Search Results (264)

Although cryoablation of tumor tissue is known to induce systemic antitumor immune responses, the underlying mechanisms remain poorly understood, and therapeutic efficacy is often unpredictable. During cryoablation, the cryoprobe creates a temperature gradient, resulting in a central zone of lethal tissue destruction and a periphery characterized by sub-lethal injury. In this peripheral zone, cells undergo programmed cell death triggered by thermal shock and ischemia. A widely cited, yet debated, hypothesis suggests that these apoptotic events in the tumor periphery may suppress the antitumor immune response. The purpose of this study was to experimentally test this hypothesis. We utilized the murine breast cancer cell line (4T1-Luc) and a panel of human cell lines (MCF-7, BT-20, BT-474, MDA-MB-231, MDA-MB-453, HBL-100). Following validation via flow cytometry using Annexin V-AF488, Rhodamine 123, TMRE, PI, and 7-AAD, we established −7 °C and −80 °C as temperature conditions that predominantly induce apoptosis and necrosis, respectively. These conditions were used to prepare cryo-treated 4T1-Luc cells for the vaccination of syngeneic mice. Ten days post-immunization, the efficacy of the treatment was evaluated in subcutaneous solid tumor and lung metastasis models by monitoring tumor growth, quantifying tumor-specific antibodies, performing histological analysis of tumor and lung tissues, and quantifying lung metastases via PCR and luciferase assays. Contrary to the initial hypothesis, our findings demonstrate that tumor cells undergoing sub-lethal cryoablation do not acquire immunosuppressive characteristics. Instead, they promote systemic antitumor immunity. Although antitumor immunity induced by apoptotic and necrotic tumor cells was comparable in a subcutaneous tumor model, vaccination with necrotic tumor cells proved more effective than vaccination with apoptotic cells in controlling lung metastasis formation. Full article

Background/Objectives: Breast cancer exhibits substantial molecular heterogeneity, resulting in diverse therapeutic vulnerabilities and limiting the efficacy of single-agent therapies. Although multi-target strategies may offer improved therapeutic benefit, the systematic identification of synergistic higher-order target combinations remains challenging. This study aimed to identify and validate effective higher-order target combinations for heterogeneous breast cancer. Methods: DeepMDS, a previously developed deep learning-based multi-compound synergy prediction model, was used to prioritize candidate target combinations for breast cancer. Exhaustive two-target and three-target combinations were ranked in the gene-expression contexts of ER-positive luminal-like MCF-7 and triple-negative MDA-MB-231 breast cancer cells. The top-ranked target combinations were evaluated using single, pairwise, and triple small interfering RNA (siRNA) perturbations. Representative inhibitors were subsequently assessed in fixed-ratio combinations in MCF-7, MDA-MB-231, and 4T1 cells and in a 4T1 syngeneic mouse experiment. Results: BIRC5-NAMPT-TOP1 ranked first in both cell lines. Triple siRNA co-transfection targeting BIRC5, NAMPT, and TOP1 produced the strongest antiproliferative effects, with inhibition rates of 62.94% in MCF-7 cells and 55.62% in MDA-MB-231 cells. The corresponding inhibitors, LQZ-7I, FK866, and topotecan, exhibited synergistic antiproliferative activity at multiple molar ratios, with combination index values below 1. The optimized 2.5:10:1 molar ratio showed strong synergy in MCF-7, MDA-MB-231, and 4T1 cells, with combination index values of 0.26, 0.19, and 0.15, respectively. In tumor-bearing mice model, the triple-inhibitor regimen achieved a tumor inhibition rate of 62.05%. Topotecan-containing groups showed hematological alterations, whereas no statistically detectable elevations were observed in the measured terminal serum hepatic or renal biomarkers. Conclusions: The BIRC5-NAMPT-TOP1 combination showed reproducible phenotypic activity in the tested genetic and pharmacological models. These findings support the use of DeepMDS as a hypothesis-generation tool for higher-order target prioritization. Full article

