High-oxygen device and high-oxygen device respectively used for preparing medicine or instrument for enhancing oncolytic virus immunotherapy effect

Abstract

The invention relates to the field of oncolytic virus immunotherapy combined synergistic preparations and instruments, in particular to a normal-pressure high-concentration oxygen / device and a high-pressure high-concentration oxygen / device used for preparing a medicine / instrument for enhancing the oncolytic virus immunotherapy effect. The invention discloses a method for improving the oxygen concentration of a tumor microenvironment by using normal-pressure high-concentration oxygen and high-pressure high-concentration oxygen so as to improve the anti-tumor immune response of oncolytic viruses and finally remarkably enhance the immunotherapy effect of the oncolytic viruses. Specifically, a mouse is placed in a normal-pressure high-concentration oxygen device or a high-pressure high-concentration oxygen device, the environment is normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen, and after being treated in the environment for a period of time and several times, the mouse can be separated from the high-pressure high-concentration oxygen device. The normal-pressure high-concentration oxygen or the high-pressure high-concentration oxygen obviously improves the tumor immunotherapy effect of the oncolytic virus. Therefore, the normal-pressure high-concentration oxygen / device and the high-pressure high-concentration oxygen / device can be used for preparing medicines / instruments for enhancing the immunotherapy effect of oncolytic viruses.

Description

Technical Field

[0001] The present invention relates to the field of oncolytic virus tumor immunotherapy, and in particular to the use of high oxygen and high oxygen devices to regulate the tumor immunotherapy effect of oncolytic viruses. Specifically, it relates to a method of using normal pressure high-concentration oxygen and high-pressure high-concentration oxygen to prepare a drug that enhances the immunotherapy effect of oncolytic viruses, and a method of using normal pressure high-concentration oxygen and high-pressure high-concentration oxygen devices to prepare an instrument that enhances the immunotherapy effect of oncolytic viruses. Background Art

[0002] Cancer immunotherapy is one of the fastest-growing areas of cancer treatment. Key approaches include immune checkpoint inhibitors, adoptive cellular immunotherapy, neoantigen tumor vaccine therapy, and oncolytic virus therapy. The FDA alone has approved over 50 immune checkpoint inhibitors for the treatment of human cancers, and their indications have expanded to many different tumor types. Adoptive cellular immunotherapy, particularly CAR-T cell therapy, has demonstrated remarkable efficacy in patients with hematologic malignancies and metastatic melanoma. Furthermore, neoantigen tumor vaccine therapy has demonstrated its clinical value in rigorous Phase I and II clinical trials for patients with melanoma and glioma. These findings demonstrate the enormous potential of cancer immunotherapy. However, numerous studies have shown that the efficacy of cancer immunotherapy for solid tumors varies across patients. Consequently, cancer immunotherapy for solid tumors suffers from significant individual variability (and therefore low overall response rates).

[0003] Oncolytic virus therapy, a key branch of tumor immunotherapy, has received increasing attention in recent years. Oncolytic viruses are developed to exploit the ability of certain viruses to selectively infect and destroy cancerous tissue without harming normal tissue, resulting in anti-tumor effects. In 2015, the FDA approved the genetically modified oncolytic herpes simplex virus T-Vec for marketing, highlighting the value of oncolytic viruses in clinical applications. Currently, a variety of oncolytic viruses have entered the clinical research stage, including adenoviruses, herpes viruses, vaccinia viruses, and reoviruses. Similar to the performance of other immunotherapies on solid tumors, the therapeutic effects of oncolytic viruses also show strong individual variability and a low overall response rate.

[0004] Currently, replicating oncolytic adenoviruses represent a significant portion of oncolytic virus drug candidates under development. Adenoviruses are widely used due to their well-researched structure and function, biological stability, and the fact that adenoviral vectors allow for specific genetic manipulation and expression of specific transgenes. The oncolytic adenovirus drug Ankorui (H101), currently marketed in China, has demonstrated some anticancer activity against various solid tumors, but its clinical efficacy still leaves room for improvement. Its overall response rate for gastric cancer is 30%, and its local control rate for gynecological malignancies is only 17.2%. ONCOS-102, an oncolytic adenovirus drug candidate still in clinical development, has an objective response rate of 21.1% for malignant pleural mesothelioma. DNX-2401 has an objective response rate of 11.9% for patients with recurrent glioblastoma. In the treatment of malignant pleural effusions, AdV-tk has only achieved responses in the high-dose group, with a response rate of only 29%. Therefore, both marketed and clinically developed oncolytic adenoviruses suffer from significant individual variability and low overall response rates.

[0005] There are no published reports demonstrating a correlation between oxygen and inter-individual differences in treatment efficacy in oncolytic virus-mediated tumor immunotherapy, nor are there reports that high-concentration oxygen enhances the efficacy of oncolytic virus immunotherapy. There are also no published reports demonstrating a correlation between oxygen and the cGAS-STING pathway in tumor immunotherapy, nor are there reports that high-concentration oxygen activates the cGAS-STING pathway in tumors.

[0006] Therefore, the present invention hopes to achieve a breakthrough through our research, using hyperoxia to intervene and regulate the oxygen concentration in the tumor microenvironment to see whether such changes in oxygen concentration will affect the immunotherapy effect of oncolytic viruses. At the same time, the present invention also hopes to achieve a breakthrough through our research, using hyperoxia to intervene and regulate the oxygen concentration in the tumor microenvironment to see whether such changes in oxygen concentration will activate the tumor cGAS-STING pathway, thereby affecting the effect of tumor immunotherapy. Summary of the Invention

[0007] In the present invention, we demonstrate that the use of hyperoxia to regulate the oxygen concentration in the tumor microenvironment can significantly change the immune status of the tumor microenvironment, thereby having a substantial impact on oncolytic virus immunotherapy of tumors.

[0008] The method disclosed in the present invention for regulating the oxygen concentration of the tumor microenvironment by hyperoxia includes normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen. Both normal-pressure high-concentration oxygen (such as oxygen inhalation through a mask, nasal oxygen inhalation, or normal-pressure oxygen chamber oxygen inhalation) and high-pressure high-concentration oxygen (such as hyperbaric oxygen) have been widely used clinically, so their safety is guaranteed.

[0009] In multiple tumor models, the tumor volume of these mouse models was significantly smaller than that of the single treatment group when treated with normal pressure high concentration oxygen or high pressure high concentration oxygen plus oncolytic virus. The use of normal pressure high concentration oxygen or high pressure high concentration oxygen plus oncolytic virus treatment significantly prolonged the survival of multiple mice.

[0010] Therefore, the specific contents of the present invention are:

[0011] 1. A method for preparing a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the high-concentration oxygen is high-concentration oxygen at normal pressure, characterized in that the oxygen concentration is above 20.9%.

[0012] 2. The use of high-concentration oxygen in the preparation of a drug that enhances the immunotherapy effect of oncolytic viruses, wherein the high-concentration oxygen is high-pressure high-concentration oxygen, characterized by an oxygen concentration of more than 20.9% and a pressure of more than one standard atmospheric pressure, i.e., 1.013*10^5Pa.

[0013] 3. The use of a high-concentration oxygen device in the preparation of an instrument for enhancing the effect of oncolytic virus immunotherapy, wherein the high-concentration oxygen device injects oxygen with a concentration higher than 20.9% into a mask or cabin through an oxygen generator, so that the oxygen concentration in the mask or cabin is higher than 20.9%.

[0014] 4. The use of a high-concentration oxygen device in the preparation of an apparatus for enhancing the effect of oncolytic virus immunotherapy. The high-concentration oxygen device injects oxygen higher than 20.9% into the cabin through an oxygen generator, so that the oxygen concentration in the cabin is higher than 20.9%; at the same time, the cabin is pressurized by an air compressor, a high-pressure air tank or a high-pressure oxygen tank, so that the pressure in the cabin is above a standard atmospheric pressure, that is, 1.013*10^5Pa.

[0015] At the same time, in the present invention, we also demonstrate that the method of using hyperoxia to regulate the oxygen concentration in the tumor microenvironment can significantly change the immune status of the tumor microenvironment, thereby activating the tumor cGAS-STING pathway, and ultimately having a substantial impact on the tumor immunotherapy.

[0016] The method disclosed in the present invention for regulating the oxygen concentration of the tumor microenvironment by hyperoxia includes normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen. Both normal-pressure high-concentration oxygen (such as oxygen inhalation through a mask, nasal oxygen inhalation, or normal-pressure oxygen chamber oxygen inhalation) and high-pressure high-concentration oxygen (such as hyperbaric oxygen) have been widely used clinically, so their safety is guaranteed.

[0017] In multiple tumor models, the use of normal pressure high concentration oxygen or high pressure high concentration oxygen can significantly activate the tumor cGAS-STING pathway.

[0018] Therefore, the specific content of the present invention also includes:

[0019] 1. A use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors, wherein the high-concentration oxygen is high-concentration oxygen at normal pressure, characterized by an oxygen concentration of greater than 20.9%.

[0020] 2. The use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors, wherein the high-concentration oxygen is high-pressure high-concentration oxygen, characterized by an oxygen concentration of more than 20.9% and a pressure of more than one standard atmospheric pressure, i.e., 1.013*10^5Pa.

[0021] 3. Use of a high-concentration oxygen device in the preparation of an instrument for activating the cGAS-STING pathway in tumors, wherein the high-concentration oxygen device injects oxygen with a concentration higher than 20.9% into a mask or cabin through an oxygen generator, so that the oxygen concentration in the mask or cabin is higher than 20.9%.

