Research On 3D Spatial Distribution Of Biological Responses Within Photodynamic Therapy-treated Mouse Breast Cancer

As a new non-invasive tumor treatment method,Photodynamic therapy(PDT)achieves the purpose of killing the tumor by irradiating the photosensitizer enriched in the tumor site with the specific wavelength of light.Its mechanism of killing tumors involves the interaction between light and biological tissues-the distribution of light in tissues,the absorption of light by tissues,the distribution of photosensitizers in tissues,and the support of oxygen in the reaction area.Therefore,PDT is a tumor treatment method that has special effect mechanism with strong spatial distribution,which is different with traditional radiotherapy and chemotherapy.The study of the mechanism of PDT is closely related to the precise treatment of tumors.The current research on the mechanism of PDT killing tumors is mainly based on its three elements and the biological responses of tumor tissues,including the distribution of photon,photosensitizers and oxygen in biological tissues,as well as the direct biological responses and indirected biological responsesc aused by PDT.Although a large number of researchers have studied the distribution of photon,photosensitizers and oxygen in tissues,as well as the local biological responses induced by PDT such as cell necrosis,apoptosis and microvascular responses,there is still a lack of research on the spatial distribution of biological responses induced by PDT in tumor tissues.Simple stree-factor model and local biological responses can not complete the guidance and assistant role of precise treatment of clinical PDT.Objective:to establish picture sets of direct and indirect biological responses for PDT whole tumor samples,analyze the 3D spatial distribution of PDT biological responses,and construct a 3D model of PDT biological responses,so as to provide better guidance and assistance for precise treatment of clinical PDT.Methods:in order to construct a spatial distribution model of biological responses of PDT killing tumor,sequence sections(5?m)and biomarkers of EMT-6 subcutaneously transplanted tumors in BALB/C nude mice 3 days after PDT treatment were performed using three-dimensional molecular reconstruction techniques commonly used in neuroscience.H&E staining,TUNEL and CD31 immunohistochemical staining were performed to analyze the necrosis,apoptosis and blood vessels after PDT.Digital image extraction(20X)was performed on pathological sections using full-field imaging technology.Digital image tumor segmentation using Matlab code to extract necrosis(red and white regions in H&E stained samples),apoptosis(brown regions in TUNEL-labeled samples)and blood vessels(brown regions in CD31-labeled samples)signals,then Gaussian nucleus density function combining heat maps were used to visualize cell necrosis,apoptosis,and density distribution of blood vessels in tissues,while area percentages were used to quantify their distribution in tissues.Finally,three-dimensional restruction was carried out using Amira software.At the same time,the MC method was applied to establish the light energy density distrubtion model and to verify with the biological responses model.Results:compared with the control group,after 3 days of PDT treatment,tumor necrosis mainly distributed in the upper and middle layers of the tumors;apoptosis mainly distributed in the lower layers of the tumors;vascular necrosis in the upper layers of the tumors,accompanied by vascular proliferation in the middle and bottom layers of the tumors.The percentage of necrotic area decreases exponentially with the depth of the tissue.As the increase of the depth,the necrotic area contracted from the whole layer of the surface(about 100%)to the two nest centers of the deep tumor tissue(25%).Apoptosis was mainly distributed located in the deep tumors.The percentage of apoptotic area at 3800?m subcutaneously was as high as 21%.Vascular proliferation was evident and diffuse in the middle and bottom layers of tumors.The percentage of vascular area at 2900?m subcutaneously was as high as 11.1%.In the three-dimensional reconstruction model,the necrosis and apoptosis of the tumors after 3 days of PDT treatment were complementary in depth.From the top down,necrosis gradually decreased,while apoptosis gradually increased.The death of the tumors composed of necrosis and apoptosis presented an inverted trapezoid,while vascular proliferation mainly occurred in the lower and middle layers of the tumors.Conclusions:in general,under PsD-007-mediated PDT,tissue necrosis and apoptosis and vascular proliferation show obvios spatial heterogeneity.The direct response(necrosis)induced by PDT is consistent with the spatial distribution trend of light energy density,both showing an exponential decrease,while the indirect response(apoptosis and vascular proliferation)induced by PDT is not directly related to the spatial distribution of light energy density.Compared with PDT light distribution model,PDT biological effect model can better reflect the therapeutic effect of PDT,and has greater reference value for precise clinical treatment of PDT.The analysis of photodynamic effects should be based on the combination of light,photosensitizers and oxygen with the actual effects of tissue.In this paper,the spatial distribution analysis model of PDT killing tumor effect provides new evidence for the model study of photodynamic effect,and also provides theoretical support for precise clinical treatment and dosimetry of PDT.