Synergetic DNA Damage Repair Inhibition And Tumor Microenvironment Regulation For Improved Tumor Radiotherapy

Radiotherapy is one of the most cost-effective ways of treating cancer,however,although accompanied with technical development of radiation,the results of radiotherapy are still sometimes unsatisfactory.Therefore,except for the simple combination of traditional chemotherapeutic drugs with radiotherapy,there is an urgent unmet need to explore new drug-radiotherapy combinations to increase patients’radiosensitivity.To realize radiotherapy sensitization,understanding the effect and tolerance mechanism of radiotherapy is of great value.The effects of radiation therapy could be attributed to direct damage to cellular DNA caused by ionizing radiation（commonly X-rays used in clinic）,or damage to intracellular components or DNA caused by reactive oxygen species（ROS）produced by the interaction of free electrons with water molecules.In addition,radiotherapy has been proved to enhance immunogenic cell death（ICD）by increasing the release of DAMPs（damage associated molecular patterns）stimulating the mature of DC（dendritic cells）,which then deliver the processed antigens to CD8⁺T cells and then activate T cells and mediate tumor killing.There are also diverse mechanisms that mediate radiotherapy tolerance.The tolerance may come from tumor cells intrinsically,as a response of radiation exposure,for example,the DDR（DNA damage response）.Tumors have evolved pathways to repair DNA damage,especially the DSBs which are most difficult to,including homologous recombination（HR）and non-homologous end joining（NHEJ）.Radiation tolerance can also be extrinsic,mediated by the tumor microenvironment against the external environment,protecting the tumor cells from external influences.For example,soft tissues encountered in clinical radiotherapy mainly consist of elements with low atomic numbers,resulting in weak X-ray deposition,which limited the effects of radiotherapy.Furthermore,immunosuppressive chemokines as well as lymphocytes such as regulatory T cells（Treg）regulated by tumor microenvironment could impair the immune activation mediated by radiotherapy.According to the mechanisms mentioned above,we designed and constructed nanoscale coordination polymers（VGd-NCPs）via supramolecular self-assembly of Gd³⁺and vidarabine monophosphate（VMP）with a molar ratio of 2:3.With the existence of high-Z element Gd,it can play the role of amplifying radiotherapy from the dual perspective of directly increasing X-ray deposition and improving ROS level.Vidarabine,like other nucleoside analogues,usually act by incorporated into DNA and RNA strands and therefore directly inhibiting intracellular enzymes together with retarding nucleic acid synthesis.As a result,those nucleoside analogues are widely used in virus,hematolological malignancies and solid tumors treatment.Nevertheless,vidarabine has been reported the ability of impeding DNA damage response by inhibiting the homologous recombination in DNA damage repair.The assembly of vidarabine with gadolinium could not only improve the in vivo pharmacokinetics of free vidarabine by virtue of nanoparticles’own properties,but also cooperatively boosted anti-tumor immune responses.In addition,as an isomer of immunosuppressive adenosine,we hypothesized that vidarabine may play a potential role in the regulation of adenosine-mediated differentiation from CD4⁺T cells into Treg.Hence,these four areas are examined in this paper:The first part clarified the preparation and characterization of VGd-NCPs.We constructed VGd-NCPs,carrier-free nanoscale polymers by one-step coordination.The size,morphology,zeta potential,chemical composition and crystal structure analysis were characterized by ultra-high-resolution field emission transmission electron microscope（FE-TEM）,dynamic light scattering（DLS）,zeta potential analyzer,Fourier transform infrared spectrum（FT-IR）,X-ray photoelectron spectroscopy（XPS）and PXRD.These results show that VGd-NCPs formed by the coordination of Gd and Vidarabine monophosphate with good stability,maintaining the functionality of X-ray deposition similar to free gadolinium.The second part investigated the intracellular radiation sensitization functionality of VGd-NCPs.The results indicated that the combination of VGD-NCPs with radiotherapy could increase the level of ROS and enhance the intensity of DNA double-strand breaks.In the cytotoxicity test,the cell viability of CT26 tumors was most significantly inhibited by VGd-NCPs+RT.The third part evaluated the in vivo radiotherapy sensitization efficacy of VGd-NCPs.Mouse CT26 colorectal cancer model was established and the combination of VGD-NCPs with radiotherapy could significantly inhibit tumor growth（90.35%）without obvious systemic toxicity and damage to major organs.In addition,immunofluorescence and immunohistochemical staining were excuted,confirming the therapeutic effect of VGd-NCPs+RT was due to the strong apoptosis of tumor cells and the significant decrease of tumor proliferation capcity caused by the maximum production of DSBs（γ-H2AX used as biomarker）.In the fourth part,we characterized the enhancement of ICD synergized with TME modulation directed by VGd-NCPs+RT.The combination of VGd-NCPs+RT stimulated the anti-tumor immune response by inducing immunogenic cell death more intensively at intracellular level,enhancing the maturation and activation of DC cells in vivo.Surprisingly,VGd-NCPs also has the effect of down-regulating Treg,which can regulate the immunosuppression TME.In this study,based on the understanding of the mechanism of radiotherapy,nanoscale coordination polymer VGd-NCPs was prepared by one-step method to compete with the tolerance mechanisms of radiotherapy,including DNA damage repair,insufficient X-ray deposition,and immunosuppressive tumor microenvironment.Gadolinium coordinated with vidarabine can amplify the oxidative stress and inhibit DNA damage repair after radiotherapy,and those two synergistically cause stronger immunogenic cell death and initiate subsequent anti-tumor immunity.In addition,VGd-NCPs+RT exhibited a degree of Treg downregulation,therefore regulated the immunosuppressive TME.VGd-NCPs could be easily prepared and enhance the effect mechanisms of radiation together with blocking radiation tolerance.This novel“drug-radiation”combination hold the potential to act as radiosensitizer in clinic.