[0022] 4. Use of a high-concentration oxygen device in the preparation of an apparatus for activating the cGAS-STING pathway in tumors. The high-concentration oxygen device injects oxygen with a concentration exceeding 20.9% into a cabin through an oxygen generator, thereby increasing the oxygen concentration in the cabin to above 20.9%. Simultaneously, the cabin is pressurized by an air compressor, a high-pressure air tank, or a high-pressure oxygen tank, such that the pressure in the cabin is above a standard atmospheric pressure, i.e., 1.013*10^5Pa. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Responses of different solid tumors to ADV treatment and changes in the cGAS-STING pathway.

[0024] (A, B) Tumor volume changes were measured in 4T1 and B16F10 tumor models (A) and CT26 and H22 tumor models (B).

[0025] (C, D) Flow cytometry analysis of the proportions of CD4+ T cells, CD8+ T cells, NK cells, and DC cells in 4T1 and B16F10 tumors (C) and H22 and CT26 tumors (D).

[0026] (E) Heat map showing the differences in expression of genes in the cGAS-STING pathway in mice with 4T1 breast cancer.

[0027] (F, G) RT-qPCR detected the changes of cGAS, STING (F), IFN-αIFN-β (G).

[0028] (H) The expression of IFN-α and IFN-β was detected by ELISA.

[0029] (I) Expression of cGAS downstream proteins was detected by western blot.

[0030] (J) ELISA was used to detect the expression of 2'3'-cGAMP in 4T1 and B16F10 tumor TIFs.

[0031] (K) 4T1 and B16F10 cells were treated with ADV (MOI = 10) for 0, 2, 6, and 12 h, respectively, and the expression of cGAS protein was detected by western blot.

[0032] (L) The tumor volumes of wild-type and sh-cGAS CT26 and H22 were measured.

[0033] (M) IFN-α and IFN-β concentrations were determined by ELISA.

[0034] (N, O) Flow cytometry analysis of CD4+ and CD8+ T cell infiltration in wild-type and sh-cGAS-treated CT26 (N) and H22 (O) tumor tissues.

[0035] (P) Western blot was used to detect the expression of cGAS and its downstream proteins.

[0036] (Q, R) The concentrations of IFN-α and IFN-β in the supernatants of 4T1 (O) and B16F10 (P) cells were determined by ELISA.

[0037] (S) Assay of wild type and cGAS OE 4T1 and B16F10 tumor volumes.

[0038] (T, U) Flow cytometry detection of wild-type and cGAS OE Infiltration of CD4+ (T) and CD8+ T cells (U) in treated 4T1 and B16F10 tumor tissues.

[0039] Figure 2 ADV activates the cGAS-STING pathway in tumors that respond well to ADV treatment.

[0040] (A, B) Kaplan-Meier survival curves of 4T1 and B16F10 tumor models (A) and CT26 and H22 tumor models (B) were plotted.

[0041] (C, D) RT-qPCR assays were used to detect the expression of cGAS, STING (C), interferon-α, and interferon-β (D) in CT26 and H22 tumors.

[0042] (E) Expression of cGAS downstream proteins in 4T1 and B16F10 tumors was detected by Western blotting.

[0043] (F) 4T1 and B16F10 cells were treated with ADV (MOI=10) for 0, 2, 6, and 12 hours, and the expression of cGAS mRNA was detected by RT-PCR.

[0044] (G, H) The knockdown efficiency of cGAS was verified by detecting mRNA by RT-PCR (G) and protein by Western blotting (H).

[0045] (I, J) CT26 and H22 tumor models were treated with RU.521, and the volume of subcutaneous tumors was measured (I), and the concentrations of IFN-α and IFN-β in serum were determined by ELISA (J).

[0046] (K) In the TCGA database, high expression of cGAS in patients with hepatocellular carcinoma (LIHC), invasive breast cancer (BRCA), or colon adenocarcinoma (COAD) was associated with a favorable prognosis (p < 0.001).

[0047] (L) In the TCGA database, high expression of cGAS was positively correlated with tumor immunity in patients with breast invasive carcinoma (BRCA), kidney chromophobe cell carcinoma (KICH), or low-grade glioma (LGG).

[0048] Figure 3 Respiratory hyperoxia can increase the sensitivity of melanoma and triple-negative breast cancer to ADV.

[0049] (A) Diagram of BALB / c and C57BL / 6 mouse tumor-bearing models and drug administration patterns.

[0050] (B, C) 4T1 and B16F10 tumors were measured twice a week to calculate tumor volume (B), and Kaplan-Meier survival curves of tumor-bearing mice were plotted (C).

[0051] (D, E) Lung tissues of 4T1 tumor-bearing mice were photographed (D), and the number of lung metastatic nodules was quantified (E).

[0052] (F) B16F10 tumor-bearing mice were treated as described above. One hour before euthanasia, a hypoxia probe was injected into the tail vein. The mean fluorescence intensity (MFI) of hypoxia probe-labeled tumor cells was then analyzed by flow cytometry.

[0053] (G) Immunohistochemistry was used to quantify the proliferation and apoptosis of tumor tissues in different treatment groups. Proliferation index: Ki67; apoptosis index: Cl-Caspase3.

[0054] (H) 4T1 and B16F10 cells were treated with ADV and 60% O2 combined with ADV (MOI=10) for 24 hours. The mRNA expression level of Hexon, a structural protein of ADV, was measured by RT-qPCR.

[0055] (I) 4T1 and B16F10 cells were treated with ADV and 60% O2 in combination with ADV (MOI = 1, 5, 10, 25, or 50) for 72 h, and cell viability was measured by CCK-8 assay.

[0056] (J) The mRNA expression levels of Hexon in 4T1 and B16F10 tumor tissues were detected by RT-qPCR.

[0057] (K) Immunofluorescence was used to measure the intensity of GFP fluorescence in tumor tissues, indicating the presence of ADV. (L) 4T1 and B16F10 tumor-bearing mice were treated with ADV and 60% O2, either alone or in combination, for 6, 12, and 24 hours, and tumor volumes were measured.

[0058] Figure 4 The anti-tumor effect of hyperoxia-sensitized ADV depends on the activation of the cGAS STING pathway.

[0059] (A) GSEA showing enrichment of cytoplasmic DNA sensing pathway signatures in hyperoxia-sensitized ADV-treated tumors.

[0060] (B) Heat map of gene expression in cytosolic DNA sensing pathways in hyperoxia-sensitized ADV and ADV-treated tumors.

[0061] (C) ELSA was used to detect changes in 2'3'-cGAMP in TIF.

[0062] (D) Expression of cGAS downstream proteins in B16F10 tumors was detected by Western blotting.

[0063] (E) B16F10 cells were cultured in 60% O₂ for different time periods (0 h, 12 h, and 24 h). Immunofluorescence analysis showed that p-STING colocalized with the Golgi apparatus. Blue: DAPI; Green: p-STING; Red: Golgi apparatus; TD: bright field.

[0064] (F) B16F10 cells were cultured in vitro with 60% O2 for different culture times (0 h, 12 h, and 24 h). Immunofluorescence analysis showed that p-IRF3 entered the cell nucleus. Blue: DAPI, Green: p-IRF3, TD: bright field.

[0065] (G, H) Orbital blood was collected from 4T1 (G) and B16F10 (H) tumor-bearing mice, and serum was separated. ELISA was used to detect the expression of IFN-α and IFN-β.

[0066] (I, J) RT-PCR was used to detect the expression of IFN-α and IFN-β in 4T1 (I) and B16F10 (J) tumor tissues.

[0067] (KN) Flow cytometry was used to measure the proportions of CD11c+MHC-II+DCs, CD3+CD4+T cells, and CD3+CD8+T cells in 4T1 (K, L) and B16F10 (M, N) tumors.

[0068] (O, P) Wild-type and sh-cGAS 4T1 or B16F10 cells were treated with ADV and 60% O₂, alone or in combination, for 48 hours. Western blot analysis was used to detect the expression of cGAS, STING, and IRF3 (O). ELISA was used to detect the expression of IFN-α and IFN-β (P).

[0069] (Q) Tumor volume curves of 4T1 and 4T1-sh-cGAS tumor-bearing mice.

[0070] (R) Tumor volume curves of B16F10 tumor-bearing mice treated with cGAS inhibitor RU.521 (5 mg / kg) intraperitoneally.

[0071] Figure 5 Hyperoxia-sensitized ADV promoted the expression of cGAS and downstream genes, increasing the sensitivity of 4T1 and B6F10 tumors to ADV.

[0072] (A) Enrichment analysis of DEGs in the hyperoxia-sensitized ADV group and the ADV group.

[0073] (B, C) RT-PCR was used to detect changes in cGAS, STING, and ISG15 mRNA levels in 4T1 tumor models cultured in 60% O2 (B) or hyperbaric oxygen (HBO) (C).

[0074] (D, E) RT-PCR was used to detect changes in cGAS, STING, and ISG15 mRNA levels in B16F10 tumor-bearing mice cultured in 60% O2 (D) or hyperbaric oxygen (HBO) (E).

[0075] (F) Flow cytometry was used to measure the proportions of CD3-CD49b+ NK cells, CD11b+F4 / 80+ macrophages, and Gr-1+CD11b+ MDSCs in B16F10 and 4T1 tumors.

[0076] (G, H) The knockdown efficiency of cGAS was verified by detecting mRNA by RT-PCR (G) and protein by Western blotting (H).

[0077] Figure 6 Effect of cGAS on tumor-infiltrating DCs and T cells during hyperoxia-sensitized ADV therapy.

[0078] (A) Flow cytometry was used to detect IFNAR-1 expression on B16F10 tumor-infiltrating DCs 3 to 5 days after ADV treatment.

[0079] (B)eFluor TM 670 proliferation probe was used to label BMDCs, and flow cytometry was used to detect BMDC proliferation.

[0080] (C) Flow cytometry was used to verify the expression of MHC-II and CD86 on BMDCs.

[0081] (D) Flow cytometry was used to determine the percentage of DCs in tumors of 4T1-shcGAS- and 4T1-bearing mice. (E) Flow cytometry was used to determine the percentage of CD86+ DCs in tumors of 4T1-shcGAS- and 4T1-bearing mice.

