Oncolytic adenovirus synergistically treats liver metastases from colon cancer with PARPi by increasing DNA damage and activating the STING pathway

Oncolytic adenovirus synergistically treats liver metastases from colon cancer with PARPi by increasing DNA damage and activating the STING pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oncolytic adenovirus synergistically treats liver metastases from colon cancer with PARPi by increasing DNA damage and activating the STING pathway Yuting Tang, Wen Hu, Benyuan Zhou, Xinyu Feng, Si Liu, Liang-Liang Weng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8189652/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Colorectal cancer (CRC) frequently metastasizes to the liver, leading to a poor prognosis and limited therapeutic options compared to other liver cancers, such as hepatocellular carcinoma (HCC). In this study, we selected the poly (ADP-ribose) polymerase inhibitors (PARPi) and Oncolytic adenoviruses (oAdv) to treat colorectal cancer liver metastases (CRLM). Methods The transcriptional differences between CRLM and HCC were compared using the GSE243245 datasets. The synergistic potential of oAdv and the PARPi was assessed with in vitro and in vivo experiments. The therapeutic efficacy and safety of the combination therapy were evaluated in subcutaneous xenograft and colorectal liver metastasis (CRLM) mouse models. The mechanism of the combination of PARPi and oAdv was further investigated using DNA damage assays and Western blotting (WB). Immune responses to CRLM were assessed using flow cytometry, Western blotting, and ELISpot assays. Results oAdv infection selectively enhanced DNA damage in CRC cells, significantly increasing their sensitivity to AZD2461, while sparing normal hepatocytes. In murine models, the combination therapy demonstrated superior tumor growth inhibition (51.56%) compared to monotherapies. The synergistic effect was particularly pronounced in the CRLM model, correlating with activation of the STING pathway, enhanced immune cell infiltration, and a specific immune response. The therapeutic benefits on the immune response were abrogated by cGAS-Sting pathway inhibition. Conclusion The combination of oAdvand PARPi represents a safe and effective strategy for treating colorectal liver metastases, functioning through tumor-selective enhancement of DNA damage and activation of the cGAS-Sting-mediated immune response. colorectal liver metastasis poly (ADP-ribose) polymerase inhibitor oncolytic adenovirus cGAS-Sting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Colorectal cancer (CRC) is the third most common cancer worldwide, and the survival of patients with early-stage colon cancer can be significantly prolonged by traditional treatments such as surgical resection, radiotherapy, and chemotherapy[ 1 , 2 ]. Despite the availability of detection techniques, most patients with colorectal cancer (CRC) are diagnosed at an advanced, unresectable stage due to the limitations of current methods. Colorectal liver metastasis (CRLM) accounts for nearly half of unresectable metastases, and according to the National Comprehensive Cancer Network (NCCN), fewer than 25% of patients with CRLM are eligible for liver transplantation[ 3 , 4 ]. However, several first-line chemotherapy regimens have proven ineffective in controlling CRLM, and they are limited by high toxicity and the development of drug resistance[ 5 – 7 ]. A more comprehensive and safer therapeutic strategy is needed to improve the outcome for patients with CRLM. Due to the extensive vascular distribution and rich blood perfusion of the liver, immunotherapy such as anti-PD-L1 antibody has shown clinical benefit in HCC treatment[ 8 , 9 ]. However, unlike HCC, CRLM are advanced metastatic lesions of CRC. The immune microenvironment of CRLM is much “hotter” than that of HCC due to lower levels of tumor-associated macrophages and regulatory T cell infiltration[ 10 , 11 ]. Additionally, CRLM also inherit some characteristics of CRC. Microsatellite instability (MSI)/DNA mismatch repair deficiency (dMMR) serve as biomarkers for colorectal cancer (CRC) that are recommended by the NCCN guidelines[ 12 ]. The guidelines also suggest that colorectal liver metastases (CRLM) may frequently exhibit DNA damage repair (DDR) deficiency, which could make them potential beneficiaries of Poly(ADP-Ribose) Polymerase inhibitor (PARPi) therapy. Currently, multiple clinical trials are exploring the safety and preliminary efficacy of PARPi in CRC[ 13 ]. Since the mechanism of PARPi was first reported in 2004, several clinical trials have confirmed the efficacy of PARPi in cancers with BRCA1/2 mutations (BRCAm) or homologous recombination deficiency (HRD)[ 14 , 15 ]. PARPi, when combined with chemotherapy, which disrupts DNA replication and increases DNA repair damage, has demonstrated potential for the clinical treatment of CRC[ 16 ]. However, due to the low incidence of double allelic loss of genes involved in homologous recombination in CRC, patients in this study derived limited benefit from PARPi treatment and experienced significant hematologic toxicity[ 13 , 17 ]. Therefore, we selected AZD2461, which has a better safety than the approved Olaparib[ 18 – 20 ]. Furthermore, we replaced less specific chemotherapeutic agents with oncolytic adenovirus (oAdv) for combination therapy to increase DNA damage. By partially deleting the E1 region of adenovirus 5 (Ad5), oAdv was limited to replicating only in tumor cells[ 21 ]. Additionally, the coxsackie and adenovirus receptor (CAR), which is critical for adenovirus infection, is strongly expressed in the live[ 20 ]. This allowed oAdv to selectively accumulate in CRLM tumors and subsequently increase DNA damage during virus replication[ 22 ]. In this study, oAdv was administered as an adjuvant-like supplementary drug at a very low dose. In this study, we examined the efficacy of combination treatment with poly (ADP-ribose) polymerase inhibitors (PARPi) in CRLM. The efficacy and safety of the combination were evaluated in vitro and in vivo . 2. Materials and methods 2.1 Animals Thirty-five BALB/c nude mice (6–8 weeks old) and sixty-two C57BL/6 mice (6–8 weeks old) were housed in a light- and temperature-controlled room. The mice were allowed to acclimatize to their new environment for at least 7 days before the start of the experiments. All animal experimental procedures were approved by the Zhoushan Branch of Ruijin Hospital Affiliated to Shanghai under a specific pathogen-free protocol (ethics no. ZSRJA-20230703-02). 2.2. RNA-seq and analysis The RNA-seq dataset GSE243245 from GEO was selected to investigate the transcriptional differences between HCC and CRLM. The gene expression profiles of each group were further analyzed using R (version 4.3.2). The correlations between groups were analyzed using principal component analysis (PCA), and the correlation matrices were calculated using PCAtools (version 2.18.0) and pheatmap (version 1.0.12). The differentially expressed genes (DEGs) between groups were subsequently compared using DESeq2 (version 1.40.2). The DEGs between each group were defined by a P value 1. The overlap of DEGs between groups was compared using a Venn diagram (version 1.7.3). The DEGs between the HCC and CRLM groups were annotated using gene set enrichment analysis (GSEA) with clusterProfiler (version 4.8.3). GSEA was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, with a threshold of q values < 0.05. GO enrichment was performed for biological process (BP) terms. The DEGs of interest were further analyzed using protein‒protein interaction (PPI) network analysis with STRING ( https://string-db.org/ , version 12.0). 2.3. Cell lines and culture condition Three human CRC cell lines (Colo-205, HCT-116, and HT-29) and human primary liver fibroblast (HPLF) cells were purchased from iCell Bioscience (China). The mouse CRC cell line MC-38 was purchased from the National Collection of Authenticated Cell Cultures (China). All cell lines were authenticated by short tandem repeat profiling before purchase. HT-29 and HCT-116 were maintained in McCoy's 5A Medium (CAT: 12330031, Gibco, USA). Colo-205 was maintained in RPMI-1640 medium (CAT: 61870036, Gibco, USA). HPLF and MC-38 were maintained in DMEM medium (CAT: 11995065, Gibco, USA). All media were supplemented with 10% fetal bovine serum (CAT: 16000-044, Gibco, USA) and 1% penicillin/streptomycin (CAT: V900929, Sigma-Aldrich, USA). Cells were incubated in a humidified atmosphere containing 5% CO₂ at 37°C. 2.4. Drug preparation AZD2461 (HY-13536) and C-176 (HY-112906) were purchased from MCE (USA) and diluted in PBS containing 10% DMSO (CAT: D2650, Sigma-Aldrich, USA). The AZD2461 and C-176 solutions were sterilized using a 0.22 µm filter before use. The oncolytic adenovirus (oAdv) was constructed as previously reported of our laboratory[ 23 ]. Viral titers were determined by median tissue culture infectious dose (TCID₅₀) assay. 2.5. Cell viability assay The three human CRC cell lines, MC-38 cells, and HPLF cells were seeded into 96-well plates at a density of 3,000 cells per well and allowed to adhere overnight. The synergistic effect of oAdv and AZD2461 was compared in vitro. AZD2461 was added to all experimental groups at concentrations of 10, 5, 2.5, 1.25, 0.63, 0.16, 0.08, and 0.04 µM/mL in triplicate. The combination groups were further infected with oAdv at a multiplicity of infection (MOI) of 10. All drug solutions were diluted in 100 µL of medium, and 100 µL of medium was added to the negative controls. MC-38 cells were treated with the same concentration gradient of AZD2461 and oAdv. Then, 10 µL of CCK-8 solution (CAT: CK04, Dojindo) was added to each well to measure cell viability. The plates were incubated for 4 hours, and absorbance was measured at 450 nm using a microplate reader to determine the 50% inhibitory concentration (IC₅₀) for each assay. 2.6. DNA damage assay DNA damage was assessed in vitro using fluorescence-activated cell sorting (FACS). MC-38, Colo-205, and HPLF cells were seeded into six-well plates at a density of 5,000 cells per well and cultured overnight. oAdv was added at a multiplicity of infection (MOI) of 10, and AZD2461 was added at 1 µM/mL in triplicate. All cells were collected 24 hours after treatment. Cells were fixed with Phosflow™ Fix Buffer I (CAT: 557870, BD, USA) for 10 minutes at 37°C and then permeabilized with Perm Buffer III for 30 minutes on ice. The cells were washed three times with PBS and stained with an FITC-conjugated anti-phospho-H2A.X (Ser139) antibody (CAT: 613403, BioLegend, USA). DNA damage was determined by the percentage of γ-H2A.X-positive cells. 2.7. Colo-205 xenograft models Thirty-two BALB/c nude mice (6–8 weeks old) were used to establish a Colo-205 xenograft model. The xenograft model was established by subcutaneous injection of Colo-205 cells (5×10⁶ cells in 100 µL) into the right flank after confirming cell viability using Trypan blue staining (CAT: 15250061, Invitrogen, USA). When the tumors reached approximately 100 mm³, the mice were randomly divided into four groups (n = 8 per group) using Study Director software (Studylog Systems, Inc., USA). Mice in the oAdv group received intravenous injections of 100 µL oAdv (1×10⁹ PFU/mL) on days 1, 3, and 5 after grouping. Mice in the AZD2461 group received intravenous injections of AZD2461 (1 mg/kg) on days 1, 2, 3, 4, 5, 7, and 9 after grouping. Mice in the AZD2461 + oAdv group received both oAdv and AZD2461 treatments as described above. Mice in the control group received intravenous injections of 100 µL PBS on days 1, 2, 3, 4, 5, 7, and 9 after grouping. Tumor volume was calculated using the formula (length × width²)/2 and measured every 3 days. All mice were euthanized 28 days after grouping. 2.8. Biodistribution assay Three mice bearing Colo-205 xenografts received 100 µL of oAdv (1×10⁹ PFU/mL) via intravenous injection. All mice were euthanized 7 days post-injection, and samples were collected. DNA was extracted from heart, liver, blood, lung, kidney, and tumor tissues using the TIANamp Genomic DNA Kit (DP304, Tiangen, China). Quantitative PCR (qPCR) was performed using verified primers[ 24 ]. Linearized oAdv vector (1×10⁸ copies) was used as the reference control. Then, 1 µg of DNA from each sample was used, and the relative copy number in each tissue was calculated as: (total DNA extracted) × (relative DNA level). 2.9.MC-38 CRLM model Forty C57BL/6 mice (6–8 weeks old) were used to establish the colorectal liver metastasis (CRLM) model. The model was established via selective portal vein injection as previously reported[ 25 ], with subsequent splenectomy. Cell viability was confirmed as described above. Two weeks after cell injection, the mice were randomly divided into four groups (n = 10 per group). The day of grouping was designated day 0, and grouping and treatment were performed as described for the MC-38 xenograft model. The survival of all mice was monitored continuously until day 40 after grouping. In accordance with animal welfare guidelines, mice exhibiting > 20% weight loss or ataxia were considered moribund, recorded as dead, and euthanized. 