A Chimeric Oncolytic Adenovirus Carried by Macrophages for Glioma Immunotherapy

A Chimeric Oncolytic Adenovirus Carried by Macrophages for Glioma Immunotherapy | 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 A Chimeric Oncolytic Adenovirus Carried by Macrophages for Glioma Immunotherapy Fansong Tang, Zongliang Zhang, Jianguo Xu, Zeng Wang, Yongdong Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6291711/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 Purpose: Oncolytic viruses hold promise as a novel frontier in glioma immunotherapy; however, current oncolytic viruses often face challenges of inadequate glioma infiltration and limited viral persistence in clinical settings. Macrophages, with their ability to effectively infiltrate glioma tissue, have emerged as potential cell vectors; however, they naturally resist virus infection. Thus, we constructed oncolytic virus which could be delivered by macrophage carriers. Methods: We engineered a chimeric adenovirus5/35 by cloning the E2F1 promoter and adenovirus E1A gene into an adenovirus5/35 backbone. The virus was then transported by macrophages to the tumor site in xenograft glioma-bearing mice. Results: The genetically engineered adenovirus selectively eradicated tumor cells while sparing normal human cells. The virus efficiently infected macrophages and was effectively delivered to the tumor site. This therapeutic system exhibited robust infiltration of tumor tissue and prolonged the survival of mice. Conclusions: Exploiting macrophage carriers presents a promising approach to enhance the penetration and therapeutic efficacy of oncolytic adenoviruses, holding considerable potential for clinical translation. glioma macrophage oncolytic virus adenovirus immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Glioma stands out as the most prevalent and deadly malignancy within the central nervous system (CNS). Despite the utilization of conventional treatments such as surgical resection, radiotherapy, and chemotherapy, the median survival period for patients merely ranges from 14.4 to 78 months.[ 1 ] Consequently, the imperative for pioneering new therapeutic strategies to combat glioma is unequivocal. Immunotherapy stands as a promising avenue in the treatment of malignant tumors, harnessing the body's innate anti-tumor immune responses. Among these innovative approaches, oncolytic virus therapy emerges as a novel immunotherapeutic strategy, leveraging viruses engineered to selectively replicate within tumor cells, thereby inducing tumor cell lysis and eliciting immune activation.[ 2 ] Notably, clinical trials investigating oncolytic adenoviral therapy for gliomas have demonstrated promising efficacy.[ 3 ], [ 4 ], [ 5 ] However, a notable challenge in the clinical application of oncolytic viruses is the requirement for direct injection into tumor lesions. This approach presents potential hurdles including viral clearance by the host's neutralizing antibodies, limited penetration of the virus into the tumor microenvironment, and suboptimal efficacy against metastatic lesions.[ 6 ] The utilization of cellular vectors to transport oncolytic viruses presents a promising strategy for circumventing viral clearance by neutralizing antibodies, prolonging viral half-life, and facilitating enhanced viral infection of tumor cells.[ 6 ] Among these vectors, neural stem cells loaded with oncolytic adenovirus have emerged as a focal point in glioma therapy, demonstrating improved viral infiltration into tumor tissue and yielding favorable therapeutic outcomes.[ 7 ] However, the acquisition of neural stem cells poses challenges due to their limited availability and high cost, necessitating the exploration of alternative cell carriers to optimize this therapeutic approach. Macrophages, constituting the predominant immune cell population within the glioma microenvironment, typically comprise 30–50% of the solid tumor mass.[ 8 ] These macrophages are primarily recruited from the bone marrow post-tumorigenesis and predominantly exhibit a pro-tumorigenic M2-like phenotype. Their pivotal role in glioma proliferation and metastasis contributes to the establishment of an immunosuppressive 'cold' tumor microenvironment.[ 9 ], [ 10 ] Moreover, their infiltration into glioma tissue underscores their potent tropism effect. However, as immune cells, macrophages possess inherent resistance to many viral infections, potentially compromising the viability of oncolytic adenoviruses such as AD5-RGD, which poses challenges for their clinical delivery using macrophages. Hence, we developed a chimeric oncolytic adenovirus, OAd5/F35 [E2F1], capable of infecting both macrophages and tumor cells effectively. This virus demonstrates potent tumor cell-killing efficacy in vitro while sparing normal brain cells. In a xenograft glioma model, virus-laden macrophages efficiently deliver the virus to tumor sites, resulting in tumor cell death and prolonged survival in mice. These findings underscore the clinical promise of this immunotherapeutic approach for glioma. 2. Materials and methods 2.1. Ethics Approval This study was approved by the Medical Ethics Committee of Hospital of West China Hospital of Sichuan University Biomedical Ethics Committee (20210535A) and was performed according to the Institutional Guidelines. 2.2. Construction of viral plasmids E2F1 promoter and adenovirus E1 gene were obtained from 293A cells by polymerase chain reaction (PCR), and cloned to pAd5/F35[CopGFP] with I-CeuI and I-SceI.[ 11 ] Adenovirus E3 gene was synthesized and cloned to pAd5/F35[CopGFP] with SpeI and BstBI. The above steps constitute pAD5/F35[E2F1-E1Δ24-CopGFP-E3] (Fig. 1A). E2F1 promoter was cloned into pLVX[EGFP] with CliaI and XbaI to form pLVX[E2F1-EGFP]. 2.3. Virus packaging and amplification pAD5/F35[E2F1-E1D24-CopGFP-E3] was linearized by PacI and transfected into 293A cells with PEI. Primary OAd5/F35 [E2F1] were obtained from 3 freeze-thaw cycles of 293A (Fig. 1A). Subsequent amplification of the virus was performed in 20 dishes of 293A cells. After 3 freeze-thaw cycles harvested viruses were purified and concentrated by cesium chloride density gradient centrifugation, and then the cesium chloride was removed by dialysis using magnesium chloride, after which they were stored at -80°C. Lentivirus[E2F1-EGFP] was packaged by transfecting 293T with pLVX[E2F1-EGFP], psPAX2, and pMD2. G. Lentivirus was collected from the supernatant of 293T. 2.4. Cell Viability Assay Five thousand U87-MG cells were plated in each well of 96-well plate and added with ether OAd5/F35 [E2F1] (multiplicity of infection (MOI) = 0.3) or buffer. After 24, 48 or 72 hours, cell viability was measured by reading OD450 nm after adding CCK8 solution. 2.5. Cell Lines The 293A, 293T, SW620, U87-MG, and BEAS-2B were obtained from the ATCC. U87-Luc were genetically engineered to carry the luciferase gene (Luc). SW620-E2F1-GFP and U87-E2F1-GFP were genetically engineered to express GFP promoted by E2F1. All these cells were cultured in the standard protocol. Any human cell lines used in this study has been proven by DNA profiling. All human cell lines have been authenticated using STR profiling. All experiments were performed with mycoplasma-free cells 2.6. Isolation and Infection of Human Primary Macrophages Human peripheral blood mononuclear cells (PBMC) are isolated from fresh blood of healthy volunteers by density gradient centrifugation. After 3 days of induction by adding 10 ng/ml of GM-CSF to PBMC, wall-adherent human macrophages were obtained by washing away the upper layer of cells. In the study, 5 MOI of OAd5/F35 [E2F1] was added to human macrophages. After 2 days, macrophage was trypsin digested and stained by red cell tracker before adding to tumor spheroids. 2.7. 3D Tumor Spheroids Coculture Assays 1% agar was spread on the bottom of 24-well plates after sterilization and 1500 U87-MG cells per well were seeded with DMEM (Gibco) + 10% fetal bovine serum. After a week the formed tumor spheroids were divided into new wells. After the tumor spheroids reach a size of approximately 500µm, the macrophage carrying oncolytic virus was added to the spheroids. The fluorescence signal of virus or cell tracker was captured by confocal fluorescence microscopy (Zeiss 880) after 48 hours. 