[0082] (F) Flow cytometry was used to verify the expression of CD69 in CD4+ and CD8+ T cells.

[0083] (G, J) RT-PCR was performed to verify the expression of CXCL9 and CXCL10 in tumors of 4T1-bearing mice (G) and B16F10-bearing mice (H) treated with PBS, ADV, 60% O2, or 60% O2 plus ADV, or in tumors of 4T1-bearing mice (I) and B16-F10-bearing mice (J) treated with PBS, ADV, HBO, or HBO plus ADV.

[0084] (K) RT-qPCR was used to verify the expression of IFN-γ in 4T1 and B16F10 tumors.

[0085] (L, M) Flow cytometry was used to detect the percentages of CD4+ T cells (L) and CD8+ T cells (M) in tumors of 4T1-shcGAS- and 4T1-bearing mice.

[0086] (N) B16F10 tumor-bearing transgenic Cre / ROSA26iDTR mice were intraperitoneally injected with diphtheria toxin (DT, 100 ng) daily for one week before treatment to achieve DC depletion 24 h before treatment. Tumor growth of B16F10 tumors.

[0087] (O)Rag1 - / -Growth of B16F10 tumors in transgenic mice.

[0088] Figure 7 Hyperoxia promoted the response of 4T1 and B16F10 cells to ADV by recruiting DCs and promoting T cell infiltration in tumors.

[0089] (A) IFNAR-1 expression on B16F10 tumor-infiltrating CD4+ and CD8+ T cells was detected by flow cytometry 3-5 days after ADV treatment.

[0090] (B) Flow cytometry was used to verify the differentiation efficiency of DCs.

[0091] (C) Flow cytometry was used to verify the expression of MHC-II on BMDCs.

[0092] (D) Flow cytometry was used to verify the expression of CD69 in CD4+ and CD8+ T cells.

[0093] (E, F) 4T1 and B16F10 tumor models were placed in 60% O2 (E) or hyperbaric oxygen (HBO) (F). ELISA was performed to verify the expression of CXCL9 and CXCL10 in mouse serum.

[0094] (G) Flow cytometry was used to verify the efficiency of DC depletion in the blood, spleen, and tumors of mice before treatment.

[0095] (H) Flow cytometry was used to verify the percentages of CD4+ and CD8+ T cells in B16F10 tumors of Cre / ROSA26iDTR mice (n=6-8 biological replicates).

[0096] Figure 8 Hyperoxia induces ROS production in tumor cells and activates the cGAS-STING pathway in ADV treatment.

[0097] (A, B) 4T1 and B16F10 tumor models were placed in 60% O2 (A) or hyperbaric oxygen (HBO) (B). The MFI of tumor cells labeled with DCFH-DA probe was analyzed by flow cytometry.

[0098] (C) MFI of kidney, liver, lung, and tumor cells labeled with DCFH-DA probe were analyzed by flow cytometry.

[0099] (D) Immunofluorescence analysis showed colocalization of ROS and mitochondria. Blue: DAPI, Green: ROS, Red: mitochondria, TD: bright field.

[0100] (E) The mean fluorescence intensity (MFI) of calcein (Beyotime, C2009S)-labeled mPTP was analyzed by flow cytometry.

[0101] (F) Transmission electron microscopy showing mitochondrial morphology in B16F10 tumor tissue.

[0102] (G, H) 4T1 and B16F10 tumor models were placed under 60% O2 (G) or hyperbaric oxygen (HBO) (H). RT-qPCR analysis of relative mtDNA copy number in tumors.

[0103] (I) RT-qPCR analysis of relative copy number of 8-OH-DG in 4T1 and B16F10 tumors.

[0104] (J) Immunofluorescence analysis showed colocalization of cGAS and mitochondrial DNA (TFAM).

[0105] (K, L) 4T1 and B16F10 tumor models were placed in 60% O2 (K) or hyperbaric oxygen (HBO) (L). The MFI of DCFH-DA probe-labeled tumor cells was then analyzed by flow cytometry.

[0106] (M, N) RT-PCR was used to detect changes in cGAS, STING, IFN-α, and IFN-β in 4T1 (M) and B16F10 (N) tumor tissues.

[0107] (O, P) 4T1 and B16F10 tumor models were divided into 60% O2 (O) and HBO groups (P), and tumor length and short diameter were measured twice a week to calculate tumor volume.

[0108] (Q) Changes in the cGAS-STING pathway were verified by Western blotting.

[0109] Figure 9 Hyperoxia increases ROS levels in tumor cells and activates the cGAS-STING pathway in ADV treatment.

[0110] (A) Cells were incubated with ROS labeled with DCFH-DA probe (1×10 6 cells / tube) for half an hour, and then the mean fluorescence intensity (MFI) of tumor cells labeled with DCFH-DA probe was analyzed by flow cytometry.

[0111] (B) 4T1 and B16F10 cells were treated with 60% O2 in vitro for 0, 3, 6, 12, 24, and 36 hours, and the mean fluorescence intensity (MFI) of tumor cells labeled with DCFH-DA probe was analyzed by flow cytometry.

[0112] (C, D) 4T1 (C) and B16F10 (D) cells were treated with 60% O2 for 24 h, and the cell supernatants were collected. The changes in the contents of superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH-) were detected by ELISA.

[0113] (E, F) RT-PCR was performed to detect the changes of cGAS, STING, IFN-α, and IFN-β in 4T1 (E) and B16F10 (F) tumor tissues.

[0114] Figure 10 Hyperoxia promoted the effectiveness of various oncolytic viruses and ADV in mouse models with favorable responses.

[0115] (A, B) Tumor length and short diameter were measured twice a week to calculate CT26 (A) and H22 (B) tumor volumes.

[0116] (CE) Flow cytometry was used to measure the proportions of CD11c+MHC-II+DCs (C), CD3+CD4+T cells (D), and CD3+CD8+T cells (E) in CT26 and H22 tumors.

[0117] (F, G) The levels of IFN-α and IFN-β in the serum of B16F10 (F) and CT26 (G) cancer model mice were measured by ELISA.

[0118] (H) Diagram of CDX and PDX model construction. Briefly, we injected human breast cancer MDA-MB-231 cells into the third mammary fat pad of NKG mice (CDX), and human hepatocellular carcinoma HCT116 cells (CDX) and human melanoma tumor tissue into the subcutaneous area of NKG mice (PDX). These tumor-bearing mice were intravenously injected with human peripheral blood mononuclear cells (PBMCs) on day 1 and then received different immunotherapy treatments starting on day 7.

[0119] (IL) Tumor length and short diameter were measured twice a week to calculate MDA-MB-231 and HCT116 tumor volumes (I) and PDX tumor volumes (J). Kaplan-Meier survival curves were plotted for CDX mice (K) and PDX mice (L).

[0120] (M, N) The proportions of CD3+CD8+ T cells and IFN-γ+CD8+ T cells in the blood of MDA-MB-231 (M) and HCT116 (N) model mice were measured by flow cytometry.

[0121] Figure 11 Hyperoxia acts as an immune adjuvant for HSV and VV.

[0122] (A, B) RT-qPCR was performed to detect the changes of cGAS and STING in CT26 (A) and H22 (B) tumor tissues.

[0123] (C, D) Tumor length and short diameter were measured twice weekly to calculate tumor volume in B16F10 and CT26 cancer models with HSV (C) or VV (D).

[0124] (E, F) Flow cytometry was used to measure the proportions of CD3+CD4+ T cells and CD3+CD8+ T cells in B16F10 (E) and CT26 (F) tumors treated with HSV and hyperoxia alone or in combination.

[0125] (G, H) Flow cytometry was used to measure the proportions of CD3+CD4+ T cells and CD3+CD8+ T cells in B16F10 (G) and CT26 (H) tumors treated with VV or hyperoxia alone or in combination.

[0126] (I, J) Flow cytometry was used to measure the proportions of GrzmB+CD8+ T cells and INF-γ+CD8+ T cells in B16F10 (I) and CT26 (J) tumors treated with HSV and hyperoxia alone or in combination.

[0127] (K, L) Flow cytometry was used to measure the proportions of GrzmB+CD8+ T cells and INF-γ+CD8+ T cells in B16F10 (K) and CT26 (L) tumors treated with VV and hyperoxia alone or in combination.

[0128] (M, N) ELISA was used to determine the levels of IFN-α and IFN-β in the serum of B16F10 (M) and CT26 (N) model mice (N = 4 biological replicates).

[0129] Figure 12 Response and safety of humanized PDX and CDX tumors to hyperoxia-sensitized ADV therapy. (A) Flow cytometry was used to measure the number of human CD45+ cells in peripheral blood one week after PBMC injection. (B) Flow cytometry was used to measure the proportions of CD3+CD8+ T cells and IFN-γ+CD8+ T cells in the blood of PDX model mice.

[0130] (CF) Body weights of MDA-MB-231, HCT116 (C), PDX (D), B16F10 (E), and 4T1 (F) tumor-bearing mice were measured twice weekly.

[0131] (G) Hematoxylin and eosin (H&E) staining of the heart, liver, lung, and kidney of 4T1 and B16F10 tumor model mice. DETAILED DESCRIPTION

[0132] All experimental materials and methods involved in the examples

[0133] Materials and methods

[0134] 1. Mice

[0135] Six-week-old female BALB / c mice and C57BL / 6J mice were purchased from the Comparative Medicine Center of Yangzhou University. ROSA26-LSL-DTR mice, Cd11c-Cre mice, Rag1 KO mice, and NOD-Prkdc scid Il2rg em1 / Cyagen C-NKG mice were purchased from Saiye Biotechnology. These animals were housed in a SPF facility at Nanjing University School of Medicine. Housing conditions were as follows: adequate light, 6:00 AM–6:00 PM; temperature, 18–22°C; and relative humidity, 40–60%. All animal studies were approved by the guidelines of the Institutional Animal Care and Use Committee of Nanjing University School of Medicine (approval number: IACUC-D2402006).