2.10. Liver immune infiltration and Sting pathway activate detection assay Twelve mice spared from the CRLM model were used to compare immune infiltration and protein level changes in the liver using flow cytometry (FACS) and western blotting (WB). The mice were divided into two groups (n = 6 per group): the control group and the AZD2461 + oAdv group, following the COLO-205 model protocol. Mice in the C-176 group received intravenous injections of oAdv and AZD2461 in combination. Single-cell suspensions (SCS) were prepared from the entire liver. Cell viability was determined using FVS510 staining, and lymphocytes were identified by CD45 expression. Liver-infiltrating lymphocytes were defined as FVS510 (CAT: 544406, BD, USA) and CD45 (CAT: 103315, BioLegend, USA) double-positive cells. Proteins were extracted from the liver SCS. A total of 30 µg of protein per sample was loaded into each well. Western blotting (WB) analysis was performed using standard protocols. The primary antibodies used were anti-GAPDH (CAT: 5174, 1:1000), anti-STING (CAT: 13647, 1:1000), anti-phospho-TBK (pTBK, CAT: 3036, 1:1000), and anti-TBK (CAT: 36169, 1:1000), all purchased from Cell Signaling Technology (CST, USA). The secondary antibody (CAT: A0208, 1:5000) was purchased from Beyotime (China). 2.11. Sting inhibitor treatment assay Ten mice spared from the CRLM model were used to compare immune infiltration and protein level changes in the liver using FACS, WB, and ELISpot assays. The mice were divided into two groups (n = 5 per group) and treated as the AZD2461 + oAdv group according to the COLO-205 model protocol. Mice in the AZD2461 + oAdv + C-176 group received intravenous injections of oAdv and AZD2461 following the same regimen as the AZD2461 + oAdv group. Subsequently, they received daily intravenous injections of 750 nmol C-176 for 7 days, starting 3 days after the initial treatment, to inhibit STING pathway activation. FACS and WB analyses were performed as described above. PBMCs from each group were isolated using Mouse Lymphocyte Separation Medium (CAT: 7211011, Dakewe, China). PBMCs from the same group were pooled to detect tumor-specific T cells using an IFN-γ ELISpot kit (CAT: 3321, Mabtech, Sweden). Fresh PBMCs (3 × 10⁵ cells per group) were pooled and seeded into wells. Each group had three replicates, and splenocytes from C57BL/6 mice without MC-38 treatment served as the negative control. Protein (50 µg) extracted from MC-38 cells was added to each well. Cells and protein were maintained in RPMI-1640 medium and incubated for 16 h. After 16 h, cells and medium were removed, and spots were detected according to the manufacturer’s protocol. Plates were read and counted using a CTL ImmunoSpot reader (CTL Technologies, USA). 2.12. Statistical analysis The in vivo distribution analysis was performed using a paired Student's t-test. Other results were analyzed using an unpaired Student's t-test or ANOVA or multiple t-test. All data are presented as the mean ± standard deviation. IC50 values were calculated using four-parameter logistic regression. Survival curves were compared using the log-rank test. All analyses were conducted using GraphPad Prism 8.0 software (GraphPad Software, Inc., USA). 3. Results 3.1 The CRLM shown RNA-seq was performed on tumor tissues from each group in the GSE243245 dataset. The dataset contains tumor sequencing data from 123 patients, with 61 in the HCC group and 62 in the CRLM group. The gene expression profile revealed significant fluctuations both within and between groups. Despite this heterogeneity, CRLM and HCC groups remained well distinguished overall. (Fig. 1 A, 1 B). A total of 3826 differentially expressed genes (DEGs) were identified between the HCC and CRLM groups. The DEGs between the CRLM and HCC groups were further analyzed using Gene Set Enrichment Analysis (GSEA) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. A total of 1278 GO biological process (BP) terms were significantly enriched ( P < 0.05). Compared to the CRLM group, the HCC group showed 622 activated pathways and 656 inhibited pathways. The GO analysis revealed that the most significantly enriched BP terms included positive regulation of response to jasmonic acid, cellular response to jasmonic acid stimulus, and chylomicron remnant clearance. Synaptic signaling, nervous system process, and monoatomic ion transmembrane transport were the main terms associated with negatively regulated BP (Fig. 1 C). A total of 98 KEGG pathways were significantly enriched (P < 0.05). The HCC group showed 58 activated pathways and 40 inhibited pathways. KEGG analysis indicated that pathways primarily involving primary bile acid biosynthesis, lipoic acid metabolism, and biosynthesis of unsaturated fatty acids were activated, whereas pathways involving taste transduction, muscle cell cytoskeleton organization, and cornified envelope formation were inhibited (Fig. 1 D). A. PCA analysis between HCC group and CRLM group; B. Correlation analysis between HCC group and CRLM group; C. GSEA for GO terms between HCC group and CRLM group; D. GSEA for KEGG terms between HCC group and CRLM group. Significant changes were observed between the CRLM and HCC groups in pathways related to cell replication and DNA replication, including positive regulation of cell population proliferation, nucleotide biosynthetic process, and ATP biosynthetic process. The normalized enrichment scores (NES) for these three pathways were − 1.26, 1.33, and 1.47 (Fig. 2 A). In contrast, immune-related pathways showed few significant differences between HCC and CRLM, with only complement activation exhibiting a significant difference (Fig. 2 B). Expression profiling and PPI analysis of DEGs of interest revealed that PARP and several related genes exhibited lower expression in CRLM. Key proteins in immune-related pathways, such as cGAS and TBK in the cGAS-STING pathway and B2M in antigen presentation and processing pathways, demonstrated divergent expression trends (Fig. 2 C-E). A. GSEA of cell proliferation related pathway; B. GSEA of immunity related pathway. C. Heatmap of 79 selected DEGs; D. Protein-protein interaction (PPI) network analysis via STRING of 79 selected DEGs; E. Compared read count of key gene between HCC group and CRLM group. * P < 0.05, *** P < 0.001. 3.1. oAdv selective improved the PARPi effect via increased DNA damage in CRC cell The potency of oAdv and AZD2461 was measured in three different CRC cell lines of human by single and combination. The IC50 of AZD2461 in HT-29 and HCT-116 was 2.20 and 0.65 µM/mL, respectively. And Colo-205 was not sensitive to AZD2461 (Fig. 3 A), the IC50 of AZD2461 in Colo-205 was more than 10.00 µM/mL. The IC50 of oAdv in HT-29, Colo-205, and HCT-116 was 16.16, 402.80 and 8.05 MOI, respectively (Fig. 3 B). 10 MOI was selected as the combined dose of oAdv at a concentration which as a poor effect on tumor cell viability. The IC50 of combination in HT-29, Colo-205, and HCT-116 was 0.20, 0.92, and 0.24 µM/mL, respectively (Fig. 3 C). The potency of the combination group was higher than that of the two single-drug groups, indicating the synergistic effect of oAdv supplementation with AZD2461. Then the safety of combination treatment was tested with the normal liver cell line HPLF. As the result, the high dose of AZD2461 didn't kill HPLF and the oAdv supplementation also not reduced the cell viability (Fig. 3 D). In addition, H2X.A phosphorylation levels in infected Colo-205 and HPLF cells at the same concentrations were detected by FACS. Infection with oAdv significantly increased the DNA damage in the Colo-205, but didn`t affect HPLF (Fig. 3 D, 3 E). These suggests that the combination of AZD2461 and oAdv had the potential to treat CRLM in vivo by selectively affecting CRC cells but not hepatocytes. The potency of oAdv and AZD2461 was assessed in three human CRC cell lines using single-agent and combination treatments. The IC50 values for AZD2461 in HT-29 and HCT-116 cell lines were 2.20 and 0.65 µM/mL, respectively. Colo-205 cells were insensitive to AZD2461 (Fig. 3 A), with an IC50 value exceeding 10.00 µM/mL. The IC50 values for oAdv in HT-29, Colo-205, and HCT-116 cell lines were 16.16, 402.80, and 8.05 MOI, respectively (Fig. 3 B). An oAdv concentration of 10 MOI was selected as the combined dose, as it had a minimal effect on tumor cell viability. The IC50 values for the combination treatment in HT-29, Colo-205, and HCT-116 cell lines were 0.20, 0.92, and 0.24 µM/mL, respectively (Fig. 3 C). The combination treatment exhibited greater potency than either single-drug treatment, suggesting a synergistic effect of oAdv in combination with AZD2461. The safety of the combination treatment was evaluated using the normal liver cell HPLF. The results showed that high doses of AZD2461 did not affect HPLF viability, nor did oAdv supplementation reduce cell viability (Fig. 3 D). Additionally, H2AX phosphorylation levels were measured in infected Colo-205 and HPLF cells at equivalent concentrations using FACS. oAdv infection significantly induced DNA damage in Colo-205 cells but had no effect on HPLF cells (Fig. 3 D, 3 E). These findings suggest that the combination of AZD2461 and oAdv has potential for in vivo treatment of CRLM by selectively targeting CRC cells without affecting hepatocytes. A. The endpoint cell viability was determined at 48h after pre-seeded CRC cell lines were treated with different concentrations of AZD2461. B. The endpoint cell viability was determined at 72h after pre-seeded CRC cell lines were treated with different concentration of oAdv. C. The endpoint cell viability was determined at 48h after pre-seeded CRC cell lines were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv. D. The endpoint cell viability was determined at 48h after pre-seeded HPLF were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv. D, E. FACS was used to detect intracellular phosphorylation levels of γH2A.X at 24 h post oAdv infection. *** P < 0.001. Given that mice are not highly sensitive to Ad5, the MC-38 cell line was chosen to perform an in vitro assay to assess the sensitivity of the combination of oAdv and PARPi. The IC50 of oAdv in MC-38 cells was 2013 MOI, which was 5 to 20 times higher than in human-derived CRC cell lines (Fig. 4 A). Subsequently, MC-38 cells were treated with AZD2461 and the combination of AZD2461 and oAdv. The IC50 values for AZD2461 alone and in combination with oAdv in MC-38 cells were 2.06 and 0.37 µM/mL, respectively (Fig. 4 B). Phosphorylation of H2A.X was also measured by FACS at 24 hours. The mean fluorescence intensity (MFI) of phosphorylated H2A.X was 56.90 ± 6.38, 5052.00 ± 1150.74, and 51.97 ± 2.68 in the AZD2461, oAdv, and control groups, respectively (Fig. 4 C and 4 D). The DNA damage in the oAdv group was significantly more intense than in the control and AZD2461 groups (P < 0.01), indicating that oAdv infection enhances the sensitivity of infected cells to PARPi by increasing DNA damage in mouse CRC cell lines. 3.3. The combination treatment of oAdv and PARPi treated CRLM in vivo We initially confirmed the efficacy and safety of the combination of AZD2461 and oAdv using the Colo-205 xenograft model (Fig. 5 A). At 28 days, tumor size in the control group increased to 1 710.34 ± 317.82 mm3, compared to 1 466.59 ± 376.44 mm3, 1175.65 ± 272.16 mm3, and 832.36 ± 540.85 mm3 in the oAdv, AZD2461, and combination groups, respectively (Fig. 5 B). The tumor growth inhibition (TGI) values for the oAdv, AZD2461, and combination groups were 13.70%, 25.16%, and 51.56%, respectively. Tumor weights were consistent with the tumor growth curves (Fig. 5 C). The combination treatment demonstrated stronger inhibition of tumor growth compared to the other groups, confirming the synergistic effect between oAdv and AZD2461. We first confirmed the efficacy and safety of the combination of AZD2461 and oAdv with the Colo-205 xenograft model (Fig. 5 A). Tumor size was increased by 1710.34 ± 317.82 mm 3 at 28 days in the control group, compared with 1466.59 ± 376.44, 1175.65 ± 272.16, and 832.36 ± 540.85 mm 3 in the oAdv, AZD2461, and combination groups, respectively (Fig. 5 B). The tumor growth inhibition (TGI) value of oAdv, AZD2461, and combination groups were 13.70%, 25.16%, and 51.56%, respectively. The tumor weights were consistent with the tumor growth curves (Fig. 5 C). Inhibition of tumor growth was stronger in the combination compared to the other groups, which confirmed the synergistic effect between oAdv and AZD2461. The safety of the combination treatment was assessed by monitoring body weight changes post-administration and conducting an oAdv biodistribution assay. The results showed no significant changes in body weight for any group following administration. The body weights of the control, oAdv, AZD2461, and combination groups were 21.97 ± 1.53, 21.93 ± 1.54, 22.54 ± 1.30, and 21.44 ± 1.45 g, respectively (Fig. 5 D). The biodistribution data further supported the finding that oAdv primarily replicated in tumors 7 days post-administration, with minimal or no detection in healthy tissues. oAdv was detected in blood, liver, and tumor tissues, with relative copy numbers of 11.81 ± 0.35, 16.96 ± 0.30, and 1 148.103 ± 59.70, respectively (Fig. 5 E). The genomic titer of oAdv in tumor tissue was over one hundred times higher than in blood and liver (P < 0.001). These findings provide preliminary evidence of the potential efficacy and safety of the combination of AZD2461 and oAdv for the treatment of CRLM. The efficacy of the combination of AZD2461 and oAdv was further investigated in the MC-38-established CRLM murine model (Fig. 5 A, 5 B). The mean survival times for mice in the AZD2461, oAdv, and control groups were 26.5, 22.5, and 20 days, respectively, whereas those in the AZD2461 + oAdv group exceeded 40 days (Fig. 5 C). Ultimately, all mice in the control group succumbed or were euthanized before the end of the experiment, compared to 8 of 10 in the AZD2461 group (survival rate: 20%), 7 of 10 in the oAdv group (survival rate: 30%), and 2 of 10 in the AZD2461 + oAdv group (survival rate: 80%). The AZD2461 + oAdv group exhibited a significantly higher survival rate compared to the AZD2461 (P < 0.01), oAdv (P < 0.05), and control groups (P 0.05). Additional CRLM model mice from the control and AZD2461 + oAdv groups were utilized to investigate the mechanism of the combination treatment at day 14 post-treatment. In the control group, 4 of 6 mice survived, while all 6 mice in the AZD2461 + oAdv group survived. Notably, the combination of AZD2461 and oAdv significantly upregulated the expression of Sting and phosphorylation of TBK, which are classical markers of Sting pathway activation (Fig. 5 D, 5 E). Moreover, the percentage of viable CD45 + cells in the AZD2461 + oAdv group was significantly elevated (Fig. 5 F, 5 G). These findings indicate that the combination of AZD2461 and oAdv selectively eliminates tumor cells and enhances immune infiltration in the liver through activation of the cGAS-Sting pathway. 