2.8. Animal Study Six-week-old female C57 nude mice were purchased from Gempharmatech. Inc. Mice were kept in pathogen-free conditions and on a typical 12 h:12 h light-dark cycle. For xenograft GBM model, mice were anesthetized and stereotaxically immobilized. After aseptically dissecting the scalp and drilling holes in the skull, 2*10 5 U87-luc cells in a volume of 5µl of PBS was injected 2 mm lateral to the sagittal suture and 0.5 mm posterior to the bregma, a depth of 2.5 mm from the surface of the brain. [ 12 ] Seven days later, the animals were randomly divided into 4 groups (n = 5) to be injected with ①1*10 5 macrophage loaded with adenovirus, ②2*10 6 PFU adenovirus, ③1*10 5 macrophage, ④3µl PBS after tumorigenesis was examined by animal lkgeq;wfxw. Tumor bioluminescence signals were then captured every few days to assess changes in tumor size and to observe survival. When a weight loss of > 5 g or a significant decrease in activity was observed, the mice were executed and the brain tissue was removed and fixed. 2.9. Immunoblotting Assay SW620, U87, BEAS-2B and human macrophages were lysed with RIPA and ultrasound, after which they were quantified using the BCA method. After PAGE electrophoresis and membrane transfer, the corresponding proteins were incubated with anti-RB(AB_3069784) and anti-E1A (ab204123) antibodies, followed by incubation with secondary antibodies. Picture were captured with e-BLOT Touch Imager. 2.10. Flow Cytometry Infection efficiency of AD5/F35[E2F1-E1D24-CopGFP-E3] was confirmed by detecting CopGFP in the viral genome using flow cytometry. Immunophenotypic changes in macrophages was measured with PE-anti-CD86(AB_1727518) antibody. 2.11. Laboratory Pathology Sections of tumor tissue were incubated with anti-mouse F4/80 antibody (AB_3072636), anti-human CD11B antibody (AB_3070630), anti IFITM1 antibody (protein tech 6074-1) and anti-CD206 antibody (AB_2935558) followed by incubation with HRP secondary antibody plus DAB staining or 594 nm fluorescent secondary antibody. 2.12. Bulk RNA Sequencing Data Generation and Analysis Total RNA was extracted using the TRIzol Reagent (Invitrogen), and Illumina sequencing was performed by Tsingke ( https://www.tsingke.com.cn/ ). Raw sequencing reads were demultiplexed by Tsingke and provided in FASTQ format. The sequencing reads were then aligned to the human reference transcriptome (GRCh38.87), and gene names were assigned based on Ensembl gene IDs. Sequencing coverage and quality statistics are described in Supplementary Materials 1 and Supplementary Table 1 The processed data were used as input for differential gene expression (DEG) analysis using the DESeq2 software package. P-value adjustment was performed using the false discovery rate (FDR) correction. Heatmaps were generated using the heatmap package. Gene Ontology (GO) and hallmark gene sets from the GO database ( http://www.geneontology.org/ ) were used for functional enrichment analysis, with GO terms mapped to the Generic GO subset. 2.13. Statistics Statistical software Prism (GraphPad) was used. We perform comparison of multiple data sets using one-way ANOVA test, comparison of two data sets using t-test, survival analysis using Log-rank (Mantel-Cox) test. 3. Results 3.1. Construction of OAd5/F35 [E2F1] Oncolytic Adenovirus We engineered a chimeric adenovirus, OAd5/F35 [E2F1-E1Δ24-EF1α-CopGFP-E3], where the E1Δ24 region of the adenoviral replication gene is driven by the tumor-specific promoter E2F1, integrated into the Ad5/F35 adenovirus backbone (Fig. 1A). CopGFP promoted by a stable promoter EF1α was introduced into the adenovirus genome to facilitate stable visualization under fluorescence microscopy. We anticipated that the E2F1 promoter, negatively regulated by the RB oncogene which is commonly mutated in human glioma cells[ 13 ], would selectively trigger viral replication in RB-mutated tumor cells while sparing RB-wildtype cells such as normal cells. As illustrated in Fig. 1B, the U87-MG cell line, characterized by low RB gene expression, and normal astrocytes, SW620 and BEAS-2B cells, characterized by high RB expression, served as representative model. To test the specificity of the E2F1 promoter for mutations in the RB gene. We first generated lentiviruses utilizing the E2F1 promoter to transcribe EGFP (pLVX[E2F1-EGFP]) (Fig. 1C). After infection with this lentivirus, EGFP intensity in these stable cell strains can reflect the specificity of the E2F1 promoter. We found that U87-E2F1-EGFP exhibited higher EGFP intensity than SW620-E2F1-EGFP and BEAS-2B-E2F1-EGFP (Fig. 1D, E). Subsequently, following the administration of OAd5/F35 [E2F1] to various cell lines, we observed the specifical expression of adenovirus E1A protein in U87-MG cells but not in normal astrocytes, SW620 and BEAS-2B cells (Fig. 1F). These results suggest that the E2F1 promoter was more powerful in RB-mutated cells (U87) than in RB-wild-type cells (SW620, BEAS-2B and astrocyte). Furthermore, to evaluate the safety of OAd5/F35 [E2F1], we exposed U87, SW620, BEAS-2B and human astrocytes to 15 MOI of the virus. By detecting the GFP intensity of the virus 36 and 72 hours post administration, we ensured that the virus infected both types of cells. However, compared to controls, OAd5/F35 [E2F1] causes complete death of U87-MG tumor cells with little effect on astrocytes (Fig. 1G). Collectively, these findings highlight OAd5/F35 [E2F1] as a safe oncolytic virus capable of specifically replicating in RB-mutated glioma cells while sparing normal brain cells. Fig. 1 Construction and validation of OAd5/F35 [E2F1]. A. The genome of OAd5/F35 [E2F1-E1D24-CopGFP-E3] contains the mutant adenovirus E1A gene, which is driven by the E2F1 promoter, along with the CopGFP reporter gene and the adenovirus E3 gene, all integrated into the Ad5/F35 backbone. The virus was packaged by transfecting 293A cells with pOAd5/F35 [E2F1-E1D24-CopGFP-E3], and subsequently collected through freeze-and-thaw cycles. B. Immunoblotting results show that the expression of RB in U87-MG cells is lower compared to normal astrocytes, SW620, and BEAS-2B cell lines. C. The E2F1 promoter drives the transcription of EGFP from the pLVX[E2F1-EGFP] lentivirus. D and E. Stable U87-MG and SW620 cell lines were generated using the LVX[E2F1-EGFP] lentivirus. EGFP expression was detected by flow cytometry, demonstrating the activity of the E2F1 promoter. F. Replication of OAd5/F35 [E2F1] in normal astrocytes, SW620, BEAS-2B, and U87-MG cells was assessed by immunoblotting for the virus protein E1A, 48 hours post-infection. G. Changes in cell morphology were observed 36 and 72 hours after the addition of 15 MOI of OAd5/F35 [E2F1] to human astrocytes, U87, BEAS-2B, and SW620 cells (****: P < 0.0001). 3.2. OAd5/F35 [E2F1] Effectively Infect both Tumor Cells and Macrophages The widely used oncolytic viruses AD5-RGD[ 5 ], featuring RGD domains on their fiber, encounter difficulties in infecting macrophages due to the absence of requisite receptors for viral entry. However, the AD5/F35 adenovirus, a chimeric variant combining features of both AD5 and AD35, demonstrates the capability to utilize the CD16 receptor on macrophages, enabling superior infection efficacy than AD5-RDG (Fig. 2A, B and C). Additionally, examination of E1A protein expression confirmed that OAd5/F35 [E2F1] selectively replicates in tumor cells but not in macrophages (Fig. 2D). Furthermore, CCK-8 assays demonstrated that virus can cause the death of tumor cells without killing macrophages. (Fig. 2E). In summary, while the virus efficiently infects macrophages and glioma cells, it demonstrates selective replication exclusively within tumor cells, exerting a potent cytotoxic effect. Fig. 2 OAd5/F35 [E2F1] infection of macrophages and tumor cells. A. Bright-field and fluorescence images of human macrophages, 24 hours post-infection with OAd5/F35 [E2F1] or OAd5-RGD (MOI = 5). B. The percentage of virus-infected human macrophages 24 hours post-infection, detected by flow cytometry (MOI = 5). C. Infection efficiency of OAd5/F35 [E2F1] and OAd5-RGD in human macrophages. D. Virus replication in macrophages and U87-MG cells, reflected by immunoblotting for E1A expression, 48 hours after OAd5/F35 [E2F1] infection. E. Relative cell viability of U87-MG and macrophage cells post-oncolytic virus infection (MOI = 0.3), measured by CCK-8 assay at three different time points (*: p < 0.05, **: p < 0.01, ****: P < 0.0001). 