[0136] 2. Cell Culture, Viruses, and Reagents

[0137] CT26, H22, B16F10, and 4T1 cancer cells and HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT116 and MDA-MB-231 cells were purchased from ATCC. All cells were cultured in a 37°C incubator with 5% CO₂ in DMEM (Gibco) supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. All cells tested negative for mycoplasma contamination.

[0138] 1. Cell Lines

[0139] The cell lines used in this project include: 4T1 / 4T1-luciferase (mouse breast cancer cells), H22 (liver cancer cells derived from Balb / C mice), CT-26 (colorectal cancer cell line derived from Balb / C mice), B16F10 (melanoma cell line derived from C57BL / 6 mice), LLC (Lewis lung cancer cell line derived from Balb / C mice), PANC-02 (pancreatic cancer cell line derived from C57BL / 6 mice), GL261 (glioma cell line derived from C57BL / 6 mice), MB49 (bladder cancer cell line derived from C57BL / 6 mice), (renal cell carcinoma cell line), MDA-MB-231 (human triple-negative breast cancer cell line), HEK293 (human embryonic kidney cells), and Hela-S3 (human cervical cancer cell line). All cell lines were maintained in the laboratory. 4T1, B16F10, LLC, GC261, HEK293, CT-26, and Hela-S3 cells were purchased from ATCC. H22, PANC-02, MB49, and MDA-MB-231 cells were purchased from China Center for Type Culture Collection (CCTCC; China). 4T1-luciferase cells were purchased from Ubigene Biosciences. 4T1, B16F10, LLC, GC261, HEK293, CT-26, PANC-02, MB49, and MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Cat#11965092, Gibco-Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Cat#10099158, Gibco-Thermo Fisher Scientific) and 1% penicillin-streptomycin (PS; Cat#450-201-EL, WISENT). H22 and Hela-S3 cells were suspended in serum-free medium (Cat#H740KJ, Basalmedia, Shanghai) and cultured in spinner flasks (Cat#TCB002002, Jetbiofil, Guangzhou, China). All cells were cultured at 37°C and 5% CO2.

[0140] Oncolytic adenovirus (Type V adenovirus), oncolytic orthoreovirus (Oncolytic orthoreovirus), oncolytic coxsackievirus (Coxsackievirus A21), oncolytic Newcastle disease virus (NDV Herts / 33 strain), oncolytic measles virus (Oncolytic measles virus Edmonston strain), oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), and oncolytic poliovirus type 1 (Sabin strain) were obtained from laboratory-stored frozen virus stocks. Oncolytic herpes simplex virus type 1 (Strain F) and oncolytic vaccinia virus (Elstree strain) were purchased from Wuhan Binhui Biotechnology Co., Ltd.

[0141] cGAS(E5V3W)rabbit mAb(79978),STING(D2P2F)rabbit mAb(13647),phospho-STING(Ser366)(E9A9K)rabbit mAb(50907),IRF-3(D83B9)rabbit mAb(4302),phospho-IRF-3(Ser396)(D6O1M)rabbit mAb(29047),PPID(E7Y8I)rabbit mAb(78247),GAPDH(D16H11) rabbit mAb (5174), anti-mouse IgG HRP-linked antibody (7076), and anti-rabbit IgG HRP-linked antibody (7074) were purchased from Cell Signaling Technology (CST). goat anti-rabbit IgG H&L (Alexa 488) (ab150077) was purchased from Abcam. APC anti-mouseCD45 recombinant antibody(157606),APC / Cyanine7 anti-mouse CD3 antibody(100222),PerCP / Cyanine5.5 anti-mouse CD8a antibody(162310),PE / Cyanine7 anti-mouse CD4 antibody(100422),PE anti-mouse IFN-γantibody(163504),PE anti-human / mouse Granzyme B recombinant antibody (372208), FITC anti-mouse / humanCD11b antibody (101206), PE / Cyanine7 anti-mouse CD11c antibody (117318), and APC / Cyanine7 anti-mouse I-Ab antibody (116426) were purchased from BioLegend. 7-AAD viability staining solution (00-6993-50) was purchased from eBioscience. DAPI (P0131), MitoTracker Red CMXRos (C1049B), and Golgi Tracker Red were purchased from Bio-Tech. Poly(dA:dT) / LoyoVec TM Purchased from InvivoGen.

[0142] 3. Tumor Model

[0143] To establish solid tumors, BALB / c mice were intradermally injected with 2×106 CT26 cells or 1×107 H22 tumor cells suspended in 100 μl of HBSS. C57BL / 6J mice were intradermally injected with 1×106 B16F10 tumor cells suspended in 100 μl of HBSS. To study orthotopically grown mammary tumors, BALB / c mice were injected with 2.5×105 4T1 cells suspended in 100 μl of HBSS into the third mammary fat pad. When tumors reached approximately 50–100 mm3, mice were randomized and treated with either 5×107 pfu of ADV intratumorally or hyperoxia. The first dose was recorded as the first day, with injections occurring every other day for a total of three doses. Hyperoxia treatment began on the first day of ADV treatment. Animal survival and tumor size were observed every other day. Tumor or normal tissue samples were collected for further analysis. Mice were intraperitoneally injected daily with the cGAS inhibitor RU.521 (5 mg / kg). For in vivo DC depletion, DC reduction was achieved by intraperitoneal injection of diphtheria toxin (DT) (100 ng) in Cre / ROSA26iDTR transgenic mice 24 h before other treatments.

[0144] 4. Hyperoxia therapy

[0145] The two hyperoxia devices used in the present invention are a normal-pressure, high-concentration oxygen device and a high-pressure, high-concentration oxygen device. In the normal-pressure hyperoxia device, mice are placed in a closed box with a well-controlled gas composition to simulate the oxygen supplementation regimen for humans. A separate oxygen generator (Haier, ZY-3BW) is used to ensure that the required oxygen level is maintained within each unit. The composition of the inhaled gas within the device is determined by analyzing the PCO2 (partial pressure of CO2) and PO2 (partial pressure of O2) values in the equilibrium atmosphere. A model CY-12C oxygen analyzer is used to measure the fractional concentration of O2. The oxygen analyzer is calibrated using dry, carbon dioxide-free air with an assumed oxygen content of 20.95%. 60% oxygen was chosen because it is used in clinical protocols for respiratory hyperoxia and is non-toxic when inhaled for 24 hours. In clinical use, continuous inhalation of 60% oxygen (supplied by an oxygen concentrator) can also be achieved through nasal or face mask oxygenation.

[0146] Hyperbaric oxygen therapy is performed in a 250L chamber, using a pure oxygen tank to deliver a pressure of 2.5 ATA. The pressure is slowly increased to 2.5 ATA over 15 minutes, then allowed to equilibrate. If the pressure drops, a valve automatically opens to increase pressure. After maintaining the pressure at 2.5 ATA for 1.5 hours, the pressure is slowly deflated over 15 minutes to return to normal atmospheric pressure. The 2.5 ATA pressure and 1.5-hour treatment duration were chosen because they are commonly used in hyperbaric oxygen clinical protocols.

[0147] Similarly, hyperbaric oxygen therapy can be performed using another method: a 250L hyperbaric oxygen chamber is simultaneously pumped with air using a high-pressure air pump and pressurized with 90% oxygen using an oxygen concentrator. The high-pressure air pump pumps air for 15 minutes, slowly increasing the chamber pressure to 2.5 ATA. After maintaining the pressure at 2.5 ATA for 1.5 hours, the chamber is slowly deflated over 15 minutes to restore it to normal atmospheric pressure. Simultaneously, the oxygen concentrator pumps 90% oxygen at a flow rate of 3L / minute. Once the chamber pressure reaches 2.5 ATA, it is maintained at 2.5 ATA by adjusting the air pump's flow and automatic deflation.

[0148] 5. OVs amplification

[0149] OVs were propagated in HEK293T cells. Briefly, ADV and HEK293T cells were mixed at a ratio of 2:1 in serum-free medium supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. The cells were cultured in CD293S (H74OKJ, Basal Media) medium for 48-60 hours. Cytopathic effect (CPE) was observed during the culture. Cells that developed CPE were collected and subjected to three cycles of repeated freeze-thaw cycles. Viral titer was determined by TCID50 calculation.

[0150] 6. Viral Oncolysis and Replication

[0151] 3×10 4T1 and B16F10 cells were seeded in 96-well plates and treated with different MOIs for 72 hours. Following the prescribed treatment, cells were placed in 10 μL of CCK-8 Cell Counting Kit (A311-01, Vazyme) solution, diluted with culture medium, and incubated at 37°C for 1.5 hours. After the incubation period, the absorbance of the samples was measured at 450 nm using a microplate reader according to the manufacturer's protocol. Adenovirus replication rates were calculated using the TCID50 assay. Briefly, cells were seeded in 24-well plates and infected with adenovirus at different MOIs. The virus-containing medium was removed, and fresh medium was added 2 hours after infection. Cells were harvested 24 hours after infection. Adenovirus titers were then determined using the TCID50 assay.

[0152] 7. RNA-seq and related data analysis

[0153] To analyze the differences in gene expression, total RNA was extracted by TRIzol according to the manufacturer’s instructions and then sent to Majorbio for RNA sequencing analysis by an Illumina NovaSeq 6000 instrument.

[0154] 8. Transmission Electron Microscopy

[0155] Three days after the completion of oncolytic adenovirus and hyperoxia treatment, mice were sacrificed by cervical dislocation, and tumor tissue was removed and stored in electron microscopy fixative at 4°C in the dark. Samples were transported on ice and processed by Servicebio according to previously reported techniques. Images were acquired using a Hitachi HT 7800 120 kV and a JEM-2100 Plus.

