Regulation Of Tumor Microenvironment With AZD1775 Combined With Stereotactic Body Radiotherapy

The growth,differentiation,and metastasis of the tumor,as a neoplasm of the organism,is influenced not only by its some special genes but the environment,which called “Tumor microenvironment”,including various kinds of immune lymphocytes,epithelial cells,interstitial cells,and inflammatory factors.CD8+T lymphocytes and nature killer lymphocytes(NKs)play important roles in killing tumor cells.The tumor-associated macrophage cells(TAMs),myeloid-derived suppressor cells(MDSCs)and T regulatory cells(Tregs)play suppressive effects on tumor cells killing.Universally,all those lymphocytes keep a status of dynamic equilibrium in normal organisms.The ratio of the two and their activity directly affects the various biological behaviors of the tumor and is closely related to the patient’s prognosis.Radiotherapy(RT)plays an increasingly important role in the treatment of cancer.Previous studies have shown that the killing effect of radiotherapy on tumor cells is mainly divided into the direct killing and indirect killing.In addition to the physical death of cells caused by the break of the DNA chain,RT can also cause a series of changes inside and outside the cell,leading to apoptosis,pyroptosis,and necrosis.Among them,RT,especially high-dose and shortterm radiation therapy,can alleviate or delay tumor growth by regulating each immune lymphocytes and its inflammatory factors in the tumor microenvironment(TME).This discovery provides new ideas for the combined application of clinical segmented radiation therapy and immunotherapy.WEE1 kinase belongs to the WEE family.Its main function is to block damaged cells in the G2-M phase by negatively regulating the cdk1-cyclin complex related to the cell cycle.During this period,damaged DNA enters normal cell mitosis after repair.As the upstream of the WEE1 molecule,ATR has been shown to upregulate the expression of PD-L1 in cancer cells through the STAT1/3-IRF1-PD-L1 pathway.AZD1775,as an inhibitor of WEE1 kinase,can promote the rapid passage of damaged cells through the cell cycle,leading to the accumulation of a large number of erroneously replicated cells and ultimately cell death.The regulation of AZD1775 on immune cells and immune checkpoints in the tumor microenvironment has not been described in detail.This study found that AZD1775 can down-regulate the expression of PD-L1 of cancer cells after radiation therapy through the STAT3-IRF1-PD-L1 pathway.At the same time,the combination of AZD1775 and large saegmentation radiation therapy can increase the expression of dendritic cells(DCs)increased the number of CD8 + T cells and their toxic factors Granzyme B and INF-γ.Additionally,the combination therapy inhibited the expression of MDSC.This study provides excellent evidence for the clinical application of the combination of AZD1775 and IR.Part I1.To study the effects of WEE1 inhibitors and large segmented stereotactic body radiotherapy on the immune microenvironment in mouse models;2.To investigate the mechanism of WEE1 inhibitors combined with hypofractionatedstereotactic body radiotherapy to inhibit tumors in mouse models.Methods3.The Western blotting and Flow Cytometry experiments were used to detect the changes of PD-L1 expression in different cancer cell lines after receiving the treatment of WEE1 inhibitor combined with radiotherapy;1.Western blotting was used to detect the internal mechanism of PD-L1 changes after combined therapy;2.By constructing a mouse tumor model,observe and record changes in tumor volume of mice in different treatment groups,2-3 times a week;3.Flow Cytometry were used to detect the changes of different immune lymphocytes and immune factors in the tumor microenvironment in mouse tumor models.Results1.WEE1 kinase inhibitor could down-regulate PD-L1 up-regulated induced by highdose radiation therapy;2.WEE1 kinase inhibitor regulated the expression level of PD-L1 mainly through the STAT3-IRF1-PD-L1 pathway;3.WEE1 kinase inhibitor combined with radiotherapy could significantly delay tumor growth in mice;4.WEE1 kinase inhibitor combined with radiotherapy could strengthen killing effect of tumor cells mainly through two aspects: 1)Up-regulate cytotoxic CD8 + T cells and their toxic factors Granzyme B;2)Down-regulate immunosuppressive MDSCs,Tregs.TAMs have no difference between each groups.Conclusion1.The combination of WEE1 kinase inhibitor and radiotherapy could reduce PD-L1 expression in tumor cells by inhibiting the STAT3-IRF1-PD-L1 pathway.2.AZD1775 combined with radiation could increase the expression of CD8 + T cells and their toxic factors by increasing the expression of DCs;meanwhile,combined therapy could down-regulate inhibitory MDSCs and Tregs expression.Part IIPurpose: To investigate the curative effect and toxicity of patients with primary UCLC after the treatment of 56 Gy dose of stereotactic radiation therapy(SBRT).Methods:We retrospectively studied patients with primary UCLC who received stereotactic body radiotherapy at a dose of 56 Gy between 2010 and 2018.The Kaplan-Meier method were used to evaluated overall survival(OS),local tumor control(LC),and progression-free survival(PFS).Univariate log-rank test and multivariate Cox regression analysis were used to evaluated prognostic variables.Relevant toxicity is graded according to NCI-CTCAE version 5.0.Results:The gross of 58 patients were included in our study whose median age was V68 years(range,43-85),74.6% of them did not accept any therapy before.The median PTV dose was 77.8Gy(ranger,43.3-91.8)and the median PTV volume was 60.2 cm3(range,12.9-257.4).With a median follow-up of 57 months(range,6-90 months),the median cumulative OS was 58 months(range,2-105).In addition,1-year,2-year and 5-year OS and LC was 94.7%and 91.5%,75.0%and78.0%,45.0% and 58.6% respectively.On our univariable analysis(p=0.020)and multivariate analysis(p=0.028),OS was associated with PTV.The 5-year OS of patients whose PTV <53.0 cm3 and PTV ≥ 53.0 cm3 were 61.6% and 37.4%,respectively.On the aspect of toxicity after SBRT,there were three cases(5.2%)with grade ≥3 dyspnea and one died of sudden severe unexplained hemoptysis.Conclusions: Our results show that SBRT with a dose of 56 Gy delivered in 7 or 8 fractions,was an appropriate dose-fraction scheme for UCLC of smaller PTV.