3.3. Macrophages Deliver Virus to Tumor Cells in vitro Concerned about the potential immunomodulatory effects of macrophages on viral infection, we treated virus and tumor cells with macrophage-conditioned media (CM) and found no reduction in viral infection efficacy (Fig. 3A and B). To assess the capacity of macrophages to deliver virus to tumor cells, we co-cultured OAd5/F35 [E2F1]-laden human macrophages with U87-MG cells (Fig. 3C) and observed the expression of virus-expressed fluorescent proteins in both macrophages and tumor cells (Fig. 3D and E). Additionally, employing a 3D tumor spheroid model of U87-MG cells, we validated the ability of macrophages to deliver OAd5/F35 [E2F1], observing infiltration of both viruses and macrophages into the tumor spheroid (Fig. 3E). In summary, macrophages demonstrate the capability to release and deliver viruses to tumor cells, with no discernible reduction in viral infection attributed to the immunological properties of macrophages. Fig. 3 Macrophages Deliver Virus to Tumor Cells in vitro. A, B. OAd5/F35[E2F1] infects U87-MG cells either alone (U87 + V) or with the supernatant from macrophage-conditioned medium. OAd5/F35[E2F1] was added simultaneously with the supernatant (U87 + CM + V) or pre-incubated with the supernatant for 8 hours (U87+(CM + V)). Viral infection efficiency was examined by flow cytometry after 24 hours. C. Schematic diagram of the experiment. D, E. Virus-laden macrophages (MOI = 5) were co-cultured with U87-MG cells. Virus released from macrophages was detected by fluorescence microscopy and flow cytometry after 24 hours. Red arrows indicate macrophages, while white arrows indicate infected tumor cells. F. Virus released from oncolytic virus-carrying macrophages (MOI = 5), stained with live cell dye red, was detected in U87-MG spheroids after 48 hours of co-culture, as observed by confocal fluorescence microscopy. 3.4. Macrophages Deliver Virus to Tumor Cells in Xenograft Model To evaluate the therapeutic efficacy of macrophage-delivered oncolytic viruses in glioblastoma multiforme (GBM), we established xenograft GBM model mice (U87-luc) and administered virus-loaded macrophages, macrophages alone, virus alone, and PBS to these mice (Fig. 4A). Monitoring tumor bioluminescence signals, we observed a reduction in tumor bioluminescence signals in mice treated with virus-loaded macrophages and those treated with virus alone at the early stages of treatment. Conversely, mice injected with PBS or macrophages alone exhibited consistently increasing tumor signals. However, tumor signals in all groups showed re-elevation at a later stage of treatment (Fig. 4B, C). Notably, among all treatment groups, mice receiving virus-loaded macrophages demonstrated the longest survival (Fig. 4D) (P = 0.0038). In conclusion, macrophage-delivered oncolytic viruses exhibit in vivo efficacy against GBM cells and significantly prolong the survival of tumor-bearing mice. Fig. 4 Macrophages Deliver Virus to Tumor Cells in Xenograft Model. A. Schematic diagram of the animal experiment. B, C. Bioluminescence signals in tumor-bearing mice at different time points post injection. (n = 5 ~ 6) D. Survival curves of different groups of mice (**: P < 0.01). 3.5. Oncolytic Viruses Alter the Tumor Microenvironment To visualize the presence of macrophages and microglia in mouse normal brain tissue and tumor tissue, we employed immunohistochemistry (IHC) with an anti-mouse F4/80-specific antibody, revealing distinct morphological differences in macrophages between the tumor and brain tissues (Fig. 5A). Additionally, we detected human macrophages and OAd5/F35 [E2F1] used for GBM treatment via immunofluorescence staining with anti-human CD11b-specific antibodies and virally expressed CopGFP. This system penetrates tumor tissue more efficiently than injecting the virus alone (Fig. 5B). To investigate whether OAd5/F35 [E2F1] influences the immunophenotype of macrophages, we assessed the expression levels of CD206 (Fig. 5C) and CD86 (Fig. 5D) post-viral infection using flow cytometry and immunofluorescence of pathological tissues (Fig. 5E, F). These findings suggest that oncolytic viruses promote the polarization of macrophages, the predominant immune cell type within the tumor microenvironment, towards a pro-inflammatory M1-like phenotype. We also observed distinct gene expression patterns (Fig. 5F, Supplemental Table 2) and a significantly higher number of differentially expressed genes between the macrophage and virus-laden macrophage groups (Fig. 5G, Supplemental Table 3). This suggests that viruses transported to the tumor site exert additional synergistic effects on macrophages. Interferon-inducible transmembrane protein 1 (IFITM1) plays a crucial role in the cellular response to various viruses [ 14 ]. and is also involved in the signal transduction pathways that regulate monocyte recruitment and tumor-associated macrophage (TAM) polarization [ 15 ]. The differential expression of IFITM1 in tumor tissues (Fig. 5H) may contribute to the enhanced recruitment and infiltration of virus-laden macrophages. 4. Discussion The use of macrophages as carriers for delivering immunotherapeutic agents or drugs in glioma treatment is a widely explored therapeutic strategy.[ 16 ], [ 17 ], [ 18 ] However, a significant challenge lies in effectively transfecting desired therapeutic substances into macrophages, which inherently possess resistance to exogenous materials. Leveraging the chimeric AD5/F35 adenovirus backbone, which efficiently infects macrophages via the CD16 receptor, holds promise. This approach not only facilitates the construction and delivery of oncolytic viruses but also enables genetic modification of macrophages to exert additional therapeutic effects on gliomas.[ 11 ] In our xenograft GBM model experiment, the virus effectively killed tumor cells early in treatment. However, after 42 days of treatment, tumor signals rebounded, suggesting that viral persistence may only extend to this point in time. Therefore, in clinical practice, oncolytic viruses are better utilized as adjuncts to surgical treatment. Additionally, the survival of mice injected solely with the virus did not significantly prolong, despite its effectiveness in tumor cell eradication during the early stages. Furthermore, although we have demonstrated that the virus only replicates in tumor cells in vitro, its toxicity mechanism is still under investigation in our research. As an immunotherapy, understanding the changes in the immune microenvironment following the administration of macrophage-delivered oncolytic viruses is of paramount importance to our research. However, due to the species-specific nature of oncolytic virus infection, even the chimeric AD5/F35 virus proved incapable of infecting mouse macrophages, let alone replicating in murine tumor cells. Consequently, our current validation of this therapy has been limited to a nude mouse xenograft GBM model, where we focus on discerning phenotypic alterations within the tumor immune microenvironment. Looking ahead, we are planning clinical studies and endeavoring to engineer viruses capable of infecting murine-derived cells for further investigation. The clinical application of oncolytic viruses for brain tumor treatment typically involves local injection into the tumor cavity, a procedure challenging to implement in many institutions. Given that a significant proportion of tumor-infiltrating macrophages originate from the bone marrow (Fig. 5A),[ 9 ] systemic administration of macrophages has been explored to achieve tropism for brain tissue.[ 19 ] We are endeavoring to establish a more convenient delivery method for oncolytic viruses by employing a similar systemic approach, utilizing macrophages or their precursor cells as carriers. Declarations Ethics Statement This study was approved by the Medical Ethics Committee of Hospital of West China Hospital of Sichuan University Biomedical Ethics Committee (20210535A) and animal experiments was performed according to the Institutional Guidelines. Conflict of Interests No conflict of interest exists in the submission of this manuscript Funding This work was supported by General Program of the National Natural Science Foundation of China (82173175), National Natural Science Foundation of China (82073404), 1·3·5 project for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (2020HXFH036), Key research and development project of science and technology department of Sichuan Province (2022YFS0321), Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJC2023010006). Competing Interests No conflict of interest exists in the submission of this manuscript Author Contributions Linrui Cai and Aiping Tong: supervision. Fansong Tang: conceptualization, methodology, investigation, writing - original draft. Zongliang Zhang: methodology, investigation. Tao Huang and Yuelong Wang and Jianguo Xu: conceptualization and writing - review & editing. Zeng Wang and Yongdong Chen and Tao Huang: methodology and Software. Some figures in this review article were created with BioRender.com. Data Availability The data will be provided upon reasonable request to corresponding author. Ethics approval This study was approved by the Medical Ethics Committee of Hospital of West China Hospital of Sichuan University Biomedical Ethics Committee (20210535A) and animal experiments was performed according to the Institutional Guidelines. 