3.4. oAdv increased the specific T cell response through activated the Sting pathway Additionally, C-176, an inhibitor of the Sting pathway, was chosen to validate the mechanism of the AZD2461 and oAdv combination treatment in the MC-38-established CRLM murine model (Fig. 7 A). All mice survived to the end of the trial. The infiltration of CD45 + cells in the liver was significantly reduced following C-176 administration (Fig. 7 B, 7 C). Moreover, we employed ELISpot to assess the proportion of T cells specifically targeting MC-38 in spleenocytes at day 14 post-treatment (Fig. 7 D, 7 E). Consequently, in line with the liver CD45 + cell infiltration, AZD2461 + oAdv treatment enhanced the proportion of T cells specific for MC-38 in the periphery. Conversely, C-176 supplementation inhibited immune recognition. The spot-forming units for the control, AZD2461 + oAdv, and AZD2461 + oAdv + C-176 groups were 74.00 ± 5.35, 273.67 ± 44.29, and 48.67 ± 20.37, respectively (Fig. 6 E). In summary, the combination of AZD2461 and oAdv enhanced the specific T cell response via activation of the cGAS-Sting pathway. 4. Discussion In this study, we sought to compare the transcriptomic differences between two prevalent types of liver cancer. RNA-seq analysis revealed that CRLM displayed reduced proliferative capacity and significant suppression of pathways associated with cellular energy metabolism and DNA replication relative to HCC. Given the epithelial origin of CRLM, its diminished proliferative capacity relative to hepatocyte-derived tumors is well-documented. Reduced proliferative potential leads to diminished accumulation of intracellular DNA damage, thereby reducing sensitivity to synthetic lethality in CRC. Furthermore, key genes involved in DNA damage response, such as PARP1 and BRCA1/2, exhibited high transcriptional activity in CRLM. Conversely, key genes involved in immune responses, such as IL-6, IL-2, and TNF-α, were rarely expressed or undetectable, whereas genes associated with antigen processing and presentation, such as cGAS and B2M, were expressed normally. These findings suggest that CRLM may benefit from therapies that enhance immune responses and/or increase DNA damage accumulation. Guided by this hypothesis, PARPi, a conventional targeted therapy approach, and oncolytic adenovirus (oAdv), a classic gene therapy vector, were chosen to selectively target CRLM. This strategy has previously been employed in a pre-clinical glioblastoma model[ 26 ]. The traditional clinical development strategy for PARPi is based on the principle of synthetic lethality. Consequently, breast, ovarian, prostate, and pancreatic cancers with BRCA-deficient tumors were chosen for treatment. Due to limited evidence suggesting a relationship between BRCA mutations and CRC carcinogenesis [ 11 , 20 – 22 ], and the low prevalence (15%) of BRCA mutations or HR defects in CRC patients, only a few clinical trials have been conducted for CRC treatment [ 12 ]. The results of RNA-seq analysis and CRC ex vivo killing assays also confirmed the limited efficacy of AZD2461 in CRC treatment. However, the sensitivity of CRC to PARPi was dramatically improved with the supplementation of oAdv at a very low dose. Liu et al. reported a similar effect in glioblastoma when Olaparib was combined with oAdv [ 27 ]. The traditional PARPi clinical develop strategy based on the principle of synthetic lethality. Thus, breast, ovarian, prostate, and pancreatic cancers with BRCA-deficient tumors were selected. Due to few evidence to presume the relationship between BRCAm and CRC carcinogenesis and the lower 15% BRCAm or HR percentage of CRC patients, only few clinical trials performed on CRC treatment[ 12 ]. The result of RNA-seq and CRC ex vivo killing assay also confirmed the poor effect of AZD2461 in CRC treatment. However, the sensitive of CRC to PARPi were dramatic improved after the oAdv supplementation oAdv at very low dose. Liu et al had reported the same effect in glioblastoma with Olaparib comminated with oAdv[ 27 ]. Subsequently, the mechanism underlying the tumor-specific synergistic effect of AZD2461 and oAdv was investigated both in vivo and in vitro . We further analyzed cellular DNA damage following oAdv infection. Wild-type adenovirus infection is known to disrupt the host cell cycle and inhibit its DNA replication, keeping cells in the S-phase for an extended period to facilitate large-scale replication of the viral genome. This disruption of the cell cycle and the rapid replication of large amounts of viral genome led to the accumulation of DNA damage in the host cell. Moreover, oAdv was engineered to delete a portion of the E1 region, limiting its replication to abnormal intracellular environments with disrupted cell cycles or blocked apoptotic pathways, such as those found in tumors. Based on these findings, oAdv was employed as a tumor-specific adjuvant treatment to enhance the sensitivity of CRLM to PARPi without affecting hepatocytes, which was the one of the most susceptible normal cells to adenovirus infection. The in vitro assays and biodistribution studies also confirmed this. The efficacy of oAdv combined with AZD2461 was evaluated in the Colo-205 xenograft model, which was the most resistant to combination therapy among the three cell lines. The results also indicated that the efficacy of PARPi was limited in the COLO-205 xenograft model compared to the ovarian cancer-derived xenograft model. However, the COLO-205 xenograft model, with its incomplete immune system and significant differences in tumor microenvironment compared to CRLM, poses challenges for fully evaluating the therapeutic efficacy of combination therapies. Fully evaluating the therapeutic efficacy of combination therapies in the COLO-205 model is challenging. Consequently, the survival benefit of combined treatment with AZD2461 and oAdv was more pronounced in the CRLM model established by MC-38 than in the COLO-205 xenograft model. Given the higher proportion of infiltrating immune cells, which is associated with the richness of hepatic blood flow, and the fact that MC-38 typically responds well to immunotherapy, notable immune activation was observed in the CRLM model concurrent with cGAS-Sting pathway activation. Previous studies have reported that PARPi can activate the cGAS-Sting pathway in mouse models of BRCA mutations[ 28 , 29 ]. Additionally, the activation of the STING pathway by adenovirus infection has also been frequently reported [ 30 , 31 ]. Moreover, administration of C-176, a reported cGAS-Sting pathway inhibitor[ 32 , 33 ], significantly suppressed immune infiltration in the liver and immune recognition following AZD2461 + oAdv treatment. These findings demonstrated that the combination of PARPi and oAdv represents an immune-enhancing approach and a common targeted therapy strategy for CRLM treatment through tumor-specific increased DNA damage and activation of the cGAS-Sting pathway. 5. Conclusions In summary, the efficacy of the combination therapy was thoroughly validated both in vivo and in vitro . We demonstrated that oAdv specifically induced DNA damage and activated the cGAS-Sting pathway, thereby enhancing the sensitivity of CRLM to PARPi. The combination therapy of oAdv and PARPi holds promise for the treatment of CRLM. Declarations Funding This work was supported by the Competitive Research Project of Quzhou Science & Technology Bureau (grant number 2025K002). Author Contribution Authors' contributions:Conceptualization, Liang-Liang Weng, Xinyu Feng and Si Liu; Methodology, Yuting Tang; Software, Xinyu Feng; Validation, Yuting Tang and Wen Hu; Formal Analysis, Yuting Tang, Benyuan Zhou; Investigation, Yuting Tang; Resources, Wen Hu; Data Curation, Wen Hu; Writing – Original Draft Preparation, Yuting Tang, Wen Hu; Writing – Review & Editing, Liang-Liang Weng, Si Liu ; Visualization & Supervision, Xinyu Feng; Project Administration, Liang-Liang Weng and Si Liu; Funding Acquisition, Liang-Liang Weng Clinical trial number: not applicable Consent to Publish declaration: not applicable Consent to Participate declaration: not applicable References C. Eng, T. Yoshino, E. Ruiz-Garcia, N. Mostafa, C.G. Cann, B. O'Brian, A. Benny, R.O. Perez, C. Cremolini, Colorectal cancer, Lancet 404(10449) (2024) 294-310. D. Moris, A. Martinino, S. Schiltz, P.J. Allen, A. Barbas, D. Sudan, L. King, C. Berg, C. Kim, M. Bashir, M. Palta, M.A. Morse, M.E. Lidsky, Advances in the treatment of hepatocellular carcinoma: An overview of the current and evolving therapeutic landscape for clinicians, CA Cancer J Clin 75(6) (2025) 498-527. Y. Ni, Y. Liang, M. Li, Y. Lin, X. Zou, F. Han, J. Cao, L. Li, The updates on metastatic mechanism and treatment of colorectal cancer, Pathol Res Pract 251 (2023) 154837. E. Abbaspour, S. Karimzadhagh, A. Monsef, F. Joukar, F. Mansour-Ghanaei, S. Hassanipour, Application of radiomics for preoperative prediction of lymph node metastasis in colorectal cancer: a systematic review and meta-analysis, Int J Surg 110(6) (2024) 3795-3813. T. Ohishi, M.K. Kaneko, Y. Yoshida, A. Takashima, Y. Kato, M. Kawada, Current Targeted Therapy for Metastatic Colorectal Cancer, Int J Mol Sci 24(2) (2023). A.E. Kusumaningrum, S. Makaba, E. Ali, M. Singh, M.N. Fenjan, I. Rasulova, N. Misra, S.G. Al-Musawi, A. Alsalamy, A perspective on emerging therapies in metastatic colorectal cancer: Focusing on molecular medicine and drug resistance, Cell Biochem Funct 42(1) (2024) e3906. H. Bando, A. Ohtsu, T. Yoshino, Therapeutic landscape and future direction of metastatic colorectal cancer, Nat Rev Gastroenterol Hepatol 20(5) (2023) 306-322. Q. Li, J. Han, Y. Yang, Y. Chen, PD-1/PD-L1 checkpoint inhibitors in advanced hepatocellular carcinoma immunotherapy, Front Immunol 13 (2022) 1070961. J.M. Llovet, R. Pinyol, M. Yarchoan, A.G. Singal, T.U. Marron, M. Schwartz, E. Pikarsky, M. Kudo, R.S. Finn, Adjuvant and neoadjuvant immunotherapies in hepatocellular carcinoma, Nat Rev Clin Oncol 21(4) (2024) 294-311. Z. Chen, G. Zhang, X. Ren, Z. Yao, Q. Zhou, X. Ren, S. Chen, L. Xu, K. Sun, Q. Zeng, M. Kuang, D.M. Kuang, S. Peng, Cross-talk between Myeloid and B Cells Shapes the Distinct Microenvironments of Primary and Secondary Liver Cancer, Cancer Res 83(21) (2023) 3544-3561. S. Santagata, G. Rea, D. Castaldo, M. Napolitano, A. Capiluongo, C. D'Alterio, A.M. Trotta, C. Ierano, L. Portella, S. Di Maro, F. Tatangelo, V. Albino, R. Guarino, C. Cutolo, F. Izzo, S. Scala, Hepatocellular carcinoma (HCC) tumor microenvironment is more suppressive than colorectal cancer liver metastasis (CRLM) tumor microenvironment, Hepatol Int 18(2) (2024) 568-581. G. Corti, K. Buzo, E. Berrino, M. Miotto, M.C. Aquilano, M. Lentini, S.E. Bellomo, A. Lorenzato, A. Bartolini, G. Mauri, L. Lazzari, M. Russo, F. Di Nicolantonio, S. Siena, S. Marsoni, C. Marchio, A. Bardelli, S. Arena, Prediction of homologous recombination deficiency identifies colorectal tumors sensitive to PARP inhibition, NPJ Precis Oncol 8(1) (2024) 231. F.H. Wu, H.Z. Wei, H.Y. Deng, G.H. Xiao, Y.C. Zhang, PARP in colorectal cancer: Molecular mechanisms, immunity, clinical trials, and drug combinations, Neoplasma 70(1) (2023) 