[0156] 9. mtDNA identification

[0157] On the third day after the oncolytic adenovirus and hyperoxia treatment, mice were killed by cervical dislocation and tumor tissue was removed. Tumor tissue was initially destroyed using a tissue grinder, and mitochondria were extracted according to the kit (Tissue Mitochondrial Isolation Kit, Thermo Fisher Scientific Inc., 89801). DNA was purified from these cytoplasmic components using a DNA purification kit (Deoxyribonucleic Acid Blood and Tissue Kit, Qiagen, 69504). RT-qPCR was performed on the purified cytoplasmic DNA using mtDNA primers (Dloop1-3, Cytb, ND1, and ND4), and all genes were normalized to GAPDH.

[0158] 10. Detection of immune cell activation levels in mouse tumor tissues by flow cytometry

[0159] Tumor tissue was minced and digested in RPMI-1640 supplemented with type IA collagenase and DNase I in a 37°C incubator with shaking (220 rpm). The tissue was then filtered through a 40 μm cell strainer (Thermo Fisher) and resuspended in PBS with 2 mM EDTA. The cells were then incubated with Fcγ receptor blocking antibodies (BD Biosciences) for 10 minutes at 4°C and then surface stained for 25 minutes at 4°C. The cells were then washed and flow cytometry was performed on a FACSCanto (BD Biosciences). Data analysis was performed using Flowjo v10.5.3 (Treestar).

[0160] 11. Generation of BMDCs

[0161] Bone marrow cells were collected from the tibia and femur of C57BL / 6J mice and cultured in RPMI-1640 medium supplemented with 10% FBS, 20 ng / ml mGM-CSF (315–03–250, PeproTech), and 10 ng / ml mIL-4 (214–14–100, PeproTech). Fresh medium supplemented with mGM-CSF and mIL-4 was added on day 3, and half of the medium was replaced with fresh medium on day 5. DCs were harvested on day 7.

[0162] 12. Isolation and Culture of Splenic T Cells

[0163] Mouse splenic CD4+ and CD8+ T cells were isolated using a mouse CD4+ T cell isolation kit (Selleck, B90001) and a mouse CD8+ T cell isolation kit (Selleck, B90011). T cells were stimulated with 1 μg / mL CD3 mAb and 5 μg / mL CD28 mAb (BD Biosciences) for 72 hours in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco), 50 U / mL penicillin, and 50 mg / mL streptomycin. Cells were cultured in a 37°C, 5% CO2 incubator.

[0164] 13. Humanized CDX Model

[0165] PBMCs were isolated from blood using a Ficoll (C0025, Beyotime, China) density gradient. NKG mice were intravenously injected with 5 × 106 human peripheral blood mononuclear cells. After reaching the appropriate humanization ratio, 5 × 106 HCT116 cells and 5 × 106 MDA-MB-231 cells were subcutaneously injected into recipient mice in a volume of 100 μl PBS.

[0166] 14. Humanized PDX Models

[0167] PBMCs were isolated by Ficoll (C0025, Biyuntian, China) density gradient separation and injected into NKG mice to reconstitute the human immune system. Primary tumor samples were resected from melanoma patients stored on ice and transported to the laboratory within 1 hour. The tumor samples were fragmented into small pieces (approximately 1 mm3) and implanted into NKG mice for three generations. The PDX samples were then subcutaneously transplanted into the axilla of humanized mice to establish a humanized PDX model. Human melanoma samples were collected from patients undergoing melanoma resection at Nanjing Drum Tower Hospital, affiliated with Nanjing University Medical School. All patients were informed of the purpose of tissue sample collection and consented to the collection. This study was approved by the Ethics Committee of Nanjing Drum Tower Hospital (approval number: 2016-05-17).

[0168] 15. RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)

[0169] Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis for qPCR was subsequently performed using HiScript III RT SuperMix (+ gDNA wiper) (Vazyme, R323). For mRNA quantification, qRT-PCR was performed using ChamQ SYBR Color qPCR Master Mix (Vazyme, Q431). GAPDH expression levels were used as an internal control for mRNA detection.

[0170] 16. Quantification and Statistical Analysis

[0171] Statistical significance was determined using the Mann-Whitney test, paired t-test, unpaired t-test, one-way / two-way ANOVA, or log-rank test using GraphPad Prism 9. Differential gene expression and pathway enrichment analyses of RNA-seq data were performed using R4.2.1. Sample size was not predetermined by statistical methods. In the absence of statistical data, experiments were performed at least three times to ensure reproducibility unless otherwise stated. All data are presented as mean ± standard deviation. P < 0.05 was considered significant.

[0172] Example 1 ADV treatment downregulates the cGAS-STING pathway, limiting the anti-tumor therapeutic effect of ADV

[0173] To investigate the responses of different tumors to oncolytic adenovirus (ADV) therapy, we treated various mouse tumor models with ADV via intratumoral injection. ADV treatment showed good efficacy against subcutaneous colorectal cancer (CT26) and hepatocellular carcinoma (H22). However, ADV treatment was not effective against orthotopic triple-negative breast cancer (4T1) and subcutaneous melanoma (B16F10). Figure 1 A, B; Figure 2 A, B). In 4T1 and B16F10 tumor tissues, the numbers of antigen-presenting dendritic cells (DCs), CD4+ T cells, CD8+ T cells, and natural killer (NK) cells were not significantly different between the ADV-treated and untreated groups ( Figure 1 C). However, in the CT26 and H22 tumor tissues treated with ADV, the infiltration of DCs, CD4+ T cells, CD8+ T cells, and NK cells into the tumors was significantly greater than that in the PBS group ( Figure 1D). These findings suggest that 4T1 and B16F10 tumors respond poorly to ADV treatment and do not transform from "cold tumors" to "hot tumors" after ADV treatment. The different responses of different tumors to ADV treatment prompted us to investigate the underlying mechanisms. Transcriptomic analysis (RNA-seq) of 4T1 tumors was performed, and differentially expressed genes (DEGs) showed that cGAS-STING pathway-related genes were significantly downregulated in the ADV group compared with the PBS group ( Figure 1 E). cGAS, as a key innate immune sensor, can induce type I interferon (IFN-I) responses, and IFN-I (IFN-α and IFN-β) has significant immunostimulatory effects. This may be an important reason for the poor immune effect of ADV treatment on 4T1 and B16F10 tumors. Consistent with these findings, cGAS, STING, IFN-α, and IFN-β gene expression were significantly downregulated in 4T1 and B16F10 tumors treated with ADV compared with the PBS group ( Figure 1 F, G), IFN-α and IFN-β secretion in TME was also significantly reduced ( Figure 1 H). At the protein level, ADV treatment downregulated the expression of cGAS-STING pathway-related proteins, including cGAS, phosphorylated STING (p-STING), and phosphorylated IRF3 (p-IRF3) ( Figure 1 I). In contrast, the cGAS-STING pathway was significantly upregulated at both mRNA and protein levels in ADV-treated CT26 and H22 tumors ( Figure 2 CE). Regarding 2'3'-cGAMP, a marker of cGAS pathway activation, 2'3'-cGAMP in 4T1 and B16F10 tumor interstitial fluid (TIF) was significantly decreased after ADV treatment ( Figure 1 J). Next, we treated tumor cells with ADV in vitro. As time increased from 0 to 24 hours, cGAS transcript and protein levels in 4T1 and B16F10 cells infected with ADV were significantly reduced ( Figure 2 F; Figure 1 K). To further verify the role of cGAS in ADV treatment, we downregulated the expression of cGAS in H22 and CT26 cells ( Figure 2 G, H), ADV's positive therapeutic effect disappears ( Figure 1 L), and the content of IFN-Ⅰ in TME was also significantly reduced ( Figure 1 M). The same results were also obtained using the cGAS inhibitor RU.521 ( Figure 2 I, J). In addition, the infiltration of CD4+ and CD8+ T cells into the tumor was significantly reduced ( Figure 1N, O). In addition, analysis of The Cancer Genome Atlas (TCGA) data showed that cGAS expression is associated with survival in various types of cancer ( Figure 2 K) and immune cell infiltration ( Figure 2 These results indicate that cGAS expression is an important factor affecting the therapeutic effect of ADV.

[0174] To determine whether the downregulation of cGAS after ADV treatment could be reversed, we transfected 4T1 cells with poly(dA:dT), a repetitive synthetic double-stranded DNA sequence that has been shown to activate cGAS. Transfection of poly(dA:dT) alone activated cGAS and downstream components such as STING and IRF3. Interestingly, cells transfected with poly(dA:dT) reversed the downregulation of cGAS, p-STING, and p-IRF3 after ADV treatment ( Figure 1 We also examined the production of IFN-I in 4T1 and B16F10 cells after ADV treatment. Consistent with the above results, poly(dA:dT) transfection reversed the downregulation of IFN-α and IFN-β production induced by ADV treatment ( Figure 1 Next, we overexpressed cGAS in 4T1 and B16F10 cells (cGAS OE ) and performed mouse tumor implantation. The results showed that overexpression of cGAS significantly promoted the therapeutic effect of ADV ( Figure 1 S), and significantly increased the number of tumor-infiltrating CD4+ and CD8+ T cells ( Figure 1 T and U).

[0175] These results indicate that different solid tumors respond differently to ADV treatment, and that downregulation of the cGAS-STING-IFN-I pathway has been shown to be a key factor limiting the efficacy of ADV-mediated immunotherapy. Furthermore, in tumors that responded poorly to ADV treatment, additional artificial administration of a cGAS activator reversed the downregulation of the cGAS-STING pathway, increased cGAS expression, and sensitized tumors to ADV treatment. This suggests that the cGAS-STING-IFN-I pathway can be reversed, and that reversing the cGAS-STING-IFN-I pathway can induce responses in tumors that were previously insensitive to ADV treatment.