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Nanomed 13(2):157–178. 10.2217/nnm-2017-0266 De Palma M et al (2008) Tumor-Targeted Interferon-α Delivery by Tie2-Expressing Monocytes Inhibits Tumor Growth and Metastasis, Cancer Cell , vol. 14, no. 4, pp. 299–311, Oct. 10.1016/j.ccr.2008.09.004 Gardell JL et al (Oct. 2020) Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma. J Immunother Cancer 8(2):e001202. 10.1136/jitc-2020-001202 Shibuya Y et al (2022) Treatment of a genetic brain disease by CNS-wide microglia replacement, Sci. Transl. Med. , vol. 14, no. 636, p. eabl9945, Mar. 10.1126/scitranslmed.abl9945 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials1.docx SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplementaryTable3.xlsx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6291711","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436488611,"identity":"03435d36-cb69-4817-bceb-b8018f2f4f78","order_by":0,"name":"Fansong Tang","email":"","orcid":"","institution":"Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu","correspondingAuthor":false,"prefix":"","firstName":"Fansong","middleName":"","lastName":"Tang","suffix":""},{"id":436488612,"identity":"46a2a2fb-3d2c-4b4d-9901-aade5632ffb1","order_by":1,"name":"Zongliang Zhang","email":"","orcid":"","institution":"State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China","correspondingAuthor":false,"prefix":"","firstName":"Zongliang","middleName":"","lastName":"Zhang","suffix":""},{"id":436488613,"identity":"e9eaca1d-320d-410f-a4de-faa30c951a0f","order_by":2,"name":"Jianguo Xu","email":"","orcid":"","institution":"Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu","correspondingAuthor":false,"prefix":"","firstName":"Jianguo","middleName":"","lastName":"Xu","suffix":""},{"id":436488614,"identity":"d6ea7492-5a8d-4aaa-9f81-a4ce8a6ce5f6","order_by":3,"name":"Zeng Wang","email":"","orcid":"","institution":"State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China","correspondingAuthor":false,"prefix":"","firstName":"Zeng","middleName":"","lastName":"Wang","suffix":""},{"id":436488615,"identity":"72df7a32-1788-4169-87e9-ede6286fcb81","order_by":4,"name":"Yongdong Chen","email":"","orcid":"","institution":"State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China","correspondingAuthor":false,"prefix":"","firstName":"Yongdong","middleName":"","lastName":"Chen","suffix":""},{"id":436488616,"identity":"3fc227f2-105a-4a8f-b3a6-6a1203e4446d","order_by":5,"name":"Tao Huang","email":"","orcid":"","institution":"Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Huang","suffix":""},{"id":436488617,"identity":"e1618e93-c691-43f7-8172-ee23a1a7c9cb","order_by":6,"name":"Aiping P. Tong","email":"","orcid":"","institution":"National Drug Clinical-Trial Institution, Ministry of Education, Sichuan University, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Aiping","middleName":"P.","lastName":"Tong","suffix":""},{"id":436488618,"identity":"6ab079dc-0877-4c19-a51f-263b712ba4e6","order_by":7,"name":"Yuelong L. Wang","email":"","orcid":"","institution":"National Drug Clinical-Trial Institution, Ministry of Education, Sichuan University, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yuelong","middleName":"L.","lastName":"Wang","suffix":""},{"id":436488619,"identity":"5564f5d1-f690-435d-9c18-5eec6532d861","order_by":8,"name":"Linrui Cai","email":"data:image/png;base64,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","orcid":"","institution":"West China Second University Hospital of Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Linrui","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2025-03-24 05:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6291711/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6291711/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79707648,"identity":"e6abcebd-a19c-4aec-868d-4d70b776b00b","added_by":"auto","created_at":"2025-04-01 18:36:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":295098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and validation of OAd5/F35 [E2F1].\u003c/strong\u003e A. The genome of OAd5/F35 [E2F1-E1D24-CopGFP-E3] contains the mutant adenovirus E1A gene, which is driven by the E2F1 promoter, along with the CopGFP reporter gene and the adenovirus E3 gene, all integrated into the Ad5/F35 backbone. The virus was packaged by transfecting 293A cells with pOAd5/F35 [E2F1-E1D24-CopGFP-E3], and subsequently collected through freeze-and-thaw cycles. B. Immunoblotting results show that the expression of RB in U87-MG cells is lower compared to normal astrocytes, SW620, and BEAS-2B cell lines. C. The E2F1 promoter drives the transcription of EGFP from the pLVX[E2F1-EGFP] lentivirus. D and E. Stable U87-MG and SW620 cell lines were generated using the LVX[E2F1-EGFP] lentivirus. EGFP expression was detected by flow cytometry, demonstrating the activity of the E2F1 promoter. F. Replication of OAd5/F35 [E2F1] in normal astrocytes, SW620, BEAS-2B, and U87-MG cells was assessed by immunoblotting for the virus protein E1A, 48 hours post-infection. G. Changes in cell morphology were observed 36 and 72 hours after the addition of 15 MOI of OAd5/F35 [E2F1] to human astrocytes, U87, BEAS-2B, and SW620 cells (****: P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/91333cd4e3caf740d6516b04.jpg"},{"id":79707289,"identity":"e553ec01-c349-4163-a95c-29a843ced03f","added_by":"auto","created_at":"2025-04-01 18:28:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOAd5/F35 [E2F1] infection of macrophages and tumor cells.\u003c/strong\u003e A. Bright-field and fluorescence images of human macrophages, 24 hours post-infection with OAd5/F35 [E2F1] or OAd5-RGD (MOI = 5). B. The percentage of virus-infected human macrophages 24 hours post-infection, detected by flow cytometry (MOI = 5). C. Infection efficiency of OAd5/F35 [E2F1] and OAd5-RGD in human macrophages. D. Virus replication in macrophages and U87-MG cells, reflected by immunoblotting for E1A expression, 48 hours after OAd5/F35 [E2F1] infection. E. Relative cell viability of U87-MG and macrophage cells post-oncolytic virus infection (MOI = 0.3), measured by CCK-8 assay at three different time points (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ****: P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/ca8926f3c21f13210270613a.jpg"},{"id":79707294,"identity":"d72cb1a6-d3d7-42d7-9d59-86abb10349b6","added_by":"auto","created_at":"2025-04-01 18:28:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophages Deliver Virus to Tumor Cells in vitro.\u003c/strong\u003e A, B. OAd5/F35[E2F1] infects U87-MG cells either alone (U87+V) or with the supernatant from macrophage-conditioned medium. OAd5/F35[E2F1] was added simultaneously with the supernatant (U87+CM+V) or pre-incubated with the supernatant for 8 hours (U87+(CM+V)). Viral infection efficiency was examined by flow cytometry after 24 hours. C. Schematic diagram of the experiment. D, E. Virus-laden macrophages (MOI = 5) were co-cultured with U87-MG cells. Virus released from macrophages was detected by fluorescence microscopy and flow cytometry after 24 hours. Red arrows indicate macrophages, while white arrows indicate infected tumor cells. F. Virus released from oncolytic virus-carrying macrophages (MOI = 5), stained with live cell dye red, was detected in U87-MG spheroids after 48 hours of co-culture, as observed by confocal fluorescence microscopy.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/bae28c9b6157d8b709d715aa.jpg"},{"id":79707293,"identity":"ce4f8598-458e-4ee8-b760-1fd2617f2e13","added_by":"auto","created_at":"2025-04-01 18:28:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":190449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophages Deliver Virus to Tumor Cells in Xenograft Model.\u003c/strong\u003e A. Schematic diagram of the animal experiment. B, C. Bioluminescence signals in tumor-bearing mice at different time points post injection. (n = 5~6) D. Survival curves of different groups of mice (**: P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/9abf7246504719513e1755c2.jpg"},{"id":79708327,"identity":"62bbb375-e528-43b4-b8d3-9614417fc5f1","added_by":"auto","created_at":"2025-04-01 18:52:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":307546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of Tumor Microenvironment.