1-14. S. Lheureux, Z. Lai, B.A. Dougherty, S. Runswick, D.R. Hodgson, K.M. Timms, J.S. Lanchbury, S. Kaye, C. Gourley, D. Bowtell, E.C. Kohn, C. Scott, U. Matulonis, T. Panzarella, K. Karakasis, J.V. Burnier, C.B. Gilks, M.J. O'Connor, J.D. Robertson, J. Ledermann, J.C. Barrett, T.W. Ho, A.M. Oza, Long-Term Responders on Olaparib Maintenance in High-Grade Serous Ovarian Cancer: Clinical and Molecular Characterization, Clin Cancer Res 23(15) (2017) 4086-4094. D. Dibitetto, C.A. Widmer, S. Rottenberg, PARPi, BRCA, and gaps: controversies and future research, Trends Cancer 10(9) (2024) 857-869. J.S. Frenel, J.W. Kim, N. Aryal, R. Asher, D. Berton, L. Vidal, P. Pautier, J.A. Ledermann, R.T. Penson, A.M. Oza, J. Korach, T. Huzarski, S. Pignata, N. Colombo, T.W. Park-Simon, K. Tamura, G.S. Sonke, A.E. Freimund, C.K. Lee, E. Pujade-Lauraine, Efficacy of subsequent chemotherapy for patients with BRCA1/2-mutated recurrent epithelial ovarian cancer progressing on olaparib versus placebo maintenance: post-hoc analyses of the SOLO2/ENGOT Ov-21 trial, Ann Oncol 33(10) (2022) 1021-1028. C.C. Tzang, E.S. Huang, H.W. Wu, W.C. Lin, Y.T. Lee, C.A. Luo, Y.H. Chen, Z.Y. Chang, Z.T. Chen, V.F. Kuo, B.S. Tzang, T.C. Hsu, Effectiveness and safety of PARP inhibitors in ovarian cancer: An umbrella review of systematic reviews and meta-analyses, Crit Rev Oncol Hematol 215 (2025) 104893. L. Oplustil O'Connor, S.L. Rulten, A.N. Cranston, R. Odedra, H. Brown, J.E. Jaspers, L. Jones, C. Knights, B. Evers, A. Ting, R.H. Bradbury, M. Pajic, S. Rottenberg, J. Jonkers, D. Rudge, N.M. Martin, K.W. Caldecott, A. Lau, M.J. O'Connor, The PARP Inhibitor AZD2461 Provides Insights into the Role of PARP3 Inhibition for Both Synthetic Lethality and Tolerability with Chemotherapy in Preclinical Models, Cancer Res 76(20) (2016) 6084-6094. M.A. Romeo, M.S. Gilardini Montani, R. Benedetti, A. Arena, M. Maretto, E. Bassetti, R. Caiazzo, G. D'Orazi, M. Cirone, Anticancer effect of AZD2461 PARP inhibitor against colon cancer cells carrying wt or dysfunctional p53, Exp Cell Res 408(2) (2021) 112879. A. Koski, E. Karli, A. Kipar, S. Escutenaire, A. Kanerva, A. Hemminki, Mutation of the fiber shaft heparan sulphate binding site of a 5/3 chimeric adenovirus reduces liver tropism, PLoS One 8(4) (2013) e60032. C. Xu, L. Chen, G. Liu, J. Xu, W. Lv, X. Gao, P. Xu, M. Tang, Y. Wang, X. Zhao, G. Nie, K. Cheng, F. Liu, Tailoring an intravenously injectable oncolytic virus for augmenting radiotherapy, Cell Rep Med 6(5) (2025) 102078. K. Nebenzahl-Sharon, R. Sharf, J. Amer, H. Shalata, H. Khoury-Haddad, S.Y. Sohn, N. Ayoub, P. Hearing, T. Kleinberger, An Interaction with PARP-1 and Inhibition of Parylation Contribute to Attenuation of DNA Damage Signaling by the Adenovirus E4orf4 Protein, J Virol 93(19) (2019). K.L. Zhang, R.P. Li, B.P. Zhang, S.T. Gao, B. Li, C.J. Huang, R. Cao, J.Y. Cheng, X.D. Xie, Z.H. Yu, X.Y. Feng, Efficacy of a new oncolytic adenovirus armed with IL-13 against oral carcinoma models, Onco Targets Ther 12 (2019) 6515-6523. M. Gao, E. Yngve, D. Yu, C. Jin, A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products, Front Mol Biosci 9 (2022) 883249. P. Limani, N. Borgeaud, M. Linecker, C. Tschuor, E. Kachaylo, A. Schlegel, J.H. Jang, U. Ungethum, M. Montani, R. Graf, B. Humar, P.A. Clavien, Selective portal vein injection for the design of syngeneic models of liver malignancy, Am J Physiol Gastrointest Liver Physiol 310(9) (2016) G682-8. J. Ning, H. Wakimoto, C. Peters, R.L. Martuza, S.D. Rabkin, Rad51 Degradation: Role in Oncolytic Virus-Poly(ADP-Ribose) Polymerase Inhibitor Combination Therapy in Glioblastoma, J Natl Cancer Inst 109(3) (2017) 1-13. Y. Liu, S. Fang, P. Wang, J. Zhang, F. Liu, Olaparib Enhances the Efficacy of Third-Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc-Induced Apoptosis, CNS Neurosci Ther 30(11) (2024) e70124. G. Giordano, A. Merlini, F. Capozzi, G. Ferrero, C. Tucciarello, S. Majidi, S. Comparato, G. Mesiano, E. Liuzzi, F. Galvagno, A. Massa, S. Brusco, V. Leuci, C. Riganti, S. Cutrupi, M. De Bortoli, L. D'Ambrosio, D. Sangiolo, G. Grignani, Y. Pignochino, The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes, Cancer Immunol Immunother 74(10) (2025) 312. C. Mao, W. Fan, J. Liu, F. Yang, W. Li, L. Li, Z. Shi, Q. Li, Z. Yuan, Y. Jiang, B. Chu, Targeting HDAC and PARP Enhances STING-Dependent Antitumor Immunity in STING-Deficient Tumor, Adv Sci (Weinh) 12(41) (2025) e07904. T. Cao, T. Hao, X. Zhang, B. Chen, H. Zhang, L. Shao, W. He, B. Li, Q. Zheng, P. Ji, K. Tao, TAMs-specific ARF1 inhibition reprograms glioma microenvironment and enhances the therapeutic effect of oncolytic adenovirus, iScience 28(6) (2025) 112696. F. Sun, Y. Xu, Z. Deng, P. Yang, A recombinant oncolytic influenza virus expressing a PD-L1 antibody induces CD8(+) T-cell activation via the cGas-STING pathway in mice with hepatocellular carcinoma, Int Immunopharmacol 120 (2023) 110323. X. Leng, Q. Li, W. Chen, H. Feng, L. Li, L. Yu, P. Huang, P. Ma, F. Xie, C-176 inhibits macrophage polarization towards M1-subtype and ameliorates LPS induced acute kidney injury, Eur J Pharmacol 984 (2024) 177028. S.M. Yang, Y.B. Li, H.X. Si, Y. Wei, F.J. Ma, J. Wang, T. Chen, K. Chen, C-176 reduces inflammation-induced pain by blocking the cGAS-STING pathway in microglia, Int J Neurosci 135(11) (2025) 1193-1207. Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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14:14:32","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94561,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/6b2cf50f47ae24c5def953b0.png"},{"id":96898002,"identity":"c7eebe03-c9fd-4bab-8b26-d6f555d7f02b","added_by":"auto","created_at":"2025-11-27 10:46:18","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122949,"visible":true,"origin":"","legend":"","description":"","filename":"36e8d5735a1548f998ea772f8301a1ff1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/09f791d4f8d9d97fb12536fa.xml"},{"id":96898004,"identity":"c9100176-c4a0-40c7-b5d2-c5bfdf88c8cd","added_by":"auto","created_at":"2025-11-27 10:46:18","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133253,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/5eed5258d0fe2466da48aabc.html"},{"id":96919920,"identity":"04f1ba36-4a3c-438a-8a9d-c1c856e115e5","added_by":"auto","created_at":"2025-11-27 14:14:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2028598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of expression profile connections between HCC group (n=61) and CRLM group (n=63)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. PCA analysis between HCC group and CRLM group; B. Correlation analysis between HCC group and CRLM group; C. GSEA for GO terms between HCC group and CRLM group; D. GSEA for KEGG terms between HCC group and CRLM group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/4c5248863dab2632499541b9.png"},{"id":96897982,"identity":"e2cde282-bf91-4729-8b20-33781dfc5879","added_by":"auto","created_at":"2025-11-27 10:46:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1582824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional Analysis of Key DEGs between HCC group (n=61) and CRLM group (n=63)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. GSEA of cell proliferation related pathway; B. GSEA of immunity related pathway. C. Heatmap of 79 selected DEGs; D. Protein-protein interaction (PPI) network analysis via STRING of 79 selected DEGs; E. Compared read count of key gene between HCC group and CRLM group. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/436a689dc9db01b3692f4843.png"},{"id":96897978,"identity":"43ccdfe0-1e7e-4f70-87a9-404ebcf96c42","added_by":"auto","created_at":"2025-11-27 10:46:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":555911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the potency of AZD2461, oAdv, and the combination of both in three different human-derived CRC cell lines (Colo-205, HCT-116, and HT-29).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The endpoint cell viability was determined at 48h after pre-seeded CRC cell lines were treated with different concentrations of AZD2461. B. The endpoint cell viability was determined at 72h after pre-seeded CRC cell lines were treated with different concentration of oAdv. C. The endpoint cell viability was determined at 48h after pre-seeded CRC cell lines were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv. D. The endpoint cell viability was determined at 48h after pre-seeded HPLF were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv. D, E. FACS was used to detect intracellular phosphorylation levels of γH2A.X at 24 h post oAdv infection. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/83af93098f626115775f5c68.png"},{"id":96920396,"identity":"98aa4edc-b9b9-4ad0-a58f-00e6a0bec15d","added_by":"auto","created_at":"2025-11-27 14:15:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":559688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe synergistic effect of oAdv with AZD2461 was confirmed in a murine-derived CRC cell line (MC-38).\u003c/strong\u003e A. The endpoint cell viability was determined at 72h after pre-seeded MC-38 were treated with different concentration of oAdv; B. The endpoint cell viability was determined at 48h after pre-seeded MC-38 cell lines were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv; C, D. To further confirm whether DNA damage was increased after infection in murine-derived cell lines, FACS was used to detect intracellular phosphorylation levels of γH2A.X at 24 h post-infection. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/d455cc5699dfdac090be6f52.png"},{"id":96897981,"identity":"5bb52363-e64e-41c3-8ff2-5cda70d313f0","added_by":"auto","created_at":"2025-11-27 10:46:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1004479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e potency and safety assay in Colo-205 xenograft model\u003c/strong\u003e \u003cstrong\u003e(n=8).\u003c/strong\u003e A. Schematic diagram of Colo-205 xenograft model. B. Colo-205 xenograft model`s tumor growth curve of each group. C. Comparison of tumor weight of each group in Colo-205 xenograft model at D28. D. Colo-205 xenograft model`s body weight change of each group. E. Additional groups (n=3) were used to investigate the biodistribution of oAdv il at 7 days after \u003cem\u003ei.v\u003c/em\u003e. DNA extracted from each tissue (heart, liver, blood, lung, kidney, and tumor) was used to quantify the relative copy number of oAdv by Q-PCR with a linearized plasmid control (1×10\u003csup\u003e8\u003c/sup\u003e copy number). ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/d4214962f781edad45ac71c8.png"},{"id":96897992,"identity":"834e2641-d61d-4ff6-99e3-79c044dd22fe","added_by":"auto","created_at":"2025-11-27 10:46:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1895476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe potency assay in MC-38 CRLM model (n=10).\u003c/strong\u003e A. Schematic diagram of MC-38 CRLM model. B. The liver metastasis of MC-38 CRLM model at D14 after grouping. C. The survival curves of the MC-38 CRLM model after treatment. D, E. Additional two groups of MC-38 CRLM model(n=6) for WB at D14 after treatment. The survival mice of each group were mixed and detected with mixture at three repeats. F,G. Additional two groups of MC-38 CRLM model(n=6) for liver immunocytes infiltration detection at D14 after treatment. Four mice (4/6) of control group and six mice (6/6) were detected by individual. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/c1932e45acc6ef4928747398.png"},{"id":96897983,"identity":"8b4e602d-700c-4432-a9fc-dc355776a9e3","added_by":"auto","created_at":"2025-11-27 10:46:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2143999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe C-176 supplement assay in MC-38 CRLM model (n=5). \u003c/strong\u003eA. Schematic diagram of MC-38 CRLM model with C-176 supplement. B, C. MC-38 CRLM model for liver immunocytes infiltration detection at D14 after treatment. All mice from each group were detected by individual. D, E. The MC-38 specific T cell was evaluated at the D14 after treatment with INF-γ ELISpot. All mice from each group were mixed and detected with mixture at three repeats. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/b468767c60acccf4cc90bf52.png"},{"id":102906130,"identity":"8906fd5e-6f46-42f0-b71b-fbf364b36118","added_by":"auto","created_at":"2026-02-18 09:12:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10624052,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/bd136155-ea50-4b37-9af7-f8943962eae3.pdf"},{"id":96920712,"identity":"7bfca794-3f19-437d-8171-7ef13822eccd","added_by":"auto","created_at":"2025-11-27 14:15:22","extension":"pptx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1371081,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8189652/v1/c9ec8e52aa1643a036cd80a6.