[0176] Example 2: For solid tumors that initially respond poorly to ADV treatment, hyperoxia can effectively improve the therapeutic effect of ADV.

[0177] After understanding the reasons why some tumors respond poorly to ADV therapy, we sought to find a method that could be directly applied clinically to address the low response to ADV therapy. Hyperoxia therapy is safe and reliable, widely recognized in clinical practice, and is highly likely to activate the cGAS-STING pathway, so we introduced it into our research ( Figure 3 A). There are two types of hyperoxia therapy. One is hyperbaric oxygen (HBO), with a treatment of 2.5 ATA, 100% O2, for 1.5 hours. The other is normal pressure high concentration oxygen (60% O2), with a treatment of 1.0 ATA, 60% O2, for 24 hours. Hyperoxia therapy combined with ADV is called hyperoxia-sensitized ADV therapy. Compared with ADV or hyperoxia alone, hyperoxia-sensitized ADV therapy significantly reduced the growth of 4T1 and B16F10 tumors that initially responded insufficiently to ADV therapy and prolonged the survival of tumor-bearing mice ( Figure 3 B, C). 4T1 orthotopic tumors are ADV-low-responding tumors with a strong malignant phenotype of lung metastasis. We found that hyperoxia-sensitized ADV treatment significantly reduced the number of lung tumor nodules ( Figure 3 D, E). In addition, we observed that the hypoxic area of the tumor was significantly reduced after hyperoxia treatment ( Figure 3 F). Immunohistochemical staining showed that the expression of proliferation marker Ki67 was decreased and the expression of apoptosis marker cleaved-caspase3 (Cl-Caspase3) was increased in tumors treated with hyperoxia-sensitized ADV ( Figure 3 G), indicating that the tumor suppression was most significant in the hyperoxia-sensitized ADV group. Next, we investigated whether hyperoxia had any effect on the behavior of ADV itself. In vitro, hyperoxia did not affect the replication of ADV in 4T1 and B16F10 cells ( Figure 3 H) or oncolytic activity ( Figure 3 I). To our surprise, hyperoxia significantly promoted the in vivo replication of ADV in 4T1 or B16F10 tumor tissues ( Figure 3 J), and analysis of frozen tissue sections showed that hyperoxia significantly promoted the replication of ADV in 4T1 and B16F10 tumors ( Figure 3 K). Due to the challenges of receiving 60% O2 treatment for 24 hours per day, we investigated whether we could reduce the duration of 60% O2 treatment to achieve similar antitumor effects as 24 hours of 60% O2-sensitized ADV treatment. We set the duration of 60% O2 treatment to 6, 12, and 24 hours. The results showed that although 24 hours of 60% O2 treatment had the greatest effect on 4T1 and B16F10 tumors, 12 hours of 60% O2 treatment also significantly improved the efficacy of ADV treatment ( Figure 3L). Taken together, these findings suggest that hyperoxia treatment can alleviate hypoxia in the TME and enhance ADV replication in tumors. Importantly, hyperoxia effectively augments the therapeutic efficacy of ADV in solid tumors that initially respond poorly to ADV therapy.

[0178] Example 3: Hyperoxia sensitizes ADV immunotherapy by rescuing cGAS expression and activating downstream pathways in solid tumors that initially respond poorly to ADV therapy

[0179] Next, we explored how hyperoxia-sensitized ADV works in tumors that initially lack a response to ADV treatment. RNA sequencing was performed on orthotopic 4T1 tumors to assess differences in gene expression between the hyperoxia-sensitized ADV-treated group and the ADV-treated group. Pathways related to immune responses were significantly upregulated in the hyperoxia-sensitized ADV group compared to the ADV group. Figure 5 A). Gene set enrichment analysis (GSEA) showed that genes related to cytoplasmic DNA sensing pathways were enriched in the hyperoxia-sensitized ADV group ( Figure 4 A). Genes enriched in the cytoplasmic DNA sensing pathway include cGAS, STING, and IRF3 ( Figure 4 B). Compared with ADV alone, hyperoxia-sensitized ADV significantly upregulated the expression of cGAS, STING, and ISG15 genes ( Figure 5 BE). Hyperoxia-sensitized ADV also significantly increased 2'3'-cGAMP in 4T1 and B16F10 tumor TIFs ( Figure 4 C). Notably, hyperoxia-sensitized ADV also significantly increased the levels of p-STING and p-IRF3 ( Figure 4 D) Activation of cGAS promotes the translocation of p-STING to the Golgi apparatus, the nuclear translocation of p-IRF3, and the production of IFN-I in tumor cells. Consistent with the above results, immunofluorescence microscopy confirmed that the colocalization of p-STING with the Golgi apparatus increased over time after hyperoxia culture ( Figure 4 E), the localization of p-IRF3 in the cell nucleus was significantly increased ( Figure 4 F). Compared with ADV alone, hyperoxia-sensitized ADV significantly increased serum levels of 4T1 and B16F10 tumor-bearing mice ( Figure 4 G, H) and tumors ( Figure 4I, J) levels of IFN-I in the tumor tissues. These findings indicate that hyperoxia-sensitized ADV treatment significantly activated the cGAS-STING-IFN-I pathway in ADV-insensitive tumors. The cGAS-STING-IFN-I pathway is known to activate innate immunity, and specific antigen-presenting DCs can be activated by IFN-I, which is required for T cell initiation. We detected tumor-infiltrating immune cells to study the interaction between hyperoxia-sensitized ADV treatment and immune cells. Compared with ADV alone, hyperoxia-sensitized ADV treatment significantly promoted the infiltration of DCs, CD4+ and CD8+ T cells into tumors ( Figure 4 KN), but did not affect the infiltration of NK cells, macrophages, or myeloid-derived suppressor cells (MDSCs) ( Figure 5 F). To further verify the role of cGAS in hyperoxia-sensitized ADV treatment, we knocked out cGAS in tumors to study whether the anti-tumor effect of hyperoxia-sensitized ADV was inhibited. We transfected 4T1 and B16F10 cells with cGAS knockdown lentivirus to reduce the expression of cGAS ( Figure 5 G, H). When cGAS expression was downregulated, 60% O2 could not reverse the ADV-induced downregulation of cGAS, p-STING, or p-IRF3 expression ( Figure 4 O). In addition, after hyperoxia sensitization ADV (ADV + 60% O2) treatment, the activation of IFN-I was impaired after artificially reducing the expression of cGAS ( Figure 4 Next, we constructed a cGAS knockdown 4T1 tumor model (4T1-shcGAS), in which mice were treated with hyperoxia-sensitized ADV as previously described. The results showed that the antitumor effect of hyperoxia-sensitized ADV treatment was significantly reversed ( Figure 4 Q). The cGAS inhibitor RU.521 also produced the same results ( Figure 4 These findings suggest that hyperoxia-sensitized ADV treatment exerts its therapeutic effects in a cGAS-dependent manner. Overall, hyperoxia reversed the hyporesponsiveness of 4T1 and B61F10 tumors to ADV and promoted the therapeutic effects of ADV by increasing cGAS expression and activating downstream pathways. Hyperoxia-sensitized ADV treatment effectively increased tumor infiltration by DCs, CD4+ T cells, and CD8+ T cells.

[0180] Example 4 Hyperoxia activates tumor-infiltrating T cells in solid tumors that initially respond poorly to ADV treatment via DCs, promoting ADV responsiveness

[0181] It is generally believed that sufficient IFN-I signaling is necessary for the recruitment and activation of proinflammatory immune cells. We confirmed that in the hyperoxia-sensitized ADV treatment group, the tumor infiltration of DCs, CD4+ T cells, and CD8+ T cells was higher. Therefore, it is necessary to further elucidate how DCs and T cells are activated. Compared with the ADV treatment group alone, the expression of type I interferon receptor (IFNAR-1) on DCs in the hyperoxia-sensitized ADV treatment group was significantly increased ( Figure 6 A). However, no similar pattern was observed in CD4+ T cells and CD8+ T cells ( Figure 7 A). Next, we extracted mouse bone marrow cells and induced them to differentiate into dendritic cells (BMDCs) in vitro ( Figure 7 B). We treated BMDCs with PBS, ADV, 60% O2, or ADV combined with 60% O2, but none of them promoted the maturation of BMDCs ( Figure 7 C). We then isolated the 4T1 cell culture (4T1 CM ), ADV-infected 4T1 cells (4T1 ADV ), 4T1 cells cultured at 60% O2 (4T1 60％O2 ) and 4T1 cells cultured with ADV+60% O2 (4T1 ADV+60％O2 ) to obtain conditioned medium and stimulate BMDCs in vitro. ADV Compared with the 4T1 ADV+60％O2 The proliferation of BMDCs in group A was significantly increased ( Figure 6 B) In addition, CD86 and MHC-II were expressed in 4T1 ADV+60％O2 The high expression of the group indicated that BMDCs were more mature ( Figure 6 C). These findings indicate that dendritic cells mature and can effectively activate T cells. When we artificially downregulated the expression of cGAS in 4T1 cells (4T1-shcGAS), the 60% O2 in the conditioned medium (4T1-shcGAS 60％O2 ) and ADV+60% O2(4T1-shcGAS ADV+60％O2 ) cultured 4T1-shcGAS cells did not promote high expression of CD86 or MHC-II ( Figure 6 C). We obtained the same results in vivo. We established 4T1 and 4T1-shcGAS tumor models. Downregulation of cGAS expression in tumors reversed the DC tumor infiltration promoted by hyperoxia-sensitized ADV treatment ( Figure 6 D), the maturity of DC also decreases ( Figure 6 E) These results collectively indicate that the promotion of DC tumor infiltration by hyperoxia-sensitized ADV treatment depends on activation of the tumor cGAS pathway.