Background/Objectives: Tumor-draining lymph nodes (TDLNs) play a critical role in shaping a pre-metastatic and immunosuppressive niche that facilitates tumor dissemination. However, a robust in vivo model that recapitulates regional lymph node and distant metastasis remains elusive. We sought to establish and characterize an animal model that reliably mimics spontaneous human melanoma progression from primary tumor to regional lymph node and distant organ metastasis. Methods: B16F10-Luc2 melanoma cells were subcutaneously implanted from the hock area into the lower medial thigh of C57BL/6J mice to establish a metastatic melanoma mouse model with reliable regional lymph node metastasis and distant metastasis. Tumor growth was monitored over time. ¹⁸F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) and bioluminescence imaging (BLI) were used to track lymph node and lung metastases in vivo. Immunocytochemistry (IHC) was performed to confirm the metastatic melanoma cells in tissues. The immune landscape of metastatic lymph nodes was characterized by Cytometry by Time-Of-Flight (CyTOF). Results: This implantation strategy resulted in consistent primary tumor growth accompanied by reproducible metastasis to ipsilateral inguinal lymph nodes (IILN) and subsequent lung metastasis. Multi-modal imaging (PET/CT and BLI) enabled non-invasive monitoring of metastatic progression in vivo. Histological analyses confirmed melanoma cell infiltration in lymph nodes and lung tissues. Notably, CyTOF profiling revealed a distinct immunological remodeling within the TDLN, characterized by immunosuppressive phenotypes in the tumor-draining ipsilateral inguinal lymph node (IILN) compared with naïve nodes, indicative of a potential immunotolerant environment in tumor-bearing mice associated with metastatic progression. Conclusions: We present a reproducible and clinically relevant syngeneic melanoma mouse model that recapitulates lymphatic dissemination to regional lymph nodes and distant lung metastasis, and mimics the immunosuppressive phenotypes in the tumor-draining lymph node microenvironment seen in human melanoma patients. This model provides a valuable platform for investigating the mechanisms of lymphatic metastasis and the immunological dynamics of TDLNs, with potential applications in evaluating therapeutic strategies targeting metastatic progression. Full article

Background/Objective: Persistent infection with human papillomavirus type 16 (HPV-16) is the principal cause of cervical high-grade squamous intraepithelial lesions (cHSIL) and cervical cancer, yet the established treatments remain limited to ablative or excisional procedures that carry reproductive risk and do not eliminate the underlying infection. We report the preclinical development of ARV-2001, a messenger RNA (mRNA)–lipid nanoparticle (LNP) immunotherapeutic encoding mutated, non-oncogenic HPV-16 E6 and E7 fused to a SARS-CoV-2 spike S2 subdomain enriched in human CD4 helper epitopes, formulated in a novel cholesterol-derived ionizable lipid (ARV-T1). Methods: Interactions of ARV-2001-expressed antigens with p53 and retinoblastoma (Rb) were evaluated in human cervical carcinoma cell line C33A, in lentiviral constructs in primary human keratinocytes, and in soft-agar colony-formation assays. ARV-2001 was administrated by intramuscular (IM) or intradermal (ID) injection in naive mice or in the TC-1 tumor models. Tumor size and survival were monitored over time and tumor-infiltrated lymphocytes were characterized by flow cytometry. Intracellular cytokine staining and Elispot were used to evaluate immunogenicity. Results: In vitro, the mutated E6/E7–S2 antigen lost the ability to degrade p53, to deregulate the retinoblastoma (Rb) pathway, and to support anchorage-independent growth, suggesting abrogation of oncogenic activity. The S2 domain and imiquimod administration each augmented antitumor activity and intratumoral CD8⁺ T-cell infiltration while reducing myeloid-derived suppressor cells in the syngeneic HPV-16 E6/E7 TC-1 tumor models. In addition, ID administration of ARV-2001 into TC-1 tumor-bearing mice was superior to IM administration in terms of both tumor growth inhibition and survival. ID vaccination with ARV-2001 in mice consistently elicited a more potent E6/E7-specific T-cell response than the same dose given IM. Dose-escalation studies showed a dose-dependent T cell response against E6/E7 in ID-injected mice. Conclusions: This study supports future human evaluation of intradermally administrated ARV-2001 for treatment of HPV-16+ cHSIL in clinical trials. Full article