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oncolytic adenovirus synergistically treats liver metastases from colon cancer with PARPi by increasing DNA damage and activating the STING pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third most common cancer worldwide, and the survival of patients with early-stage colon cancer can be significantly prolonged by traditional treatments such as surgical resection, radiotherapy, and chemotherapy[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the availability of detection techniques, most patients with colorectal cancer (CRC) are diagnosed at an advanced, unresectable stage due to the limitations of current methods. Colorectal liver metastasis (CRLM) accounts for nearly half of unresectable metastases, and according to the National Comprehensive Cancer Network (NCCN), fewer than 25% of patients with CRLM are eligible for liver transplantation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, several first-line chemotherapy regimens have proven ineffective in controlling CRLM, and they are limited by high toxicity and the development of drug resistance[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A more comprehensive and safer therapeutic strategy is needed to improve the outcome for patients with CRLM.\u003c/p\u003e\u003cp\u003eDue to the extensive vascular distribution and rich blood perfusion of the liver, immunotherapy such as anti-PD-L1 antibody has shown clinical benefit in HCC treatment[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, unlike HCC, CRLM are advanced metastatic lesions of CRC. The immune microenvironment of CRLM is much \u0026ldquo;hotter\u0026rdquo; than that of HCC due to lower levels of tumor-associated macrophages and regulatory T cell infiltration[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, CRLM also inherit some characteristics of CRC. Microsatellite instability (MSI)/DNA mismatch repair deficiency (dMMR) serve as biomarkers for colorectal cancer (CRC) that are recommended by the NCCN guidelines[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The guidelines also suggest that colorectal liver metastases (CRLM) may frequently exhibit DNA damage repair (DDR) deficiency, which could make them potential beneficiaries of Poly(ADP-Ribose) Polymerase inhibitor (PARPi) therapy. Currently, multiple clinical trials are exploring the safety and preliminary efficacy of PARPi in CRC[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSince the mechanism of PARPi was first reported in 2004, several clinical trials have confirmed the efficacy of PARPi in cancers with BRCA1/2 mutations (BRCAm) or homologous recombination deficiency (HRD)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. PARPi, when combined with chemotherapy, which disrupts DNA replication and increases DNA repair damage, has demonstrated potential for the clinical treatment of CRC[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, due to the low incidence of double allelic loss of genes involved in homologous recombination in CRC, patients in this study derived limited benefit from PARPi treatment and experienced significant hematologic toxicity[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, we selected AZD2461, which has a better safety than the approved Olaparib[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, we replaced less specific chemotherapeutic agents with oncolytic adenovirus (oAdv) for combination therapy to increase DNA damage.\u003c/p\u003e\u003cp\u003eBy partially deleting the E1 region of adenovirus 5 (Ad5), oAdv was limited to replicating only in tumor cells[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, the coxsackie and adenovirus receptor (CAR), which is critical for adenovirus infection, is strongly expressed in the live[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This allowed oAdv to selectively accumulate in CRLM tumors and subsequently increase DNA damage during virus replication[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this study, oAdv was administered as an adjuvant-like supplementary drug at a very low dose.\u003c/p\u003e\u003cp\u003eIn this study, we examined the efficacy of combination treatment with poly (ADP-ribose) polymerase inhibitors (PARPi) in CRLM. The efficacy and safety of the combination were evaluated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eThirty-five BALB/c nude mice (6\u0026ndash;8 weeks old) and sixty-two C57BL/6 mice (6\u0026ndash;8 weeks old) were housed in a light- and temperature-controlled room. The mice were allowed to acclimatize to their new environment for at least 7 days before the start of the experiments. All animal experimental procedures were approved by the Zhoushan Branch of Ruijin Hospital Affiliated to Shanghai under a specific pathogen-free protocol (ethics no. ZSRJA-20230703-02).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. RNA-seq and analysis\u003c/h2\u003e\u003cp\u003eThe RNA-seq dataset GSE243245 from GEO was selected to investigate the transcriptional differences between HCC and CRLM. The gene expression profiles of each group were further analyzed using R (version 4.3.2). The correlations between groups were analyzed using principal component analysis (PCA), and the correlation matrices were calculated using PCAtools (version 2.18.0) and pheatmap (version 1.0.12). The differentially expressed genes (DEGs) between groups were subsequently compared using DESeq2 (version 1.40.2). The DEGs between each group were defined by a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and a |log2fold change| \u0026gt;1. The overlap of DEGs between groups was compared using a Venn diagram (version 1.7.3). The DEGs between the HCC and CRLM groups were annotated using gene set enrichment analysis (GSEA) with clusterProfiler (version 4.8.3). GSEA was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, with a threshold of q values\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GO enrichment was performed for biological process (BP) terms. The DEGs of interest were further analyzed using protein‒protein interaction (PPI) network analysis with STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 12.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Cell lines and culture condition\u003c/h2\u003e\u003cp\u003eThree human CRC cell lines (Colo-205, HCT-116, and HT-29) and human primary liver fibroblast (HPLF) cells were purchased from iCell Bioscience (China). The mouse CRC cell line MC-38 was purchased from the National Collection of Authenticated Cell Cultures (China). All cell lines were authenticated by short tandem repeat profiling before purchase. HT-29 and HCT-116 were maintained in McCoy's 5A Medium (CAT: 12330031, Gibco, USA). Colo-205 was maintained in RPMI-1640 medium (CAT: 61870036, Gibco, USA). HPLF and MC-38 were maintained in DMEM medium (CAT: 11995065, Gibco, USA). All media were supplemented with 10% fetal bovine serum (CAT: 16000-044, Gibco, USA) and 1% penicillin/streptomycin (CAT: V900929, Sigma-Aldrich, USA). Cells were incubated in a humidified atmosphere containing 5% CO₂ at 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Drug preparation\u003c/h2\u003e\u003cp\u003eAZD2461 (HY-13536) and C-176 (HY-112906) were purchased from MCE (USA) and diluted in PBS containing 10% DMSO (CAT: D2650, Sigma-Aldrich, USA). The AZD2461 and C-176 solutions were sterilized using a 0.22 \u0026micro;m filter before use. The oncolytic adenovirus (oAdv) was constructed as previously reported of our laboratory[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Viral titers were determined by median tissue culture infectious dose (TCID₅₀) assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cell viability assay\u003c/h2\u003e\u003cp\u003eThe three human CRC cell lines, MC-38 cells, and HPLF cells were seeded into 96-well plates at a density of 3,000 cells per well and allowed to adhere overnight. The synergistic effect of oAdv and AZD2461 was compared in vitro. AZD2461 was added to all experimental groups at concentrations of 10, 5, 2.5, 1.25, 0.63, 0.16, 0.08, and 0.04 \u0026micro;M/mL in triplicate. The combination groups were further infected with oAdv at a multiplicity of infection (MOI) of 10. All drug solutions were diluted in 100 \u0026micro;L of medium, and 100 \u0026micro;L of medium was added to the negative controls. MC-38 cells were treated with the same concentration gradient of AZD2461 and oAdv. Then, 10 \u0026micro;L of CCK-8 solution (CAT: CK04, Dojindo) was added to each well to measure cell viability. The plates were incubated for 4 hours, and absorbance was measured at 450 nm using a microplate reader to determine the 50% inhibitory concentration (IC₅₀) for each assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. DNA damage assay\u003c/h2\u003e\u003cp\u003eDNA damage was assessed in vitro using fluorescence-activated cell sorting (FACS). MC-38, Colo-205, and HPLF cells were seeded into six-well plates at a density of 5,000 cells per well and cultured overnight. oAdv was added at a multiplicity of infection (MOI) of 10, and AZD2461 was added at 1 \u0026micro;M/mL in triplicate. All cells were collected 24 hours after treatment. Cells were fixed with Phosflow\u0026trade; Fix Buffer I (CAT: 557870, BD, USA) for 10 minutes at 37\u0026deg;C and then permeabilized with Perm Buffer III for 30 minutes on ice. The cells were washed three times with PBS and stained with an FITC-conjugated anti-phospho-H2A.X (Ser139) antibody (CAT: 613403, BioLegend, USA). DNA damage was determined by the percentage of γ-H2A.X-positive cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Colo-205 xenograft models\u003c/h2\u003e\u003cp\u003eThirty-two BALB/c nude mice (6\u0026ndash;8 weeks old) were used to establish a Colo-205 xenograft model. The xenograft model was established by subcutaneous injection of Colo-205 cells (5\u0026times;10⁶ cells in 100 \u0026micro;L) into the right flank after confirming cell viability using Trypan blue staining (CAT: 15250061, Invitrogen, USA). When the tumors reached approximately 100 mm\u0026sup3;, the mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;8 per group) using Study Director software (Studylog Systems, Inc., USA). Mice in the oAdv group received intravenous injections of 100 \u0026micro;L oAdv (1\u0026times;10⁹ PFU/mL) on days 1, 3, and 5 after grouping. Mice in the AZD2461 group received intravenous injections of AZD2461 (1 mg/kg) on days 1, 2, 3, 4, 5, 7, and 9 after grouping. Mice in the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group received both oAdv and AZD2461 treatments as described above. Mice in the control group received intravenous injections of 100 \u0026micro;L PBS on days 1, 2, 3, 4, 5, 7, and 9 after grouping. Tumor volume was calculated using the formula (length \u0026times; width\u0026sup2;)/2 and measured every 3 days. All mice were euthanized 28 days after grouping.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Biodistribution assay\u003c/h2\u003e\u003cp\u003eThree mice bearing Colo-205 xenografts received 100 \u0026micro;L of oAdv (1\u0026times;10⁹ PFU/mL) via intravenous injection. All mice were euthanized 7 days post-injection, and samples were collected. DNA was extracted from heart, liver, blood, lung, kidney, and tumor tissues using the TIANamp Genomic DNA Kit (DP304, Tiangen, China). Quantitative PCR (qPCR) was performed using verified primers[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Linearized oAdv vector (1\u0026times;10⁸ copies) was used as the reference control. Then, 1 \u0026micro;g of DNA from each sample was used, and the relative copy number in each tissue was calculated as: (total DNA extracted) \u0026times; (relative DNA level).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9.MC-38 CRLM model\u003c/h2\u003e\u003cp\u003eForty C57BL/6 mice (6\u0026ndash;8 weeks old) were used to establish the colorectal liver metastasis (CRLM) model. The model was established via selective portal vein injection as previously reported[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], with subsequent splenectomy. Cell viability was confirmed as described above. Two weeks after cell injection, the mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;10 per group). The day of grouping was designated day 0, and grouping and treatment were performed as described for the MC-38 xenograft model. The survival of all mice was monitored continuously until day 40 after grouping. In accordance with animal welfare guidelines, mice exhibiting\u0026thinsp;\u0026gt;\u0026thinsp;20% weight loss or ataxia were considered moribund, recorded as dead, and euthanized.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Liver immune infiltration and Sting pathway activate detection assay\u003c/h2\u003e\u003cp\u003eTwelve mice spared from the CRLM model were used to compare immune infiltration and protein level changes in the liver using flow cytometry (FACS) and western blotting (WB). The mice were divided into two groups (n\u0026thinsp;=\u0026thinsp;6 per group): the control group and the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group, following the COLO-205 model protocol. Mice in the C-176 group received intravenous injections of oAdv and AZD2461 in combination. Single-cell suspensions (SCS) were prepared from the entire liver. Cell viability was determined using FVS510 staining, and lymphocytes were identified by CD45 expression. Liver-infiltrating lymphocytes were defined as FVS510 (CAT: 544406, BD, USA) and CD45 (CAT: 103315, BioLegend, USA) double-positive cells.