Visualization Of Tumor Microenvironment Based On Mouse Dorsal Skin Fold Window Chamber

The morbidity and mortality of malignant tumors are always high in the world,so the prevention and treatment of malignant tumors has become especially important.In recent years,with the development of new technologies,great progress has been made in the study of the pathogenesis and treatment of tumors.Researchers have also come to realize that to achieve a comprehensive understanding and therapeutic control of tumors,we need to understand and understand the extremely complex microenvironment of tumors.Tumor microenvironment(TME)is a multi-factor,complex and dynamic interaction network,which plays an important role in the process of Tumor cell proliferation,invasion,metastasis or Tumor angiogenesis.The understanding of tumor microenvironment is of great academic and clinical significance for the understanding of tumor evolution and the study of tumor therapy.However,the internal changes in the treatment of the tumor are still unknown.In vivo optical imaging technology has the advantages of non-contact,small disturbance,nondestructive and intuitive,so it has been widely used in the study of tumor and tumor microenvironment.But is affected by the depth of imaging and optical imaging methods in the traditional mouse tumor model and unable to get the detailed information of local microenvironment,therefore,this paper constructed the mouse Dorsal Skin Fold Window Chamber(DSFC)tumor model for optical imaging in vivo provides a suitable observation and Window,this model can be on the same local tumor cell proliferation,tumor expansion,deformation,the change of blood flow velocity of local blood vessels for a long time,dynamic,continuous monitoring,It can provide the most intuitive picture information for tumor research and efficacy evaluation of anti-tumor drugs.In this study,techniques such as white light imaging,fluorescence imaging and scatter imaging were used to obtain the image information of tumor microenvironment of DSFC mice,so as to realize the study of tumor development and the evaluation of tumor treatment effect.The paper is mainly divided into the following parts:Part i: Screening tumor cell lines.In order to achieve a high rate of tumor,fluorescence imaging,and fast into tumor DSFC tumor model,we first to screening laboratory existing tumor cell lines,in the same condition of 4T1(breast cancer cells in mice),B16(mouse melanoma cells),Hepa1-6(liver cancer cells in mice)three types of cells,and use the inverted microscope to observe the 24 h,48 h,72 h,96 h after cell growth situation,estimate the growth rate;The transfection efficiency of Green Fluorescent Protein(GFP)in 4T1 cells was qualitatively analyzed by fluorescence microscope,and the transfection efficiency of GFP in 4T1 cells was quantitatively analyzed by flow cytometry.The results showed that under the same conditions,the growth rate of 4T1 cells was the fastest,and the transfection efficiency of GFP was 86.4%,which was better than the other two types of cells.Therefore,4T1 cells transfected with GFP were selected as tumor cell lines for subsequent animal experiments.Part ii: Mouse DSFC breast cancer tumor model was constructedFemale BALB/c-nu nude mice(22-25 g)aged 6-8 weeks were selected and DSFC imaging window was constructed on their backs.On the day when the window was constructed,GFPtransfected 4T1 cells were injected into the center of the window,and the mice were kept under sterile conditions.The tumor and neovascularization were observed under a stereoscopic microscope.The results showed that after 3-5 days,the tissue thickening,the increase of capillaries and the increase of vascular curvature in the injection area were observed by stereomicroscope.In conclusion,we believe that the mouse DSFC breast cancer tumor model can be successfully constructed in 3-5 days,which can be used for subsequent treatment and microenvironmental imaging experiments.Part iii: Carry out in vivo imaging experiment of tumor microenvironmentIn the early stage,the laboratory successfully designed and prepared the nano-gene therapy drug PCL-PDEM-si RNA.In this paper,we verified the effectiveness of the treatment method on the tumor model of common mice.At the same time,we used mouse DSFC breast cancer tumor model and optical in vivo imaging method to observe the process of tumor growth inhibition by nano gene therapy drugs.The mouse DSFC breast cancer tumor model was randomly divided into a blank group,a Phosphate Buffer Saline(PBS)group,a tumor group and a tumor treatment group.Imaging observation of the tumor microenvironment in mice was performed every other day by using fluorescence microscope,fluorescence microscope and laser speckle imaging system.Imaging was performed for a total of 20 days.The results showed that the tumor volume of the untreated tumor group continued to increase,while the tumor volume of the tumor treatment group remained stable and no longer increased from the 12 th day.The perfusion volume of the tumor group gradually increased with time,and by the 12 th day,the perfusion volume was 395% of the initial perfusion volume.However,the blood perfusion volume of the tumor treatment group did not increase from the 12 th day,which was consistent with the change of tumor volume.The experimental results were consistent with the terminal trend of the normal mouse tumor model,but the changes of multiple parameters such as tumor,blood vessel and blood perfusion volume could be dynamically observed in the whole treatment process,which could provide more intuitive and fine graphical information for the study of tumor treatment.From what has been discussed above,we use transfection GFP mouse breast cancer cells(4T1),the mouse Dorsal Skin Fold Window Chamber breast cancer tumors in mice model was established by combining postures fluorescence microscope and Laser Speckle Imaging(LSI)and in vivo tumor growth in mice to observe the changes and tumor local status and the formation of blood vessels,and verify the gene therapy drugs PCL-PDEM-si RNA treatment effect.This study provides a powerful platform for in vivo imaging of local blood vessels in tumor and microenvironment,and is of great significance to the basic research of cancer biology and the development of anti-tumor drugs.