Background: IL-15 is a promising cytokine for cancer immunotherapy. IL-15 promotes differentiation and homeostasis of innate and adaptive immune cells, and their cytolytic effector functions. The IL-15 receptor is composed of IL-15Rα, IL-15Rβ and the common γ_c chains. IL-15 is also trans-presented as IL-15Rα:IL-15 complex to IL-15Rβ:γ_c on neighboring cells. IL-15Rα is dispensable for early immune response to infections and in autoimmune diabetes. The role of IL-15Rα in antitumor immune responses remains unclear. Methods: In WT, Il15^−/− and Il15ra^−/− mice, we studied the growth of syngeneic tumor cell lines and tumor immune surveillance against endogenous fibrosarcoma induced by methylcholanthrene (MCA). Immune gene signature and total proteome analysis were performed on MCA-induced tumors. Results: Lack of IL-15 or IL-15Rα did not enhance the growth of implanted tumor cell lines, despite reduced immune cell infiltration. MCA-induced tumor incidence was reduced in mice lacking IL-15Rα but not IL-15, although both are required for efficient tumor immunoediting. Il15^−/− and Il15ra^−/− tumors showed reduced Ifng expression but displayed differential modulation of Ifng-responsive genes. Proteome profiles of Il15^−/− tumors, but not tumor-derived cell lines, showed significant reduction in antigen presentation pathways. B16-F10 melanoma cells expressing NLRC5, the IFNγ-induced transcriptional activator of tumor antigen presentation, still required IL-15 but not IL-15Rα for efficient tumor control. Conclusions: Our findings show that IL-15 plays a negligible role in immunosurveillance against spontaneous tumor development, whereas IL-15Rα restrains immunosurveillance. Neither IL-15 nor IL-15Rα have a significant impact on implanted tumor models, although IL-15 facilitates efficient control of highly immunogenic tumors for which IL-15Rα is dispensable. Full article

APG157DS is a multi-component investigational drug which is currently the subject of multiple cancer trials. It has shown promising efficacy in patients with head and neck cancer. We report its immuno-modulatory effect in a syngeneic mouse model of glioblastoma (GBM). Long-term treatment with APG157DS led to durable tumor remission in 50% of mice, while all vehicle-treated mice reached humane endpoints within 42 days. Mechanistically, the drug induced selective repolarization of tumor-associated macrophages (TAMs) from an immunosuppressive Arg1^high/iNOS^low phenotype to a tumoricidal Arg1^low/iNOS^high state, accompanied by increased intratumoral recruitment of activated (NKp46+) natural killer cells and CD8+ cytotoxic T-cells. APG157DS also suppressed vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor 1-alpha (HIF-1α) expression in tumors, further impairing tumor growth. Notably, APG157DS did not elicit off-target macrophage activation in peripheral tissues such as the spleen, highlighting its selective immune targeting. These findings provide a mechanistic rationale for further clinical development of APG157DS in GBM and potentially other immune-evasive cancers. Full article

Oncolytic virotherapy offers a promising avenue for solid tumor treatment, yet single-agent approaches are frequently limited by insufficient tumor lysis and inadequate immune activation. Here we report that combined therapy with two recombinant variants of the oncolytic vaccinia virus, armed with either herpes simplex virus thymidine kinase (VV-HSVtk) or the interleukin 15 receptor subunit alpha (VV-mIL15Rα), leads to enhanced cytotoxicity and immune stimulation in a murine mammary adenocarcinoma model (4T1). In vitro, VV-HSVtk exhibited dose-dependent cytotoxicity markedly potentiated by ganciclovir (GCV) through HSVtk-mediated phosphorylation into a cytotoxic nucleoside analog, and co-culture of VV-infected tumor cells with donor-derived NK cells further increased oncolytic efficiency. In vivo, combined treatment with VV-HSVtk, VV-mIL15Rα, and GCV resulted in significant tumor regression and extended survival relative to monotherapy controls in 4T1 syngeneic mice. Histological examination revealed increased lymphocytic infiltration at tumor sites and absence of hepatic or splenic toxicity. Together, these data indicate that integrating direct viral cytotoxicity, HSVtk/GCV-mediated suicide gene therapy, and IL-15-pathway-targeted immunomodulation within an oncolytic vaccinia platform can improve antitumor efficacy in a stringent breast cancer model. Full article

Background: Toxoplasma gondii (T. gondii) is one of the most prevalent parasitic zoonoses worldwide, and the host’s immunological state significantly influences its clinical manifestations, which can be potentially fatal in immunocompromised hosts. The unavailability of a vaccine, combined with the considerable toxicity of existing medications, necessitates the urgent search for new therapies or adjunctive techniques, including regenerative and immunomodulatory approaches. Hence, the present study investigated, for the first time, the therapeutic potential of syngeneic platelet rich plasma (PRP) against T. gondii ME49 strain-induced chronic toxoplasmosis in both immunocompetent and immunosuppressed mouse models. Methods: 72 albino mice were divided into two sections, immunocompetent and immunosuppressed. Each section contained six groups: healthy, model, cotrimoxazole (CTZ)-treated, PRP-treated, half-dose of both CTZ and PRP-treated, and full-dose of both CTZ and PRP-treated. Treatment efficacy was assessed via parasitological, histological, immunohistochemical, and immunological analyses. Results: PRP, especially when coadministered with the CTZ, mitigated the consequences of toxoplasmosis by significantly reducing brain cyst counts (p < 0.0001), restoring brain tissue architecture, modulating apoptotic pathways by restoring caspase-3 expression in the brain, and normalizing systemic IFN-γ, TNF-α, and IL-10 cytokine profiles. Conclusions: The findings highlight PRP as an adjunct to the reference treatment, CTZ, for controlling toxoplasmosis in both immunocompetent and immunosuppressed conditions via anti-infective, neuroprotective, and immunomodulatory activities. Full article