\u003c/p\u003e\u003cp\u003eProteins were extracted from the liver SCS. A total of 30 \u0026micro;g of protein per sample was loaded into each well. Western blotting (WB) analysis was performed using standard protocols. The primary antibodies used were anti-GAPDH (CAT: 5174, 1:1000), anti-STING (CAT: 13647, 1:1000), anti-phospho-TBK (pTBK, CAT: 3036, 1:1000), and anti-TBK (CAT: 36169, 1:1000), all purchased from Cell Signaling Technology (CST, USA). The secondary antibody (CAT: A0208, 1:5000) was purchased from Beyotime (China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Sting inhibitor treatment assay\u003c/h2\u003e\u003cp\u003eTen mice spared from the CRLM model were used to compare immune infiltration and protein level changes in the liver using FACS, WB, and ELISpot assays. The mice were divided into two groups (n\u0026thinsp;=\u0026thinsp;5 per group) and treated as the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group according to the COLO-205 model protocol. Mice in the AZD2461\u0026thinsp;+\u0026thinsp;oAdv\u0026thinsp;+\u0026thinsp;C-176 group received intravenous injections of oAdv and AZD2461 following the same regimen as the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group. Subsequently, they received daily intravenous injections of 750 nmol C-176 for 7 days, starting 3 days after the initial treatment, to inhibit STING pathway activation. FACS and WB analyses were performed as described above.\u003c/p\u003e\u003cp\u003ePBMCs from each group were isolated using Mouse Lymphocyte Separation Medium (CAT: 7211011, Dakewe, China). PBMCs from the same group were pooled to detect tumor-specific T cells using an IFN-γ ELISpot kit (CAT: 3321, Mabtech, Sweden). Fresh PBMCs (3 \u0026times; 10⁵ cells per group) were pooled and seeded into wells. Each group had three replicates, and splenocytes from C57BL/6 mice without MC-38 treatment served as the negative control. Protein (50 \u0026micro;g) extracted from MC-38 cells was added to each well. Cells and protein were maintained in RPMI-1640 medium and incubated for 16 h. After 16 h, cells and medium were removed, and spots were detected according to the manufacturer\u0026rsquo;s protocol. Plates were read and counted using a CTL ImmunoSpot reader (CTL Technologies, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e distribution analysis was performed using a paired Student's t-test. Other results were analyzed using an unpaired Student's t-test or ANOVA or multiple t-test. All data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. IC50 values were calculated using four-parameter logistic regression. Survival curves were compared using the log-rank test. All analyses were conducted using GraphPad Prism 8.0 software (GraphPad Software, Inc., USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1 The CRLM shown\u003c/h2\u003e\u003cp\u003eRNA-seq was performed on tumor tissues from each group in the GSE243245 dataset. The dataset contains tumor sequencing data from 123 patients, with 61 in the HCC group and 62 in the CRLM group. The gene expression profile revealed significant fluctuations both within and between groups. Despite this heterogeneity, CRLM and HCC groups remained well distinguished overall. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A total of 3826 differentially expressed genes (DEGs) were identified between the HCC and CRLM groups.\u003c/p\u003e\u003cp\u003eThe DEGs between the CRLM and HCC groups were further analyzed using Gene Set Enrichment Analysis (GSEA) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. A total of 1278 GO biological process (BP) terms were significantly enriched (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared to the CRLM group, the HCC group showed 622 activated pathways and 656 inhibited pathways. The GO analysis revealed that the most significantly enriched BP terms included positive regulation of response to jasmonic acid, cellular response to jasmonic acid stimulus, and chylomicron remnant clearance. Synaptic signaling, nervous system process, and monoatomic ion transmembrane transport were the main terms associated with negatively regulated BP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). A total of 98 KEGG pathways were significantly enriched (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The HCC group showed 58 activated pathways and 40 inhibited pathways. KEGG analysis indicated that pathways primarily involving primary bile acid biosynthesis, lipoic acid metabolism, and biosynthesis of unsaturated fatty acids were activated, whereas pathways involving taste transduction, muscle cell cytoskeleton organization, and cornified envelope formation were inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA. PCA analysis between HCC group and CRLM group; B. Correlation analysis between HCC group and CRLM group; C. GSEA for GO terms between HCC group and CRLM group; D. GSEA for KEGG terms between HCC group and CRLM group.\u003c/p\u003e\u003cp\u003eSignificant changes were observed between the CRLM and HCC groups in pathways related to cell replication and DNA replication, including positive regulation of cell population proliferation, nucleotide biosynthetic process, and ATP biosynthetic process. The normalized enrichment scores (NES) for these three pathways were \u0026minus;\u0026thinsp;1.26, 1.33, and 1.47 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, immune-related pathways showed few significant differences between HCC and CRLM, with only complement activation exhibiting a significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Expression profiling and PPI analysis of DEGs of interest revealed that PARP and several related genes exhibited lower expression in CRLM. Key proteins in immune-related pathways, such as cGAS and TBK in the cGAS-STING pathway and B2M in antigen presentation and processing pathways, demonstrated divergent expression trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA. GSEA of cell proliferation related pathway; B. GSEA of immunity related pathway. C. Heatmap of 79 selected DEGs; D. Protein-protein interaction (PPI) network analysis via STRING of 79 selected DEGs; E. Compared read count of key gene between HCC group and CRLM group. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1. oAdv selective improved the PARPi effect via increased DNA damage in CRC cell\u003c/h2\u003e\u003cp\u003eThe potency of oAdv and AZD2461 was measured in three different CRC cell lines of human by single and combination. The IC50 of AZD2461 in HT-29 and HCT-116 was 2.20 and 0.65 \u0026micro;M/mL, respectively. And Colo-205 was not sensitive to AZD2461 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the IC50 of AZD2461 in Colo-205 was more than 10.00 \u0026micro;M/mL. The IC50 of oAdv in HT-29, Colo-205, and HCT-116 was 16.16, 402.80 and 8.05 MOI, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). 10 MOI was selected as the combined dose of oAdv at a concentration which as a poor effect on tumor cell viability. The IC50 of combination in HT-29, Colo-205, and HCT-116 was 0.20, 0.92, and 0.24 \u0026micro;M/mL, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The potency of the combination group was higher than that of the two single-drug groups, indicating the synergistic effect of oAdv supplementation with AZD2461. Then the safety of combination treatment was tested with the normal liver cell line HPLF. As the result, the high dose of AZD2461 didn't kill HPLF and the oAdv supplementation also not reduced the cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, H2X.A phosphorylation levels in infected Colo-205 and HPLF cells at the same concentrations were detected by FACS. Infection with oAdv significantly increased the DNA damage in the Colo-205, but didn`t affect HPLF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These suggests that the combination of AZD2461 and oAdv had the potential to treat CRLM in vivo by selectively affecting CRC cells but not hepatocytes.\u003c/p\u003e\u003cp\u003eThe potency of oAdv and AZD2461 was assessed in three human CRC cell lines using single-agent and combination treatments. The IC50 values for AZD2461 in HT-29 and HCT-116 cell lines were 2.20 and 0.65 \u0026micro;M/mL, respectively. Colo-205 cells were insensitive to AZD2461 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with an IC50 value exceeding 10.00 \u0026micro;M/mL. The IC50 values for oAdv in HT-29, Colo-205, and HCT-116 cell lines were 16.16, 402.80, and 8.05 MOI, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). An oAdv concentration of 10 MOI was selected as the combined dose, as it had a minimal effect on tumor cell viability. The IC50 values for the combination treatment in HT-29, Colo-205, and HCT-116 cell lines were 0.20, 0.92, and 0.24 \u0026micro;M/mL, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The combination treatment exhibited greater potency than either single-drug treatment, suggesting a synergistic effect of oAdv in combination with AZD2461. The safety of the combination treatment was evaluated using the normal liver cell HPLF. The results showed that high doses of AZD2461 did not affect HPLF viability, nor did oAdv supplementation reduce cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, H2AX phosphorylation levels were measured in infected Colo-205 and HPLF cells at equivalent concentrations using FACS. oAdv infection significantly induced DNA damage in Colo-205 cells but had no effect on HPLF cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These findings suggest that the combination of AZD2461 and oAdv has potential for in vivo treatment of CRLM by selectively targeting CRC cells without affecting hepatocytes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA. The endpoint cell viability was determined at 48h after pre-seeded CRC cell lines were treated with different concentrations of AZD2461. B. The endpoint cell viability was determined at 72h after pre-seeded CRC cell lines were treated with different concentration of oAdv. C. The endpoint cell viability was determined at 48h after pre-seeded CRC cell lines were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv. D. The endpoint cell viability was determined at 48h after pre-seeded HPLF were treated with different concentrations of AZD2461 supplemented with 10 MOI oAdv. D, E. FACS was used to detect intracellular phosphorylation levels of γH2A.X at 24 h post oAdv infection. ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003cp\u003eGiven that mice are not highly sensitive to Ad5, the MC-38 cell line was chosen to perform an in vitro assay to assess the sensitivity of the combination of oAdv and PARPi. The IC50 of oAdv in MC-38 cells was 2013 MOI, which was 5 to 20 times higher than in human-derived CRC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Subsequently, MC-38 cells were treated with AZD2461 and the combination of AZD2461 and oAdv. The IC50 values for AZD2461 alone and in combination with oAdv in MC-38 cells were 2.06 and 0.37 \u0026micro;M/mL, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Phosphorylation of H2A.X was also measured by FACS at 24 hours. The mean fluorescence intensity (MFI) of phosphorylated H2A.X was 56.90\u0026thinsp;\u0026plusmn;\u0026thinsp;6.38, 5052.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1150.74, and 51.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68 in the AZD2461, oAdv, and control groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The DNA damage in the oAdv group was significantly more intense than in the control and AZD2461 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that oAdv infection enhances the sensitivity of infected cells to PARPi by increasing DNA damage in mouse CRC cell lines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. The combination treatment of oAdv and PARPi treated CRLM in vivo\u003c/h2\u003e\u003cp\u003eWe initially confirmed the efficacy and safety of the combination of AZD2461 and oAdv using the Colo-205 xenograft model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At 28 days, tumor size in the control group increased to 1 710.34\u0026thinsp;\u0026plusmn;\u0026thinsp;317.82 mm3, compared to 1 466.59\u0026thinsp;\u0026plusmn;\u0026thinsp;376.44 mm3, 1175.65\u0026thinsp;\u0026plusmn;\u0026thinsp;272.16 mm3, and 832.36\u0026thinsp;\u0026plusmn;\u0026thinsp;540.85 mm3 in the oAdv, AZD2461, and combination groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The tumor growth inhibition (TGI) values for the oAdv, AZD2461, and combination groups were 13.70%, 25.16%, and 51.56%, respectively. Tumor weights were consistent with the tumor growth curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The combination treatment demonstrated stronger inhibition of tumor growth compared to the other groups, confirming the synergistic effect between oAdv and AZD2461.