\u003c/strong\u003e A. Anti-mouse F4/80 immunohistochemistry shows rounded macrophages in mouse GBM tumor tissue(left) and branched microglia in normal brain tissue(right). B. Immunofluorescence of anti-human CD11b and GFP of the viral genome, indicating injected human macrophages and OAd5/F35 [E2F1] in tumor tissue, respectively. C. Macrophage immunophenotype in tumor tissues after administration of cells or virus was observed by immunofluorescence of anti-CD206. D. Macrophage’s immunophenotype after viral infection was detected by PE-anti-CD86 flow cytometry. E. Expression of macrophage polarization markers in macrophages or virus-laden macrophages. F. Gene ontogeny functional significance enrichment analysis. The horizontal axis represents the proportion of genes in each entry among all genes in that entry, while the vertical axis represents different gene function entries. The size of the circles indicates the number of genes enriched in the corresponding entry. The larger the circle, the more genes are enriched in that pathway. The color represents the significance of enrichment. G. Differentially expressed genes. Red color indicates upregulated (p \u0026lt; 0.05; log2 fold change \u0026gt;) and blue downregulated (p\u0026lt; 0.05; log2 fold change \u0026lt; -1) genes. Adjusted p values derived from DESeq2 DEG analysis. (***: P \u0026lt; 0.001, ****: P \u0026lt; 0.0001). H. Anti IFITM1 immunohistochemistry shows differential expression in macrophage and virus-laden macrophage group.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/3c066500ecfb8a3b75eaba7e.jpg"},{"id":80539586,"identity":"42b85c72-29dc-4722-8613-66cb5982e740","added_by":"auto","created_at":"2025-04-14 12:38:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2028472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/27543cf7-8994-490f-b0fe-93766d6ef5b7.pdf"},{"id":79707288,"identity":"31a028aa-1f73-434c-ab79-c03e6b865d09","added_by":"auto","created_at":"2025-04-01 18:28:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20665,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/2a1a230f666713da689e772d.docx"},{"id":79707649,"identity":"73b9ed61-c896-4e8b-9656-e002bb0a3f7f","added_by":"auto","created_at":"2025-04-01 18:36:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10461,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/11fada4b223e878cfd66c54a.xlsx"},{"id":79707300,"identity":"c900c3f8-94a0-4832-a284-707f556c068f","added_by":"auto","created_at":"2025-04-01 18:28:40","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1782688,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/02d2269c2ce07fdad54c4daa.xlsx"},{"id":79707314,"identity":"fe46e543-27f7-4295-b395-4b28ba74062f","added_by":"auto","created_at":"2025-04-01 18:28:41","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25884002,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6291711/v1/7e2b296a7f8680f75e2e9345.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Chimeric Oncolytic Adenovirus Carried by Macrophages for Glioma Immunotherapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlioma stands out as the most prevalent and deadly malignancy within the central nervous system (CNS). Despite the utilization of conventional treatments such as surgical resection, radiotherapy, and chemotherapy, the median survival period for patients merely ranges from 14.4 to 78 months.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Consequently, the imperative for pioneering new therapeutic strategies to combat glioma is unequivocal.\u003c/p\u003e \u003cp\u003eImmunotherapy stands as a promising avenue in the treatment of malignant tumors, harnessing the body's innate anti-tumor immune responses. Among these innovative approaches, oncolytic virus therapy emerges as a novel immunotherapeutic strategy, leveraging viruses engineered to selectively replicate within tumor cells, thereby inducing tumor cell lysis and eliciting immune activation.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Notably, clinical trials investigating oncolytic adenoviral therapy for gliomas have demonstrated promising efficacy.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] However, a notable challenge in the clinical application of oncolytic viruses is the requirement for direct injection into tumor lesions. This approach presents potential hurdles including viral clearance by the host's neutralizing antibodies, limited penetration of the virus into the tumor microenvironment, and suboptimal efficacy against metastatic lesions.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe utilization of cellular vectors to transport oncolytic viruses presents a promising strategy for circumventing viral clearance by neutralizing antibodies, prolonging viral half-life, and facilitating enhanced viral infection of tumor cells.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Among these vectors, neural stem cells loaded with oncolytic adenovirus have emerged as a focal point in glioma therapy, demonstrating improved viral infiltration into tumor tissue and yielding favorable therapeutic outcomes.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] However, the acquisition of neural stem cells poses challenges due to their limited availability and high cost, necessitating the exploration of alternative cell carriers to optimize this therapeutic approach.\u003c/p\u003e \u003cp\u003eMacrophages, constituting the predominant immune cell population within the glioma microenvironment, typically comprise 30\u0026ndash;50% of the solid tumor mass.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] These macrophages are primarily recruited from the bone marrow post-tumorigenesis and predominantly exhibit a pro-tumorigenic M2-like phenotype. Their pivotal role in glioma proliferation and metastasis contributes to the establishment of an immunosuppressive 'cold' tumor microenvironment.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Moreover, their infiltration into glioma tissue underscores their potent tropism effect. However, as immune cells, macrophages possess inherent resistance to many viral infections, potentially compromising the viability of oncolytic adenoviruses such as AD5-RGD, which poses challenges for their clinical delivery using macrophages.\u003c/p\u003e \u003cp\u003eHence, we developed a chimeric oncolytic adenovirus, OAd5/F35 [E2F1], capable of infecting both macrophages and tumor cells effectively. This virus demonstrates potent tumor cell-killing efficacy in vitro while sparing normal brain cells. In a xenograft glioma model, virus-laden macrophages efficiently deliver the virus to tumor sites, resulting in tumor cell death and prolonged survival in mice. These findings underscore the clinical promise of this immunotherapeutic approach for glioma.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics Approval\u003c/h2\u003e \u003cp\u003e This study was approved by the Medical Ethics Committee of Hospital of West China Hospital of Sichuan University Biomedical Ethics Committee (20210535A) and was performed according to the Institutional Guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Construction of viral plasmids\u003c/h2\u003e \u003cp\u003eE2F1 promoter and adenovirus E1 gene were obtained from 293A cells by polymerase chain reaction (PCR), and cloned to pAd5/F35[CopGFP] with I-CeuI and I-SceI.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Adenovirus E3 gene was synthesized and cloned to pAd5/F35[CopGFP] with SpeI and BstBI. The above steps constitute pAD5/F35[E2F1-E1Δ24-CopGFP-E3] (Fig.\u0026nbsp;1A). E2F1 promoter was cloned into pLVX[EGFP] with CliaI and XbaI to form pLVX[E2F1-EGFP].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Virus packaging and amplification\u003c/h2\u003e \u003cp\u003epAD5/F35[E2F1-E1D24-CopGFP-E3] was linearized by PacI and transfected into 293A cells with PEI. Primary OAd5/F35 [E2F1] were obtained from 3 freeze-thaw cycles of 293A (Fig.\u0026nbsp;1A). Subsequent amplification of the virus was performed in 20 dishes of 293A cells. After 3 freeze-thaw cycles harvested viruses were purified and concentrated by cesium chloride density gradient centrifugation, and then the cesium chloride was removed by dialysis using magnesium chloride, after which they were stored at -80\u0026deg;C. Lentivirus[E2F1-EGFP] was packaged by transfecting 293T with pLVX[E2F1-EGFP], psPAX2, and pMD2. G. Lentivirus was collected from the supernatant of 293T.