[0182] Next, we determined the effect of hyperoxia-induced ADV treatment on cytotoxic T lymphocytes (CTLs). CD4+ and CD8+ T cells were isolated from mouse spleens and compared with 4T1 CM 、4T1 ADV 、4T1 60％O2 or 4T1 ADV+60％O2 The supernatant of treated 4T1 cells was co-cultured for 24 hours. There was no change in the expression of CD69 on CD4+ or CD8+ T cells ( Figure 7 D) However, when DCs and CD4+ T cells or DCs and CD8+ T cells in the co-culture system were used separately with 4T1 CM 、4T1 ADV 、4T1 60％O2 or 4T1 ADV+60％O2 When processing, 4T1 ADV +60％O2 The activity of CD4+ and CD8+ T cells in the group was significantly increased ( Figure 6 F). These findings suggest that DCs are required for T cell activation by hyperoxia-sensitized ADV therapy. One of the main factors for DCs to recruit T cells to tumors is the production of chemokines, of which chemokines CXCL9 and CXCL10 are the main chemokines. In 4T1 and B16F10 tumors, the expression of CXCL9 and CXCL10 was significantly increased in the hyperoxia-sensitized ADV treatment group ( Figure 6 GJ; Figure 7 E, F), the expression of effector IFN-γ in T cells was also significantly increased ( Figure 6 K), indicating that hyperoxia-sensitized ADV treatment leads to T cell recruitment and enhances T cell antitumor activity. Next, we aimed to demonstrate the important role of cGAS in promoting T cell tumor infiltration during hyperoxia-sensitized ADV treatment. After downregulating cGAS in tumors, the effects of combined treatment on promoting CD4+ and CD8+ T cell infiltration were reversed ( Figure 6 L, M). These results indicate that ADV therapy-mediated immune activation induced by hyperoxia is dependent on cGAS. To further verify the important role of DCs in ADV therapy induced by hyperoxia, we excised mouse DCs ( Figure 7 G), as expected, the antitumor effect of ADV was abolished after hyperoxia sensitization ( Figure 6 N). In addition, hyperoxia-sensitized ADV did not promote the infiltration of CD4+ T cells or CD8+ T cells into tumors ( Figure 7 H). Next, we treated Rag1-deficient T cells with PBS, ADV, hyperoxia, or hyperoxia-sensitized ADV. - / - In mice, the therapeutic effect of hyperoxia-sensitized ADV also disappeared ( Figure 6Taken together, these data indicate that the antitumor effects of hyperoxia-sensitized ADV depend on T cell activation by DCs, a process regulated by cGAS in tumors.

[0183] Example 5 Hyperoxia promotes ROS production, induces mtDNA release, and activates the cGAS-STING pathway, thereby sensitizing tumors to ADV treatment

[0184] Studies have shown that both normal pressure and high pressure hyperoxia can disrupt the oxidative balance of tumor cells, leading to the production of excessive reactive oxygen species (ROS). Mitochondria are the main site of ROS production. Abnormal accumulation of ROS can open the mitochondrial permeability transition pore (mPTP), damage mitochondria, and cause the release of mtDNA into the cytoplasm. Therefore, we hypothesize that excessive ROS produced by hyperoxia damages mitochondria and releases mtDNA into the cytoplasm. Once mtDNA enters the cytoplasm, it acts as a damage-associated molecular pattern (DAMP) to activate cGAS and STING. As expected, the fluorescence of the probe DCFH-DA was significantly enhanced, indicating that hyperoxia significantly induced ROS in 4T1 and B16F10 tumor tissues ( Figure 8 A, B). Due to abnormalities in the redox and metabolic systems of tumor cells, tumor cells appear to be more susceptible to the effects of oxygen. Compared with the human immortalized epidermal cell line HaCaT and the mouse normal liver cell line AML-12, tumor cell lines treated with 60% O2 appear to produce more ROS ( Figure 9 A). Under 60% O2 conditions, the longer the tumor cells were cultured, the more ROS were produced, reaching a maximum at 24 hours ( Figure 9 B). Compared with other normal tissues (heart, liver, and lung) of mice, ROS production in tumor tissues is higher ( Figure 8 C). There are many types of ROS, including hydrogen peroxide (H2O2), superoxide anion (O2 .- ) and hydroxyl radicals (OH .- The results showed that after cells were treated with 60% O2, O2 .- Significantly increased in 4T1 and B16F10 cells ( Figure 9 C, D), indicating that the type of ROS produced by hyperoxia is O2. - Immunofluorescence analysis showed that the co-localization of mitochondria and ROS increased with the prolonged O2 exposure ( Figure 8 D), while calcein fluorescence decreased significantly, indicating that mPTP was open in 4T1 and B16F10 cells ( Figure 8 E). However, exposure to 60% O2 and n-acetylcysteine (NAC), a traditional antioxidant that can scavenge intracellular ROS, reduced the degree of mitochondrial mPTP opening in 4T1 and B16F10 cells to normal levels ( Figure 8 E). In addition, we used transmission electron microscopy (TEM) to observe mitochondrial morphology and found that compared with the ADV group, the mitochondria in the hyperoxia-sensitized ADV group were swollen, and the cristae were atrophied or disappeared ( Figure 8 F) These results indicate that hyperoxia induces increased ROS production in tumor cell mitochondria, leading to mitochondrial damage.

[0185] Studies have shown that cGAS can respond to mitochondrial double-stranded DNA. We isolated mitochondria from cells and measured whether mtDNA (Dloop1, Dloop2, Dloop3, CytB, Nd1, and Nd4) leaked from mitochondria to the cytoplasm. Compared with ADV treatment alone, the amount of mtDNA in the cytoplasm increased significantly ( Figure 8 G, H), after hyperoxia-sensitized ADV treatment of 4T1 and B16F10 tumor tissues, the level of 8-hydroxydeoxyguanosine (8-OH dG), a DNA oxidation indicator, also increased ( Figure 8 I). In B16F10 cells, the co-localization of cGAS and mtDNA also became more obvious after prolonged 60% O2 treatment ( Figure 8 J). Next, we treated mice with hyperoxia-sensitized ADV and NAC. After NAC treatment, the fluorescence of DCFH-DA in tumor tissues of the hyperoxia and hyperoxia-sensitized ADV groups was significantly reduced, indicating that ROS generation was reduced ( Figure 8 K, L). Activation of the cGAS-STING-IFN-I pathway was reversed ( Figure 8 M, N). In addition, the antitumor effect of ADV sensitized by hyperoxia was significantly reversed ( Figure 8 O, P). To demonstrate that mtDNA is a DAMP that triggers cGAS activation in 4T1 and B16F10 cells, we treated 4T1 and B16F10 cells with ddC, a nucleoside reverse transcriptase inhibitor that specifically inhibits mtDNA replication and leads to mtDNA depletion. Notably, the combined treatment of hyperoxia-sensitized ADV with ddC significantly decreased the protein levels of cGAS and downstream factors in 4T1 and B16F10 cells compared with those after hyperoxia-sensitized ADV treatment ( Figure 8 Q). These results suggest that mtDNA is a trigger for cGAS activation. These experiments reveal the mechanism by which hyperoxia promotes tumor sensitivity to ADV therapy: hyperoxia leads to excessive mitochondrial ROS in tumors, causing mitochondrial damage. The mtDNA in the damaged mitochondria leaks into the cytoplasm, triggering activation of the cGAS-STING pathway.

[0186] Example 6 Hyperoxia can effectively enhance the therapeutic effect of solid tumors that initially respond well to ADV and can serve as an immune adjuvant for multiple OVs

[0187] Since hyperoxia effectively enhanced the efficacy of ADV therapy in solid tumors that had a poor response to initial ADV treatment, we wondered whether hyperoxia could further enhance the efficacy of ADV therapy in tumors that had a good response to initial ADV treatment. The synergistic effect of hyperoxia and ADV treatment on CT26 and H22 solid tumors was significantly stronger than that on ADV treatment alone ( Figure 10 A, B). cGAS and STING genes were also significantly upregulated in CT26 and H22 tumors ( Figure 11 A, B). Flow cytometry analysis showed that the synergistic effect of hyperoxia and ADV increased DC ( Figure 10 C), CD4+ T cells, and CD8+ T cells in tumor infiltration ( Figure 10 D, E). These results indicate that in tumors that initially responded well to ADV treatment, hyperoxia can still enhance the immune effect of ADV by activating the cGAS-STING pathway. To determine the universality of hyperoxia in promoting the efficacy of OVs, we selected two other OVs used in clinical studies, HSV and VV, to verify the synergistic effect of hyperoxia on B16F10 and CT26 subcutaneous tumor models. The results showed that hyperoxia significantly enhanced the anti-tumor effects of HSV and VV ( Figure 11 C, D), promote the infiltration of CD4+T cells and CD8+T cells into tumors ( Figure 11 EH). After combined treatment, the levels of effector factors granzyme B and IFN-γ in TME CD8+ T cells were also significantly increased ( Figure 11 IL). Similarly, as expected, the expression of IFN-I in mouse serum was significantly increased ( Figure 10 F, G; Figure 11 The above results confirm that hyperoxia can also promote the anti-tumor immunity of HSV and VV.