\u003c/p\u003e\u003cp\u003eWe first confirmed the efficacy and safety of the combination of AZD2461 and oAdv with the Colo-205 xenograft model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Tumor size was increased by 1710.34\u0026thinsp;\u0026plusmn;\u0026thinsp;317.82 mm\u003csup\u003e3\u003c/sup\u003e at 28 days in the control group, compared with 1466.59\u0026thinsp;\u0026plusmn;\u0026thinsp;376.44, 1175.65\u0026thinsp;\u0026plusmn;\u0026thinsp;272.16, and 832.36\u0026thinsp;\u0026plusmn;\u0026thinsp;540.85 mm\u003csup\u003e3\u003c/sup\u003e in the oAdv, AZD2461, and combination groups, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The tumor growth inhibition (TGI) value of oAdv, AZD2461, and combination groups were 13.70%, 25.16%, and 51.56%, respectively. The tumor weights were consistent with the tumor growth curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Inhibition of tumor growth was stronger in the combination compared to the other groups, which confirmed the synergistic effect between oAdv and AZD2461.\u003c/p\u003e\u003cp\u003eThe safety of the combination treatment was assessed by monitoring body weight changes post-administration and conducting an oAdv biodistribution assay. The results showed no significant changes in body weight for any group following administration. The body weights of the control, oAdv, AZD2461, and combination groups were 21.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53, 21.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54, 22.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30, and 21.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 g, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The biodistribution data further supported the finding that oAdv primarily replicated in tumors 7 days post-administration, with minimal or no detection in healthy tissues. oAdv was detected in blood, liver, and tumor tissues, with relative copy numbers of 11.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35, 16.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30, and 1 148.103\u0026thinsp;\u0026plusmn;\u0026thinsp;59.70, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The genomic titer of oAdv in tumor tissue was over one hundred times higher than in blood and liver (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings provide preliminary evidence of the potential efficacy and safety of the combination of AZD2461 and oAdv for the treatment of CRLM.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe efficacy of the combination of AZD2461 and oAdv was further investigated in the MC-38-established CRLM murine model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The mean survival times for mice in the AZD2461, oAdv, and control groups were 26.5, 22.5, and 20 days, respectively, whereas those in the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group exceeded 40 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Ultimately, all mice in the control group succumbed or were euthanized before the end of the experiment, compared to 8 of 10 in the AZD2461 group (survival rate: 20%), 7 of 10 in the oAdv group (survival rate: 30%), and 2 of 10 in the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group (survival rate: 80%). The AZD2461\u0026thinsp;+\u0026thinsp;oAdv group exhibited a significantly higher survival rate compared to the AZD2461 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), oAdv (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and control groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas the survival rates in the AZD2461 and control groups were comparable (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Additional CRLM model mice from the control and AZD2461\u0026thinsp;+\u0026thinsp;oAdv groups were utilized to investigate the mechanism of the combination treatment at day 14 post-treatment. In the control group, 4 of 6 mice survived, while all 6 mice in the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group survived. Notably, the combination of AZD2461 and oAdv significantly upregulated the expression of Sting and phosphorylation of TBK, which are classical markers of Sting pathway activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Moreover, the percentage of viable CD45\u0026thinsp;+\u0026thinsp;cells in the AZD2461\u0026thinsp;+\u0026thinsp;oAdv group was significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These findings indicate that the combination of AZD2461 and oAdv selectively eliminates tumor cells and enhances immune infiltration in the liver through activation of the cGAS-Sting pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. oAdv increased the specific T cell response through activated the Sting pathway\u003c/h2\u003e\u003cp\u003eAdditionally, C-176, an inhibitor of the Sting pathway, was chosen to validate the mechanism of the AZD2461 and oAdv combination treatment in the MC-38-established CRLM murine model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). All mice survived to the end of the trial. The infiltration of CD45\u0026thinsp;+\u0026thinsp;cells in the liver was significantly reduced following C-176 administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Moreover, we employed ELISpot to assess the proportion of T cells specifically targeting MC-38 in spleenocytes at day 14 post-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Consequently, in line with the liver CD45\u0026thinsp;+\u0026thinsp;cell infiltration, AZD2461\u0026thinsp;+\u0026thinsp;oAdv treatment enhanced the proportion of T cells specific for MC-38 in the periphery. Conversely, C-176 supplementation inhibited immune recognition. The spot-forming units for the control, AZD2461\u0026thinsp;+\u0026thinsp;oAdv, and AZD2461\u0026thinsp;+\u0026thinsp;oAdv\u0026thinsp;+\u0026thinsp;C-176 groups were 74.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35, 273.67\u0026thinsp;\u0026plusmn;\u0026thinsp;44.29, and 48.67\u0026thinsp;\u0026plusmn;\u0026thinsp;20.37, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In summary, the combination of AZD2461 and oAdv enhanced the specific T cell response via activation of the cGAS-Sting pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we sought to compare the transcriptomic differences between two prevalent types of liver cancer. RNA-seq analysis revealed that CRLM displayed reduced proliferative capacity and significant suppression of pathways associated with cellular energy metabolism and DNA replication relative to HCC. Given the epithelial origin of CRLM, its diminished proliferative capacity relative to hepatocyte-derived tumors is well-documented. Reduced proliferative potential leads to diminished accumulation of intracellular DNA damage, thereby reducing sensitivity to synthetic lethality in CRC. Furthermore, key genes involved in DNA damage response, such as PARP1 and BRCA1/2, exhibited high transcriptional activity in CRLM. Conversely, key genes involved in immune responses, such as IL-6, IL-2, and TNF-α, were rarely expressed or undetectable, whereas genes associated with antigen processing and presentation, such as cGAS and B2M, were expressed normally. These findings suggest that CRLM may benefit from therapies that enhance immune responses and/or increase DNA damage accumulation. Guided by this hypothesis, PARPi, a conventional targeted therapy approach, and oncolytic adenovirus (oAdv), a classic gene therapy vector, were chosen to selectively target CRLM. This strategy has previously been employed in a pre-clinical glioblastoma model[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe traditional clinical development strategy for PARPi is based on the principle of synthetic lethality. Consequently, breast, ovarian, prostate, and pancreatic cancers with BRCA-deficient tumors were chosen for treatment. Due to limited evidence suggesting a relationship between BRCA mutations and CRC carcinogenesis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and the low prevalence (15%) of BRCA mutations or HR defects in CRC patients, only a few clinical trials have been conducted for CRC treatment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The results of RNA-seq analysis and CRC ex vivo killing assays also confirmed the limited efficacy of AZD2461 in CRC treatment. However, the sensitivity of CRC to PARPi was dramatically improved with the supplementation of oAdv at a very low dose. Liu et al. reported a similar effect in glioblastoma when Olaparib was combined with oAdv [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe traditional PARPi clinical develop strategy based on the principle of synthetic lethality. Thus, breast, ovarian, prostate, and pancreatic cancers with BRCA-deficient tumors were selected. Due to few evidence to presume the relationship between BRCAm and CRC carcinogenesis and the lower 15% BRCAm or HR percentage of CRC patients, only few clinical trials performed on CRC treatment[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The result of RNA-seq and CRC ex vivo killing assay also confirmed the poor effect of AZD2461 in CRC treatment. However, the sensitive of CRC to PARPi were dramatic improved after the oAdv supplementation oAdv at very low dose. Liu et al had reported the same effect in glioblastoma with Olaparib comminated with oAdv[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSubsequently, the mechanism underlying the tumor-specific synergistic effect of AZD2461 and oAdv was investigated both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. We further analyzed cellular DNA damage following oAdv infection. Wild-type adenovirus infection is known to disrupt the host cell cycle and inhibit its DNA replication, keeping cells in the S-phase for an extended period to facilitate large-scale replication of the viral genome. This disruption of the cell cycle and the rapid replication of large amounts of viral genome led to the accumulation of DNA damage in the host cell. Moreover, oAdv was engineered to delete a portion of the E1 region, limiting its replication to abnormal intracellular environments with disrupted cell cycles or blocked apoptotic pathways, such as those found in tumors. Based on these findings, oAdv was employed as a tumor-specific adjuvant treatment to enhance the sensitivity of CRLM to PARPi without affecting hepatocytes, which was the one of the most susceptible normal cells to adenovirus infection. The \u003cem\u003ein vitro\u003c/em\u003e assays and biodistribution studies also confirmed this. The efficacy of oAdv combined with AZD2461 was evaluated in the Colo-205 xenograft model, which was the most resistant to combination therapy among the three cell lines. The results also indicated that the efficacy of PARPi was limited in the COLO-205 xenograft model compared to the ovarian cancer-derived xenograft model.\u003c/p\u003e\u003cp\u003eHowever, the COLO-205 xenograft model, with its incomplete immune system and significant differences in tumor microenvironment compared to CRLM, poses challenges for fully evaluating the therapeutic efficacy of combination therapies. Fully evaluating the therapeutic efficacy of combination therapies in the COLO-205 model is challenging. Consequently, the survival benefit of combined treatment with AZD2461 and oAdv was more pronounced in the CRLM model established by MC-38 than in the COLO-205 xenograft model. Given the higher proportion of infiltrating immune cells, which is associated with the richness of hepatic blood flow, and the fact that MC-38 typically responds well to immunotherapy, notable immune activation was observed in the CRLM model concurrent with cGAS-Sting pathway activation. Previous studies have reported that PARPi can activate the cGAS-Sting pathway in mouse models of BRCA mutations[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, the activation of the STING pathway by adenovirus infection has also been frequently reported [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, administration of C-176, a reported cGAS-Sting pathway inhibitor[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], significantly suppressed immune infiltration in the liver and immune recognition following AZD2461\u0026thinsp;+\u0026thinsp;oAdv treatment. These findings demonstrated that the combination of PARPi and oAdv represents an immune-enhancing approach and a common targeted therapy strategy for CRLM treatment through tumor-specific increased DNA damage and activation of the cGAS-Sting pathway.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, the efficacy of the combination therapy was thoroughly validated both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. We demonstrated that oAdv specifically induced DNA damage and activated the cGAS-Sting pathway, thereby enhancing the sensitivity of CRLM to PARPi. The combination therapy of oAdv and PARPi holds promise for the treatment of CRLM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Competitive Research Project of Quzhou Science \u0026amp; Technology Bureau (grant number 2025K002).