Background: Epithelial ovarian cancer (EOC) is characterized by aggressive peritoneal dissemination and an immunosuppressive tumor microenvironment in which tumor-associated macrophages (TAMs) play a central role. Chemokine signaling pathways regulate macrophage recruitment and function; however, the contribution of the CX3CL1–CX3CR1 axis to ovarian cancer progression and TAM-mediated effector mechanisms remains unclarified. This study aimed to clarify the role of CX3CL1–CX3CR1 signaling in ovarian cancer progression, focusing on macrophage-derived pro-tumorigenic factors. Methods: CX3CL1 and CX3CR1 expression was examined in human EOC and healthy ovarian tissues by real-time polymerase chain reaction and immunohistochemistry. Functional effects of CX3CL1 on ovarian cancer cells were evaluated via migration and proliferation assays in the murine ID8 cell line. An intraperitoneal syngeneic ovarian cancer model was established by injecting ID8 cells into wild-type and Cx3cr1-deficient mice. Tumor burden, ascites formation, survival, macrophage infiltration, and expression levels of matrix metalloprotease-2 (MMP-2) and transforming growth factor-β (TGF-β) were assessed by histological, immunohistochemical, and molecular analyses. Results: CX3CL1 and CX3CR1 expression was significantly upregulated in human EOC tissues and associated with marked macrophage infiltration. CX3CL1 stimulation enhanced migration, but not proliferation, of ID8 cells. Cx3cr1 deficiency significantly suppressed intraperitoneal tumor growth, reduced ascitic fluid volume, and prolonged survival. This was accompanied by reduced CX3CR1⁺ TAM accumulation and decreased MMP-2 and TGF-β expression, which were predominantly produced by infiltrating macrophages. Conclusions: The CX3CL1–CX3CR1 axis promotes ovarian cancer progression by recruiting MMP-2- and TGF-β-producing macrophages. Targeting CX3CR1-dependent TAM functions may represent a therapeutic strategy for limiting peritoneal dissemination in ovarian cancer. Full article

Background/Objectives: This study aimed to explore the potential value of narrow-band-imaging (NBI)-derived mean optical intensity (MOI) in monitoring the progression of oral squamous cell carcinoma (OSCC), from the normal oral mucosa through epithelial dysplasia to invasive carcinoma. We compared differences in the NBI MOI among distinct pathological stages, so as to provide preliminary evidence for its clinical application in auxiliary diagnosis and progression assessment for OSCC. Methods: A total of 40 human oral mucosal specimens (15 normal, 15 oral leukoplakia, 10 OSCC) were enrolled for NBI image acquisition and MOI measurements. A 4-nitroquinoline-1-oxide (4NQO)-induced mouse OSCC model (n = 34) was used to dynamically record MOI changes across different pathological stages. A syngeneic tongue tumor mouse model (n = 16) was further established to evaluate whether MOI could reflect tumor formation and growth. All MOI values were quantified using ImageJ software with standardized region-of-interest (ROI) selection and background correction. Results: In clinical samples, MOI values decreased progressively from the normal mucosa (129.6 ± 5.991 arbitrary units (a.u.)) to oral leukoplakia (OLK) subgroups, including mild dysplasia (104.6 ± 3.757 a.u.) and moderate-to-severe dysplasia (91.77 ± 4.345 a.u.), and further to OSCC (54.41 ± 14.40 a.u.). In the 4NQO model, the MOI of the lingual mucosa was highest in the healthy control group (167.3 ± 10.05 a.u.) and gradually declined with increasing dysplasia severity, reaching the lowest level at the OSCC stage (48.67 ± 10.07 a.u.). In the syngeneic tumor model, the MOI was significantly lower in tumor-bearing mice than in healthy controls (47.85 ± 10.44 a.u. vs. 119.7 ± 14.20 a.u., p < 0.001). Receiver operating characteristic (ROC) analysis demonstrated good diagnostic performance of the MOI in distinguishing healthy tissue from cancerous lesions. Conclusions: NBI-derived MOI may quantitatively reflect the dynamic alterations of the oral mucosa during oral carcinogenesis and could represent a potential biomarker enabling the non-invasive, repeatable early evaluation and dynamic monitoring of OSCC. Full article