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell Viability Assay\u003c/h2\u003e \u003cp\u003eFive thousand U87-MG cells were plated in each well of 96-well plate and added with ether OAd5/F35 [E2F1] (multiplicity of infection (MOI)\u0026thinsp;=\u0026thinsp;0.3) or buffer. After 24, 48 or 72 hours, cell viability was measured by reading OD450 nm after adding CCK8 solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell Lines\u003c/h2\u003e \u003cp\u003eThe 293A, 293T, SW620, U87-MG, and BEAS-2B were obtained from the ATCC. U87-Luc were genetically engineered to carry the luciferase gene (Luc). SW620-E2F1-GFP and U87-E2F1-GFP were genetically engineered to express GFP promoted by E2F1. All these cells were cultured in the standard protocol. Any human cell lines used in this study has been proven by DNA profiling. All human cell lines have been authenticated using STR profiling. All experiments were performed with mycoplasma-free cells\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Isolation and Infection of Human Primary Macrophages\u003c/h2\u003e \u003cp\u003eHuman peripheral blood mononuclear cells (PBMC) are isolated from fresh blood of healthy volunteers by density gradient centrifugation. After 3 days of induction by adding 10 ng/ml of GM-CSF to PBMC, wall-adherent human macrophages were obtained by washing away the upper layer of cells. In the study, 5 MOI of OAd5/F35 [E2F1] was added to human macrophages. After 2 days, macrophage was trypsin digested and stained by red cell tracker before adding to tumor spheroids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. 3D Tumor Spheroids Coculture Assays\u003c/h2\u003e \u003cp\u003e1% agar was spread on the bottom of 24-well plates after sterilization and 1500 U87-MG cells per well were seeded with DMEM (Gibco)\u0026thinsp;+\u0026thinsp;10% fetal bovine serum. After a week the formed tumor spheroids were divided into new wells. After the tumor spheroids reach a size of approximately 500\u0026micro;m, the macrophage carrying oncolytic virus was added to the spheroids. The fluorescence signal of virus or cell tracker was captured by confocal fluorescence microscopy (Zeiss 880) after 48 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Animal Study\u003c/h2\u003e \u003cp\u003eSix-week-old female C57 nude mice were purchased from Gempharmatech. Inc. Mice were kept in pathogen-free conditions and on a typical 12 h:12 h light-dark cycle. For xenograft GBM model, mice were anesthetized and stereotaxically immobilized. After aseptically dissecting the scalp and drilling holes in the skull, 2*10\u003csup\u003e5\u003c/sup\u003e U87-luc cells in a volume of 5\u0026micro;l of PBS was injected 2 mm lateral to the sagittal suture and 0.5 mm posterior to the bregma, a depth of 2.5 mm from the surface of the brain. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Seven days later, the animals were randomly divided into 4 groups (n\u0026thinsp;=\u0026thinsp;5) to be injected with ①1*10\u003csup\u003e5\u003c/sup\u003e macrophage loaded with adenovirus, ②2*10\u003csup\u003e6\u003c/sup\u003e PFU adenovirus, ③1*10\u003csup\u003e5\u003c/sup\u003e macrophage, ④3\u0026micro;l PBS after tumorigenesis was examined by animal lkgeq;wfxw. Tumor bioluminescence signals were then captured every few days to assess changes in tumor size and to observe survival. When a weight loss of \u0026gt;\u0026thinsp;5 g or a significant decrease in activity was observed, the mice were executed and the brain tissue was removed and fixed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Immunoblotting Assay\u003c/h2\u003e \u003cp\u003eSW620, U87, BEAS-2B and human macrophages were lysed with RIPA and ultrasound, after which they were quantified using the BCA method. After PAGE electrophoresis and membrane transfer, the corresponding proteins were incubated with anti-RB(AB_3069784) and anti-E1A (ab204123) antibodies, followed by incubation with secondary antibodies. Picture were captured with e-BLOT Touch Imager.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Flow Cytometry\u003c/h2\u003e \u003cp\u003eInfection efficiency of AD5/F35[E2F1-E1D24-CopGFP-E3] was confirmed by detecting CopGFP in the viral genome using flow cytometry. Immunophenotypic changes in macrophages was measured with PE-anti-CD86(AB_1727518) antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Laboratory Pathology\u003c/h2\u003e \u003cp\u003eSections of tumor tissue were incubated with anti-mouse F4/80 antibody (AB_3072636), anti-human CD11B antibody (AB_3070630), anti IFITM1 antibody (protein tech 6074-1) and anti-CD206 antibody (AB_2935558) followed by incubation with HRP secondary antibody plus DAB staining or 594 nm fluorescent secondary antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Bulk RNA Sequencing Data Generation and Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the TRIzol Reagent (Invitrogen), and Illumina sequencing was performed by Tsingke (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.tsingke.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.tsingke.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Raw sequencing reads were demultiplexed by Tsingke and provided in FASTQ format. The sequencing reads were then aligned to the human reference transcriptome (GRCh38.87), and gene names were assigned based on Ensembl gene IDs. Sequencing coverage and quality statistics are described in Supplementary Materials 1 and Supplementary Table\u0026nbsp;1 The processed data were used as input for differential gene expression (DEG) analysis using the DESeq2 software package. P-value adjustment was performed using the false discovery rate (FDR) correction. Heatmaps were generated using the heatmap package. Gene Ontology (GO) and hallmark gene sets from the GO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org/\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used for functional enrichment analysis, with GO terms mapped to the Generic GO subset.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistics\u003c/h2\u003e \u003cp\u003eStatistical software Prism (GraphPad) was used. We perform comparison of multiple data sets using one-way ANOVA test, comparison of two data sets using t-test, survival analysis using Log-rank (Mantel-Cox) test.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Construction of OAd5/F35 [E2F1] Oncolytic Adenovirus\u003c/h2\u003e \u003cp\u003eWe engineered a chimeric adenovirus, OAd5/F35 [E2F1-E1Δ24-EF1α-CopGFP-E3], where the E1Δ24 region of the adenoviral replication gene is driven by the tumor-specific promoter E2F1, integrated into the Ad5/F35 adenovirus backbone (Fig.\u0026nbsp;1A). CopGFP promoted by a stable promoter EF1α was introduced into the adenovirus genome to facilitate stable visualization under fluorescence microscopy. We anticipated that the E2F1 promoter, negatively regulated by the RB oncogene which is commonly mutated in human glioma cells[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], would selectively trigger viral replication in RB-mutated tumor cells while sparing RB-wildtype cells such as normal cells. As illustrated in Fig.\u0026nbsp;1B, the U87-MG cell line, characterized by low RB gene expression, and normal astrocytes, SW620 and BEAS-2B cells, characterized by high RB expression, served as representative model.\u003c/p\u003e \u003cp\u003eTo test the specificity of the E2F1 promoter for mutations in the RB gene. We first generated lentiviruses utilizing the E2F1 promoter to transcribe EGFP (pLVX[E2F1-EGFP]) (Fig.\u0026nbsp;1C). After infection with this lentivirus, EGFP intensity in these stable cell strains can reflect the specificity of the E2F1 promoter. We found that U87-E2F1-EGFP exhibited higher EGFP intensity than SW620-E2F1-EGFP and BEAS-2B-E2F1-EGFP (Fig.\u0026nbsp;1D, E). Subsequently, following the administration of OAd5/F35 [E2F1] to various cell lines, we observed the specifical expression of adenovirus E1A protein in U87-MG cells but not in normal astrocytes, SW620 and BEAS-2B cells (Fig.\u0026nbsp;1F). These results suggest that the E2F1 promoter was more powerful in RB-mutated cells (U87) than in RB-wild-type cells (SW620, BEAS-2B and astrocyte).\u003c/p\u003e \u003cp\u003eFurthermore, to evaluate the safety of OAd5/F35 [E2F1], we exposed U87, SW620, BEAS-2B and human astrocytes to 15 MOI of the virus. By detecting the GFP intensity of the virus 36 and 72 hours post administration, we ensured that the virus infected both types of cells. However, compared to controls, OAd5/F35 [E2F1] causes complete death of U87-MG tumor cells with little effect on astrocytes (Fig.\u0026nbsp;1G). Collectively, these findings highlight OAd5/F35 [E2F1] as a safe oncolytic virus capable of specifically replicating in RB-mutated glioma cells while sparing normal brain cells. Fig.\u0026nbsp;1 \u003cb\u003eConstruction and validation of OAd5/F35 [E2F1].