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors' contributions:Conceptualization, Liang-Liang Weng, Xinyu Feng and Si Liu; Methodology, Yuting Tang; Software, Xinyu Feng; Validation, Yuting Tang and Wen Hu; Formal Analysis, Yuting Tang, Benyuan Zhou; Investigation, Yuting Tang; Resources, Wen Hu; Data Curation, Wen Hu; Writing \u0026ndash; Original Draft Preparation, Yuting Tang, Wen Hu; Writing \u0026ndash; Review \u0026amp; Editing, Liang-Liang Weng, Si Liu ; Visualization \u0026amp; Supervision, Xinyu Feng; Project Administration, Liang-Liang Weng and Si Liu; Funding Acquisition, Liang-Liang Weng\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable\u003c/p\u003e\n\u003cp\u003eConsent to Publish declaration: not applicable\u003c/p\u003e\n\u003cp\u003eConsent to Participate declaration: not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eC. Eng, T. Yoshino, E. Ruiz-Garcia, N. Mostafa, C.G. Cann, B. O\u0026apos;Brian, A. Benny, R.O. Perez, C. Cremolini, Colorectal cancer, Lancet 404(10449) (2024) 294-310.\u003c/li\u003e\n\u003cli\u003eD. Moris, A. Martinino, S. Schiltz, P.J. Allen, A. Barbas, D. Sudan, L. King, C. Berg, C. Kim, M. Bashir, M. Palta, M.A. Morse, M.E. Lidsky, Advances in the treatment of hepatocellular carcinoma: An overview of the current and evolving therapeutic landscape for clinicians, CA Cancer J Clin 75(6) (2025) 498-527.\u003c/li\u003e\n\u003cli\u003eY. Ni, Y. Liang, M. Li, Y. Lin, X. Zou, F. Han, J. Cao, L. Li, The updates on metastatic mechanism and treatment of colorectal cancer, Pathol Res Pract 251 (2023) 154837.\u003c/li\u003e\n\u003cli\u003eE. Abbaspour, S. Karimzadhagh, A. Monsef, F. Joukar, F. Mansour-Ghanaei, S. Hassanipour, Application of radiomics for preoperative prediction of lymph node metastasis in colorectal cancer: a systematic review and meta-analysis, Int J Surg 110(6) (2024) 3795-3813.\u003c/li\u003e\n\u003cli\u003eT. Ohishi, M.K. Kaneko, Y. Yoshida, A. Takashima, Y. Kato, M. Kawada, Current Targeted Therapy for Metastatic Colorectal Cancer, Int J Mol Sci 24(2) (2023).\u003c/li\u003e\n\u003cli\u003eA.E. Kusumaningrum, S. Makaba, E. Ali, M. Singh, M.N. Fenjan, I. Rasulova, N. Misra, S.G. Al-Musawi, A. Alsalamy, A perspective on emerging therapies in metastatic colorectal cancer: Focusing on molecular medicine and drug resistance, Cell Biochem Funct 42(1) (2024) e3906.\u003c/li\u003e\n\u003cli\u003eH. Bando, A. Ohtsu, T. Yoshino, Therapeutic landscape and future direction of metastatic colorectal cancer, Nat Rev Gastroenterol Hepatol 20(5) (2023) 306-322.\u003c/li\u003e\n\u003cli\u003eQ. Li, J. Han, Y. Yang, Y. Chen, PD-1/PD-L1 checkpoint inhibitors in advanced hepatocellular carcinoma immunotherapy, Front Immunol 13 (2022) 1070961.\u003c/li\u003e\n\u003cli\u003eJ.M. Llovet, R. Pinyol, M. Yarchoan, A.G. Singal, T.U. Marron, M. Schwartz, E. Pikarsky, M. Kudo, R.S. Finn, Adjuvant and neoadjuvant immunotherapies in hepatocellular carcinoma, Nat Rev Clin Oncol 21(4) (2024) 294-311.\u003c/li\u003e\n\u003cli\u003eZ. Chen, G. Zhang, X. Ren, Z. Yao, Q. Zhou, X. Ren, S. Chen, L. Xu, K. Sun, Q. Zeng, M. Kuang, D.M. Kuang, S. Peng, Cross-talk between Myeloid and B Cells Shapes the Distinct Microenvironments of Primary and Secondary Liver Cancer, Cancer Res 83(21) (2023) 3544-3561.\u003c/li\u003e\n\u003cli\u003eS. Santagata, G. Rea, D. Castaldo, M. Napolitano, A. Capiluongo, C. D\u0026apos;Alterio, A.M. Trotta, C. Ierano, L. Portella, S. Di Maro, F. Tatangelo, V. Albino, R. Guarino, C. Cutolo, F. Izzo, S. Scala, Hepatocellular carcinoma (HCC) tumor microenvironment is more suppressive than colorectal cancer liver metastasis (CRLM) tumor microenvironment, Hepatol Int 18(2) (2024) 568-581.\u003c/li\u003e\n\u003cli\u003eG. Corti, K. Buzo, E. Berrino, M. Miotto, M.C. Aquilano, M. Lentini, S.E. Bellomo, A. Lorenzato, A. Bartolini, G. Mauri, L. Lazzari, M. Russo, F. Di Nicolantonio, S. Siena, S. Marsoni, C. Marchio, A. Bardelli, S. Arena, Prediction of homologous recombination deficiency identifies colorectal tumors sensitive to PARP inhibition, NPJ Precis Oncol 8(1) (2024) 231.\u003c/li\u003e\n\u003cli\u003eF.H. Wu, H.Z. Wei, H.Y. Deng, G.H. Xiao, Y.C. Zhang, PARP in colorectal cancer: Molecular mechanisms, immunity, clinical trials, and drug combinations, Neoplasma 70(1) (2023) 1-14.\u003c/li\u003e\n\u003cli\u003eS. Lheureux, Z. Lai, B.A. Dougherty, S. Runswick, D.R. Hodgson, K.M. Timms, J.S. Lanchbury, S. Kaye, C. Gourley, D. Bowtell, E.C. Kohn, C. Scott, U. Matulonis, T. Panzarella, K. Karakasis, J.V. Burnier, C.B. Gilks, M.J. O\u0026apos;Connor, J.D. Robertson, J. Ledermann, J.C. Barrett, T.W. Ho, A.M. Oza, Long-Term Responders on Olaparib Maintenance in High-Grade Serous Ovarian Cancer: Clinical and Molecular Characterization, Clin Cancer Res 23(15) (2017) 4086-4094.\u003c/li\u003e\n\u003cli\u003eD. Dibitetto, C.A. Widmer, S. Rottenberg, PARPi, BRCA, and gaps: controversies and future research, Trends Cancer 10(9) (2024) 857-869.\u003c/li\u003e\n\u003cli\u003eJ.S. Frenel, J.W. Kim, N. Aryal, R. Asher, D. Berton, L. Vidal, P. Pautier, J.A. Ledermann, R.T. Penson, A.M. Oza, J. Korach, T. Huzarski, S. Pignata, N. Colombo, T.W. Park-Simon, K. Tamura, G.S. Sonke, A.E. Freimund, C.K. Lee, E. Pujade-Lauraine, Efficacy of subsequent chemotherapy for patients with BRCA1/2-mutated recurrent epithelial ovarian cancer progressing on olaparib versus placebo maintenance: post-hoc analyses of the SOLO2/ENGOT Ov-21 trial, Ann Oncol 33(10) (2022) 1021-1028.\u003c/li\u003e\n\u003cli\u003eC.C. Tzang, E.S. Huang, H.W. Wu, W.C. Lin, Y.T. Lee, C.A. Luo, Y.H. Chen, Z.Y. Chang, Z.T. Chen, V.F. Kuo, B.S. Tzang, T.C. Hsu, Effectiveness and safety of PARP inhibitors in ovarian cancer: An umbrella review of systematic reviews and meta-analyses, Crit Rev Oncol Hematol 215 (2025) 104893.\u003c/li\u003e\n\u003cli\u003eL. Oplustil O\u0026apos;Connor, S.L. Rulten, A.N. Cranston, R. Odedra, H. Brown, J.E. Jaspers, L. Jones, C. Knights, B. Evers, A. Ting, R.H. Bradbury, M. Pajic, S. Rottenberg, J. Jonkers, D. Rudge, N.M. Martin, K.W. Caldecott, A. Lau, M.J. O\u0026apos;Connor, The PARP Inhibitor AZD2461 Provides Insights into the Role of PARP3 Inhibition for Both Synthetic Lethality and Tolerability with Chemotherapy in Preclinical Models, Cancer Res 76(20) (2016) 6084-6094.\u003c/li\u003e\n\u003cli\u003eM.A. Romeo, M.S. Gilardini Montani, R. Benedetti, A. Arena, M. Maretto, E. Bassetti, R. Caiazzo, G. D\u0026apos;Orazi, M. Cirone, Anticancer effect of AZD2461 PARP inhibitor against colon cancer cells carrying wt or dysfunctional p53, Exp Cell Res 408(2) (2021) 112879.\u003c/li\u003e\n\u003cli\u003eA. Koski, E. Karli, A. Kipar, S. Escutenaire, A. Kanerva, A. Hemminki, Mutation of the fiber shaft heparan sulphate binding site of a 5/3 chimeric adenovirus reduces liver tropism, PLoS One 8(4) (2013) e60032.\u003c/li\u003e\n\u003cli\u003eC. Xu, L. Chen, G. Liu, J. Xu, W. Lv, X. Gao, P. Xu, M. Tang, Y. Wang, X. Zhao, G. Nie, K. Cheng, F. Liu, Tailoring an intravenously injectable oncolytic virus for augmenting radiotherapy, Cell Rep Med 6(5) (2025) 102078.\u003c/li\u003e\n\u003cli\u003eK. Nebenzahl-Sharon, R. Sharf, J. Amer, H. Shalata, H. Khoury-Haddad, S.Y. Sohn, N. Ayoub, P. Hearing, T. Kleinberger, An Interaction with PARP-1 and Inhibition of Parylation Contribute to Attenuation of DNA Damage Signaling by the Adenovirus E4orf4 Protein, J Virol 93(19) (2019).\u003c/li\u003e\n\u003cli\u003eK.L. Zhang, R.P. Li, B.P. Zhang, S.T. Gao, B. Li, C.J. Huang, R. Cao, J.Y. Cheng, X.D. Xie, Z.H. Yu, X.Y. Feng, Efficacy of a new oncolytic adenovirus armed with IL-13 against oral carcinoma models, Onco Targets Ther 12 (2019) 6515-6523.\u003c/li\u003e\n\u003cli\u003eM. Gao, E. Yngve, D. Yu, C. Jin, A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products, Front Mol Biosci 9 (2022) 883249.\u003c/li\u003e\n\u003cli\u003eP. Limani, N. Borgeaud, M. Linecker, C. Tschuor, E. Kachaylo, A. Schlegel, J.H. Jang, U. Ungethum, M. Montani, R. Graf, B. Humar, P.A. Clavien, Selective portal vein injection for the design of syngeneic models of liver malignancy, Am J Physiol Gastrointest Liver Physiol 310(9) (2016) G682-8.\u003c/li\u003e\n\u003cli\u003eJ. Ning, H. Wakimoto, C. Peters, R.L. Martuza, S.D. Rabkin, Rad51 Degradation: Role in Oncolytic Virus-Poly(ADP-Ribose) Polymerase Inhibitor Combination Therapy in Glioblastoma, J Natl Cancer Inst 109(3) (2017) 1-13.\u003c/li\u003e\n\u003cli\u003eY. Liu, S. Fang, P. Wang, J. Zhang, F. Liu, Olaparib Enhances the Efficacy of Third-Generation Oncolytic Adenoviruses Against Glioblastoma by Modulating DNA Damage Response and p66shc-Induced Apoptosis, CNS Neurosci Ther 30(11) (2024) e70124.\u003c/li\u003e\n\u003cli\u003eG. Giordano, A. Merlini, F. Capozzi, G. Ferrero, C. Tucciarello, S. Majidi, S. Comparato, G. Mesiano, E. Liuzzi, F. Galvagno, A. Massa, S. Brusco, V. Leuci, C. Riganti, S. Cutrupi, M. De Bortoli, L. D\u0026apos;Ambrosio, D. Sangiolo, G. Grignani, Y. Pignochino, The cGAS-STING pathway is a master regulator of OCT4 expression in persistent sarcoma cells and enhances cellular immunotherapy with NK and CIK lymphocytes, Cancer Immunol Immunother 74(10) (2025) 312.\u003c/li\u003e\n\u003cli\u003eC. Mao, W. Fan, J. Liu, F. Yang, W. Li, L. Li, Z. Shi, Q. Li, Z. Yuan, Y. Jiang, B. Chu, Targeting HDAC and PARP Enhances STING-Dependent Antitumor Immunity in STING-Deficient Tumor, Adv Sci (Weinh) 12(41) (2025) e07904.\u003c/li\u003e\n\u003cli\u003eT. Cao, T. Hao, X. Zhang, B. Chen, H. Zhang, L. Shao, W. He, B. Li, Q. Zheng, P. Ji, K. Tao, TAMs-specific ARF1 inhibition reprograms glioma microenvironment and enhances the therapeutic effect of oncolytic adenovirus, iScience 28(6) (2025) 112696.\u003c/li\u003e\n\u003cli\u003eF. Sun, Y. Xu, Z. Deng, P. Yang, A recombinant oncolytic influenza virus expressing a PD-L1 antibody induces CD8(+) T-cell activation via the cGas-STING pathway in mice with hepatocellular carcinoma, Int Immunopharmacol 120 (2023) 110323.\u003c/li\u003e\n\u003cli\u003eX. Leng, Q. Li, W. Chen, H. Feng, L. Li, L. Yu, P. Huang, P. Ma, F. Xie, C-176 inhibits macrophage polarization towards M1-subtype and ameliorates LPS induced acute kidney injury, Eur J Pharmacol 984 (2024) 177028.\u003c/li\u003e\n\u003cli\u003eS.M. Yang, Y.B. Li, H.X. Si, Y. Wei, F.J. Ma, J. Wang, T. Chen, K. Chen, C-176 reduces inflammation-induced pain by blocking the cGAS-STING pathway in microglia, Int J Neurosci 135(11) (2025) 1193-1207.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"colorectal liver metastasis, poly (ADP-ribose) polymerase inhibitor, oncolytic adenovirus, cGAS-Sting","lastPublishedDoi":"10.21203/rs.3.rs-8189652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8189652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColorectal cancer (CRC) frequently metastasizes to the liver, leading to a poor prognosis and limited therapeutic options compared to other liver cancers, such as hepatocellular carcinoma (HCC). In this study, we selected the poly (ADP-ribose) polymerase inhibitors (PARPi) and Oncolytic adenoviruses (oAdv) to treat colorectal cancer liver metastases (CRLM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptional differences between CRLM and HCC were compared using the GSE243245 datasets. The synergistic potential of oAdv and the PARPi was assessed with \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. The therapeutic efficacy and safety of the combination therapy were evaluated in subcutaneous xenograft and colorectal liver metastasis (CRLM) mouse models. The mechanism of the combination of PARPi and oAdv was further investigated using DNA damage assays and Western blotting (WB). Immune responses to CRLM were assessed using flow cytometry, Western blotting, and ELISpot assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eoAdv infection selectively enhanced DNA damage in CRC cells, significantly increasing their sensitivity to AZD2461, while sparing normal hepatocytes. In murine models, the combination therapy demonstrated superior tumor growth inhibition (51.56%) compared to monotherapies. The synergistic effect was particularly pronounced in the CRLM model, correlating with activation of the STING pathway, enhanced immune cell infiltration, and a specific immune response. The therapeutic benefits on the immune response were abrogated by cGAS-Sting pathway inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe combination of oAdvand PARPi represents a safe and effective strategy for treating colorectal liver metastases, functioning through tumor-selective enhancement of DNA damage and activation of the cGAS-Sting-mediated immune response.\u003c/p\u003e","manuscriptTitle":"Oncolytic adenovirus synergistically treats liver metastases from colon cancer with PARPi by increasing DNA damage and activating the STING pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-27 10:46:13","doi":"10.21203/rs.3.rs-8189652/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"37036ed1-c1c5-4f97-ae04-4a005961d5c6","owner":[],"postedDate":"November 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-18T09:11:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-27 10:46:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8189652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8189652","identity":"rs-8189652","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}