[0188] Example 7 Hyperoxia can enhance the therapeutic effect of ADV in humanized PDX and CDX tumor models, preliminarily demonstrating the safety of this therapy

[0189] To further explore the potential of hyperoxia-sensitized ADV therapy in clinically relevant animal models, we used humanized tumor cell line-derived xenograft (CDX) models and patient-driven xenograft (PDX) models ( Figure 10 H; Figure 12 A). Hyperoxia-sensitized ADV treatment significantly inhibited the growth of CDX and PDX tumors ( Figure 10 I, J), and effectively prolonged the survival of tumor-bearing mice ( Figure 10 K, L). We also observed a significant increase in CD8+ T cells and IFN-γ expression ( Figure 10 M, N; Figure 12 B). Importantly, there was no significant change in the body weight of the mice after treatment ( Figure 12 C, D), including the previously mentioned B16F10 and 4T1 mouse models ( Figure 12 E, F). Hematoxylin-eosin (H&E) staining showed no obvious toxic effects on any tissue ( Figure 12 G), demonstrating the safety of the combined treatment strategy. In summary, in a clinically relevant animal model, hyperoxia-sensitized ADV therapy demonstrated superior efficacy compared to monotherapy and showed preliminary safety. Therefore, hyperoxia therapy may be a promising approach to enhance the clinical effectiveness of OV therapy and has positive prospects for clinical translation.

[0190] Example 8 Synergistic Effects of Normal Pressure Hyperoxia and High Pressure Hyperoxia on Different Oncolytic Viruses

[0191] In the tumor-bearing models of breast cancer, liver cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, glioma or bladder cancer in immune-normal mice, compared with normoxia at normal pressure, hyperoxia at normal pressure significantly improved the therapeutic effect of oncolytic adenovirus (Type V adenovirus), oncolytic reovirus (Oncolytic orthoreovirus), oncolytic Coxsackievirus (Coxsackievirus A21), oncolytic Newcastle disease virus (NDV Herts / 33 strain), oncolytic measles virus (Oncolytic measles virus Edmonston strain), oncolytic vesicular stomatitis virus (Mudd-Summer strain, Indiana serotype), oncolytic poliovirus type 1, sabin strain), oncolytic herpes simplex virus type 1, strain F or oncolytic vaccinia virus (Elstree strain), significantly inhibited tumor volume and significantly prolonged the survival time of tumor-bearing mice.

Claims

1. A use of high-concentration oxygen in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, wherein the high-concentration oxygen is normal pressure high-concentration oxygen or high pressure high-concentration oxygen.

2. The use of a high-concentration oxygen according to claim 1 in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The high-concentration oxygen at normal pressure has an oxygen concentration of more than 20.9%.

3. The use of a high-concentration oxygen according to claim 1 in the preparation of a drug for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The high-pressure and high-concentration oxygen has an oxygen concentration of more than 20.9% and a pressure of more than one standard atmospheric pressure, that is, 1.013*10^5Pa.

4. Application of a high-concentration oxygen device in the preparation of an instrument for enhancing the immunotherapy effect of oncolytic viruses, wherein the high-concentration oxygen is normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen.

5. Use of a high-concentration oxygen device according to claim 4 in the preparation of a device for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The normal pressure high concentration oxygen device injects oxygen with a concentration higher than 20.9% into a nasal inhaler, an oral inhaler, a mask or a cabin through an oxygen generator, so that the oxygen concentration in the nasal inhaler, an oral inhaler, a mask or a cabin is higher than 20.9%.

6. Use of a high-concentration oxygen device according to claim 4 in the preparation of a device for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The high-pressure, high-concentration oxygen device injects oxygen with a concentration higher than 20.9% into the cabin through an oxygen production device, so that the oxygen concentration in the cabin is higher than 20.9%; at the same time, the cabin is pressurized through an air compressor, a high-pressure air tank or a high-pressure oxygen tank, so that the pressure in the cabin is above a standard atmospheric pressure, that is, 1.013*10^5Pa.

7. A use of high-concentration oxygen in the preparation of a drug that promotes ROS production in tumors, wherein the high-concentration oxygen is normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen. The normal-pressure high-concentration oxygen has an oxygen concentration of greater than 20.9%; the high-pressure high-concentration oxygen has an oxygen concentration of greater than 20.9%, and the pressure is above one standard atmospheric pressure, i.e., 1.013*10^5 Pa.

8. Use of a high-concentration oxygen device in the preparation of an instrument for promoting ROS production in tumors, wherein the high-concentration oxygen is normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen. The normal-pressure high-concentration oxygen has an oxygen concentration of greater than 20.9%; the high-pressure high-concentration oxygen has an oxygen concentration of greater than 20.9%, and the pressure is above one standard atmospheric pressure, i.e., 1.013*10^5 Pa.

9. Use of a high-concentration oxygen device according to claim 8 in the preparation of a device for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The high-pressure, high-concentration oxygen device injects oxygen with a concentration higher than 20.9% into the cabin through an oxygen production device, so that the oxygen concentration in the cabin is higher than 20.9%; at the same time, the cabin is pressurized through an air compressor, a high-pressure air tank or a high-pressure oxygen tank, so that the pressure in the cabin is above a standard atmospheric pressure, that is, 1.013*10^5Pa.

10. Use of a high-concentration oxygen device according to claim 8 in the preparation of a device for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The normal pressure high concentration oxygen device injects oxygen with a concentration higher than 20.9% into a nasal inhaler, an oral inhaler, a mask or a cabin through an oxygen generator, so that the oxygen concentration in the nasal inhaler, an oral inhaler, a mask or a cabin is higher than 20.9%.

11. A use of high-concentration oxygen in the preparation of a drug that promotes tumor mitochondrial DNA leakage, wherein the high-concentration oxygen is normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen, wherein the normal-pressure high-concentration oxygen has an oxygen concentration of greater than 20.9%; the high-pressure high-concentration oxygen has an oxygen concentration of greater than 20.9%, and the pressure is above one standard atmospheric pressure, i.e., 1.013*10^5Pa.

12. The use of a high-concentration oxygen device in the preparation of an instrument for promoting the leakage of tumor mitochondrial DNA, wherein the high-concentration oxygen is normal-pressure high-concentration oxygen or high-pressure high-concentration oxygen, wherein the normal-pressure high-concentration oxygen has an oxygen concentration of more than 20.9%; the high-pressure high-concentration oxygen has an oxygen concentration of more than 20.9%, and the pressure is above one standard atmospheric pressure, i.e., 1.013*10^5Pa.

13. Use of a high-concentration oxygen device according to claim 12 in the preparation of a device for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The high-pressure, high-concentration oxygen device injects oxygen with a concentration higher than 20.9% into the cabin through an oxygen production device, so that the oxygen concentration in the cabin is higher than 20.9%; at the same time, the cabin is pressurized through an air compressor, a high-pressure air tank or a high-pressure oxygen tank, so that the pressure in the cabin is above a standard atmospheric pressure, that is, 1.013*10^5Pa.

14. Use of a high-concentration oxygen device according to claim 12 in the preparation of a device for enhancing the immunotherapy effect of oncolytic viruses, characterized in that: The normal pressure high concentration oxygen device injects oxygen with a concentration higher than 20.9% into a nasal inhaler, an oral inhaler, a mask or a cabin through an oxygen generator, so that the oxygen concentration in the nasal inhaler, an oral inhaler, a mask or a cabin is higher than 20.9%.

15. Use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors.

16. The use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors according to claim 15, characterized in that: The high-concentration oxygen is high-concentration oxygen at normal pressure.

17. The use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors according to claim 16, characterized in that: The high-concentration oxygen at normal pressure has an oxygen concentration of more than 20.9%.

18. The use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors according to claim 15, characterized in that: The high-concentration oxygen is high-pressure high-concentration oxygen.

19. The use of high-concentration oxygen in the preparation of a drug for activating the cGAS-STING pathway in tumors according to claim 18, characterized in that: The high-pressure and high-concentration oxygen has an oxygen concentration of more than 20.9% and a pressure of more than one standard atmospheric pressure, that is, 1.013*10^5Pa.

20. Use of a high-concentration oxygen device in the preparation of an apparatus for activating the cGAS-STING pathway in tumors.

21. Use of the high-concentration oxygen device according to claim 20 in preparing a device for activating the cGAS-STING pathway in tumors, wherein: The high-concentration oxygen device is a normal-pressure high-concentration oxygen device.

22. Use of the high-concentration oxygen device according to claim 21 in the preparation of a device for enhancing activation of the cGAS-STING pathway in tumors, wherein: The normal pressure high concentration oxygen device injects oxygen with a concentration higher than 20.9% into a nasal inhaler, an oral inhaler, a mask or a cabin through an oxygen generator, so that the oxygen concentration in the nasal inhaler, an oral inhaler, a mask or a cabin is higher than 20.9%.

23. Use of the high-concentration oxygen device according to claim 20 in preparing a device for activating the cGAS-STING pathway in tumors, wherein: The high-concentration oxygen device is a high-pressure high-concentration oxygen device.

24. Use of the high-concentration oxygen device according to claim 23 in preparing a device for activating the cGAS-STING pathway in tumors, wherein: The high-pressure, high-concentration oxygen device injects oxygen with a concentration higher than 20.9% into the cabin through an oxygen production device, so that the oxygen concentration in the cabin is higher than 20.9%; at the same time, the cabin is pressurized through an air compressor, a high-pressure air tank or a high-pressure oxygen tank, so that the pressure in the cabin is above a standard atmospheric pressure, that is, 1.013*10^5Pa.

25. Use of the high-concentration oxygen or high-concentration oxygen device according to any one of claims 1 to 24, characterized in that: The oncolytic virus includes but is not limited to oncolytic adenovirus, oncolytic reovirus, oncolytic coxsackievirus, oncolytic Newcastle disease virus, oncolytic measles virus, oncolytic vesicular stomatitis virus, oncolytic poliovirus, oncolytic herpes simplex virus or oncolytic vaccinia virus.

26. Use of the high-concentration oxygen or high-concentration oxygen device according to any one of claims 1 to 24, characterized in that: The tumor or tumour includes but is not limited to breast cancer, liver cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, glioma or bladder cancer.

27. Use of the high-concentration oxygen or high-concentration oxygen device according to claim 25, characterized in that: The tumor or tumour includes but is not limited to breast cancer, liver cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, glioma or bladder cancer.

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