\u003c/b\u003e A. The genome of OAd5/F35 [E2F1-E1D24-CopGFP-E3] contains the mutant adenovirus E1A gene, which is driven by the E2F1 promoter, along with the CopGFP reporter gene and the adenovirus E3 gene, all integrated into the Ad5/F35 backbone. The virus was packaged by transfecting 293A cells with pOAd5/F35 [E2F1-E1D24-CopGFP-E3], and subsequently collected through freeze-and-thaw cycles. B. Immunoblotting results show that the expression of RB in U87-MG cells is lower compared to normal astrocytes, SW620, and BEAS-2B cell lines. C. The E2F1 promoter drives the transcription of EGFP from the pLVX[E2F1-EGFP] lentivirus. D and E. Stable U87-MG and SW620 cell lines were generated using the LVX[E2F1-EGFP] lentivirus. EGFP expression was detected by flow cytometry, demonstrating the activity of the E2F1 promoter. F. Replication of OAd5/F35 [E2F1] in normal astrocytes, SW620, BEAS-2B, and U87-MG cells was assessed by immunoblotting for the virus protein E1A, 48 hours post-infection. G. Changes in cell morphology were observed 36 and 72 hours after the addition of 15 MOI of OAd5/F35 [E2F1] to human astrocytes, U87, BEAS-2B, and SW620 cells (****: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. OAd5/F35 [E2F1] Effectively Infect both Tumor Cells and Macrophages\u003c/h2\u003e \u003cp\u003eThe widely used oncolytic viruses AD5-RGD[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], featuring RGD domains on their fiber, encounter difficulties in infecting macrophages due to the absence of requisite receptors for viral entry. However, the AD5/F35 adenovirus, a chimeric variant combining features of both AD5 and AD35, demonstrates the capability to utilize the CD16 receptor on macrophages, enabling superior infection efficacy than AD5-RDG (Fig.\u0026nbsp;2A, B and C). Additionally, examination of E1A protein expression confirmed that OAd5/F35 [E2F1] selectively replicates in tumor cells but not in macrophages (Fig.\u0026nbsp;2D). Furthermore, CCK-8 assays demonstrated that virus can cause the death of tumor cells without killing macrophages. (Fig.\u0026nbsp;2E). In summary, while the virus efficiently infects macrophages and glioma cells, it demonstrates selective replication exclusively within tumor cells, exerting a potent cytotoxic effect. Fig.\u0026nbsp;2 \u003cb\u003eOAd5/F35 [E2F1] infection of macrophages and tumor cells.\u003c/b\u003e A. Bright-field and fluorescence images of human macrophages, 24 hours post-infection with OAd5/F35 [E2F1] or OAd5-RGD (MOI\u0026thinsp;=\u0026thinsp;5). B. The percentage of virus-infected human macrophages 24 hours post-infection, detected by flow cytometry (MOI\u0026thinsp;=\u0026thinsp;5). C. Infection efficiency of OAd5/F35 [E2F1] and OAd5-RGD in human macrophages. D. Virus replication in macrophages and U87-MG cells, reflected by immunoblotting for E1A expression, 48 hours after OAd5/F35 [E2F1] infection. E. Relative cell viability of U87-MG and macrophage cells post-oncolytic virus infection (MOI\u0026thinsp;=\u0026thinsp;0.3), measured by CCK-8 assay at three different time points (*: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **: p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ****: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Macrophages Deliver Virus to Tumor Cells in vitro\u003c/h2\u003e \u003cp\u003eConcerned about the potential immunomodulatory effects of macrophages on viral infection, we treated virus and tumor cells with macrophage-conditioned media (CM) and found no reduction in viral infection efficacy (Fig.\u0026nbsp;3A and B). To assess the capacity of macrophages to deliver virus to tumor cells, we co-cultured OAd5/F35 [E2F1]-laden human macrophages with U87-MG cells (Fig.\u0026nbsp;3C) and observed the expression of virus-expressed fluorescent proteins in both macrophages and tumor cells (Fig.\u0026nbsp;3D and E). Additionally, employing a 3D tumor spheroid model of U87-MG cells, we validated the ability of macrophages to deliver OAd5/F35 [E2F1], observing infiltration of both viruses and macrophages into the tumor spheroid (Fig.\u0026nbsp;3E). In summary, macrophages demonstrate the capability to release and deliver viruses to tumor cells, with no discernible reduction in viral infection attributed to the immunological properties of macrophages. Fig.\u0026nbsp;3 \u003cb\u003eMacrophages Deliver Virus to Tumor Cells in vitro.\u003c/b\u003e A, B. OAd5/F35[E2F1] infects U87-MG cells either alone (U87\u0026thinsp;+\u0026thinsp;V) or with the supernatant from macrophage-conditioned medium. OAd5/F35[E2F1] was added simultaneously with the supernatant (U87\u0026thinsp;+\u0026thinsp;CM\u0026thinsp;+\u0026thinsp;V) or pre-incubated with the supernatant for 8 hours (U87+(CM\u0026thinsp;+\u0026thinsp;V)). Viral infection efficiency was examined by flow cytometry after 24 hours. C. Schematic diagram of the experiment. D, E. Virus-laden macrophages (MOI\u0026thinsp;=\u0026thinsp;5) were co-cultured with U87-MG cells. Virus released from macrophages was detected by fluorescence microscopy and flow cytometry after 24 hours. Red arrows indicate macrophages, while white arrows indicate infected tumor cells. F. Virus released from oncolytic virus-carrying macrophages (MOI\u0026thinsp;=\u0026thinsp;5), stained with live cell dye red, was detected in U87-MG spheroids after 48 hours of co-culture, as observed by confocal fluorescence microscopy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Macrophages Deliver Virus to Tumor Cells in Xenograft Model\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic efficacy of macrophage-delivered oncolytic viruses in glioblastoma multiforme (GBM), we established xenograft GBM model mice (U87-luc) and administered virus-loaded macrophages, macrophages alone, virus alone, and PBS to these mice (Fig.\u0026nbsp;4A). Monitoring tumor bioluminescence signals, we observed a reduction in tumor bioluminescence signals in mice treated with virus-loaded macrophages and those treated with virus alone at the early stages of treatment. Conversely, mice injected with PBS or macrophages alone exhibited consistently increasing tumor signals. However, tumor signals in all groups showed re-elevation at a later stage of treatment (Fig.\u0026nbsp;4B, C). Notably, among all treatment groups, mice receiving virus-loaded macrophages demonstrated the longest survival (Fig.\u0026nbsp;4D) (P\u0026thinsp;=\u0026thinsp;0.0038). In conclusion, macrophage-delivered oncolytic viruses exhibit in vivo efficacy against GBM cells and significantly prolong the survival of tumor-bearing mice. Fig.\u0026nbsp;4 \u003cb\u003eMacrophages Deliver Virus to Tumor Cells in Xenograft Model.\u003c/b\u003e A. Schematic diagram of the animal experiment. B, C. Bioluminescence signals in tumor-bearing mice at different time points post injection. (n\u0026thinsp;=\u0026thinsp;5\u0026thinsp;~\u0026thinsp;6) D. Survival curves of different groups of mice (**: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Oncolytic Viruses Alter the Tumor Microenvironment\u003c/h2\u003e \u003cp\u003eTo visualize the presence of macrophages and microglia in mouse normal brain tissue and tumor tissue, we employed immunohistochemistry (IHC) with an anti-mouse F4/80-specific antibody, revealing distinct morphological differences in macrophages between the tumor and brain tissues (Fig.\u0026nbsp;5A). Additionally, we detected human macrophages and OAd5/F35 [E2F1] used for GBM treatment via immunofluorescence staining with anti-human CD11b-specific antibodies and virally expressed CopGFP. This system penetrates tumor tissue more efficiently than injecting the virus alone (Fig.\u0026nbsp;5B). To investigate whether OAd5/F35 [E2F1] influences the immunophenotype of macrophages, we assessed the expression levels of CD206 (Fig.\u0026nbsp;5C) and CD86 (Fig.\u0026nbsp;5D) post-viral infection using flow cytometry and immunofluorescence of pathological tissues (Fig.\u0026nbsp;5E, F). These findings suggest that oncolytic viruses promote the polarization of macrophages, the predominant immune cell type within the tumor microenvironment, towards a pro-inflammatory M1-like phenotype.\u003c/p\u003e \u003cp\u003eWe also observed distinct gene expression patterns (Fig.\u0026nbsp;5F, Supplemental Table\u0026nbsp;2) and a significantly higher number of differentially expressed genes between the macrophage and virus-laden macrophage groups (Fig.\u0026nbsp;5G, Supplemental Table\u0026nbsp;3). This suggests that viruses transported to the tumor site exert additional synergistic effects on macrophages. Interferon-inducible transmembrane protein 1 (IFITM1) plays a crucial role in the cellular response to various viruses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. and is also involved in the signal transduction pathways that regulate monocyte recruitment and tumor-associated macrophage (TAM) polarization [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The differential expression of IFITM1 in tumor tissues (Fig.