesis has carried out a comprehensive study on the biological effects of different compositions and doses of tTF-pHLIP in normal mice and tumor-bearing mice,providing basic data for its toxicity.The understanding of the mechanism and safety of the fusion protein has been deepened,and it provides a reference for the research of anti-tumor targeted coagulation drugs.The final conclusion of this study is that tTF-pHLIP has no serious side effects when dosed at normal treatment level,and metabolomics is an excellent tool for the study of the biological effects of complex drugs.