\u0026nbsp;5H) may contribute to the enhanced recruitment and infiltration of virus-laden macrophages. \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe use of macrophages as carriers for delivering immunotherapeutic agents or drugs in glioma treatment is a widely explored therapeutic strategy.[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] However, a significant challenge lies in effectively transfecting desired therapeutic substances into macrophages, which inherently possess resistance to exogenous materials. Leveraging the chimeric AD5/F35 adenovirus backbone, which efficiently infects macrophages via the CD16 receptor, holds promise. This approach not only facilitates the construction and delivery of oncolytic viruses but also enables genetic modification of macrophages to exert additional therapeutic effects on gliomas.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn our xenograft GBM model experiment, the virus effectively killed tumor cells early in treatment. However, after 42 days of treatment, tumor signals rebounded, suggesting that viral persistence may only extend to this point in time. Therefore, in clinical practice, oncolytic viruses are better utilized as adjuncts to surgical treatment. Additionally, the survival of mice injected solely with the virus did not significantly prolong, despite its effectiveness in tumor cell eradication during the early stages. Furthermore, although we have demonstrated that the virus only replicates in tumor cells in vitro, its toxicity mechanism is still under investigation in our research.\u003c/p\u003e \u003cp\u003eAs an immunotherapy, understanding the changes in the immune microenvironment following the administration of macrophage-delivered oncolytic viruses is of paramount importance to our research. However, due to the species-specific nature of oncolytic virus infection, even the chimeric AD5/F35 virus proved incapable of infecting mouse macrophages, let alone replicating in murine tumor cells. Consequently, our current validation of this therapy has been limited to a nude mouse xenograft GBM model, where we focus on discerning phenotypic alterations within the tumor immune microenvironment. Looking ahead, we are planning clinical studies and endeavoring to engineer viruses capable of infecting murine-derived cells for further investigation.\u003c/p\u003e \u003cp\u003eThe clinical application of oncolytic viruses for brain tumor treatment typically involves local injection into the tumor cavity, a procedure challenging to implement in many institutions. Given that a significant proportion of tumor-infiltrating macrophages originate from the bone marrow (Fig.\u0026nbsp;5A),[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] systemic administration of macrophages has been explored to achieve tropism for brain tissue.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] We are endeavoring to establish a more convenient delivery method for oncolytic viruses by employing a similar systemic approach, utilizing macrophages or their precursor cells as carriers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics Statement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of Hospital of West China Hospital of Sichuan University Biomedical Ethics Committee (20210535A) and animal experiments was performed according to the Institutional Guidelines.\u003c/p\u003e\n\u003cp\u003eConflict of Interests\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exists in the submission of this manuscript\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by General Program of the National Natural Science Foundation of China (82173175), National Natural Science Foundation of China (82073404), 1\u0026middot;3\u0026middot;5 project for disciplines of excellence\u0026ndash;Clinical Research Incubation Project, West China Hospital, Sichuan University (2020HXFH036), Key research and development project of science and technology department of Sichuan Province (2022YFS0321), Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJC2023010006).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exists in the submission of this manuscript\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eLinrui Cai and Aiping Tong: supervision. Fansong Tang: conceptualization, methodology, investigation, writing - original draft. Zongliang Zhang: methodology, investigation. Tao Huang and Yuelong Wang and Jianguo Xu: conceptualization and writing - review \u0026amp; editing. Zeng Wang and Yongdong Chen and Tao Huang: methodology and Software. Some figures in this review article were created with BioRender.com.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe data will be provided upon reasonable request to corresponding author.\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of Hospital of West China Hospital of Sichuan University Biomedical Ethics Committee (20210535A) and animal experiments was performed according to the Institutional Guidelines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOstrom QT, Gittleman H, Truitt G, Boscia A, Kruchko C, Barnholtz-Sloan JS (2018) CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2011\u0026ndash;2015, \u003cem\u003eNeuro-Oncol.\u003c/em\u003e, vol. 20, no. suppl_4, pp. iv1\u0026ndash;iv86, Oct. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/neuonc/noy131\u003c/span\u003e\u003cspan address=\"10.1093/neuonc/noy131\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelcher A, Harrington K, Vile R (2021) Oncolytic virotherapy as immunotherapy, \u003cem\u003eScience\u003c/em\u003e, vol. 374, no. 6573, pp. 1325\u0026ndash;1326, Dec. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.abk3436\u003c/span\u003e\u003cspan address=\"10.1126/science.abk3436\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLang FF et al (May 2018) Phase I Study of DNX-2401 (Delta-24-RGD) Oncolytic Adenovirus: Replication and Immunotherapeutic Effects in Recurrent Malignant Glioma. 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Med.\u003c/em\u003e, vol. 14, no. 636, p. eabl9945, Mar. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/scitranslmed.abl9945\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.abl9945\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"glioma, macrophage, oncolytic virus, adenovirus, immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6291711/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6291711/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurpose: Oncolytic viruses hold promise as a novel frontier in glioma immunotherapy; however, current oncolytic viruses often face challenges of inadequate glioma infiltration and limited viral persistence in clinical settings. Macrophages, with their ability to effectively infiltrate glioma tissue, have emerged as potential cell vectors; however, they naturally resist virus infection. Thus, we constructed oncolytic virus which could be delivered by macrophage carriers.\u003c/p\u003e\n\u003cp\u003eMethods: We engineered a chimeric adenovirus5/35 by cloning the E2F1 promoter and adenovirus E1A gene into an adenovirus5/35 backbone. The virus was then transported by macrophages to the tumor site in xenograft glioma-bearing mice.\u003c/p\u003e\n\u003cp\u003eResults: The genetically engineered adenovirus selectively eradicated tumor cells while sparing normal human cells. The virus efficiently infected macrophages and was effectively delivered to the tumor site. This therapeutic system exhibited robust infiltration of tumor tissue and prolonged the survival of mice.\u003c/p\u003e\n\u003cp\u003eConclusions: Exploiting macrophage carriers presents a promising approach to enhance the penetration and therapeutic efficacy of oncolytic adenoviruses, holding considerable potential for clinical translation.\u003c/p\u003e","manuscriptTitle":"A Chimeric Oncolytic Adenovirus Carried by Macrophages for Glioma Immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 18:28:35","doi":"10.21203/rs.3.rs-6291711/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":"c0b07567-bfa6-45d6-ab8a-6215a6bb39ad","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-14T12:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-01 18:28:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6291711","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6291711","identity":"rs-6291711","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}