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis, largely due to therapeutic resistance and the limited availability of effective targeted therapies. Aloperine (ALO), a natural alkaloid derived from Sophora alopecuroides L., exhibits anti-cancer properties in various tumor types; however, its therapeutic potential and underlying mechanism in ESCC remain unclear. Here, we report that ALO inhibited ESCC cell proliferation and colony formation in a dose- and time-dependent manner and induced caspase-dependent apoptosis, accompanied by loss of mitochondrial membrane potential and PARP1 cleavage. Mechanistically, ALO significantly suppressed inflammatory pathways, with IL-6 identified as a critical downregulated target. ALO inhibited IL-6 production by targeting the AP-1 transcription factor complex, as evidenced by reduced cFOS expression and suppressed cJUN phosphorylation. Consequently, ALO inhibited downstream IL-6/JAK-STAT3 signaling. Functionally, exogenous IL-6 rescued ALO-induced loss of cell viability. Notably, the combination of ALO with cisplatin exerted synergistic antitumor effects. In a syngeneic mice model, the combination therapy significantly reduced tumor growth and Ki67 expression while inducing apoptosis, as shown by increased TUNEL staining and cleaved caspase-3 expression, and further suppressing the IL-6/STAT3 axis compared with either monotherapy. Together, these findings demonstrate that ALO exerts potent anti-ESCC activity by targeting the AP-1/IL-6/STAT3 signaling axis. The synergistic efficacy of ALO with cisplatin highlights its potential as a promising therapeutic agent to overcome chemoresistance and improve outcomes in ESCC patients. Full article

Background: Aging profoundly alters host immunity, yet how age-associated immune changes in the liver influence the growth of metastatic tumors remains incompletely understood. Liver metastasis is a major cause of cancer-related mortality, particularly in elderly patients, for whom aggressive treatments are often not feasible. This study aimed to clarify how aging reshapes the hepatic immune microenvironment and to identify age-associated host factors that influence liver metastasis growth. Methods: Tumor-naïve and tumor-bearing young and aged mice were analyzed using a syngeneic MC38 liver metastasis model. Immune cell composition in the liver was assessed by flow cytometry, and gene expression was evaluated by quantitative reverse transcription PCR (RT–qPCR). Public transcriptomic datasets were screened to identify age-associated inflammatory factors. The functional relevance of the S100A8/A9 axis was examined using the small-molecule inhibitor paquinimod. Results: Aging was associated with a distinct baseline immune cell composition in the liver. During liver metastasis, overall growth was comparable between young and aged mice; however, metastatic lesions in aged hosts showed increased expression of multiple inflammation-related genes and prominent accumulation of Ly6G⁺ cells. In silico screening identified S100a9 as one of the most highly upregulated inflammation-related genes in aged livers, which was confirmed in both tumor-naïve and metastatic liver tissues. Pharmacological modulation of the S100A8/A9 axis with paquinimod significantly reduced liver metastasis growth in aged, but not young, mice, and was accompanied by a shift in immune cell composition, including an increased representation of CD8⁺ T cells. Conclusions: These findings indicate that aging is associated with a distinct hepatic immune context that shapes the inflammatory and cellular composition of the tumor microenvironment during liver metastasis. S100A9 emerges as a key age-associated, host-derived factor that is functionally relevant to the growth of liver metastases in aged hosts, supporting the S100A8/A9 axis as a context-specific therapeutic target. Full article

Colorectal cancer (CRC) is a digestive tract malignant tumor with a relatively high incidence and mortality rate worldwide. The occurrence and development of CRC are closely associated with disturbances in the gut microbiota. Paraclostridium tenue (synonym Eubacterium tenue) is generally considered a harmless commensal and can be isolated from fecal samples of healthy adults. However, whether this bacterium is a beneficial organism with an antitumor effect is unknown. This study systematically evaluated the anti-CRC effects of P. tenue strain Pt517 on CRC cells in vitro and in the CT26 syngeneic mouse model. Pt517 culture supernatant (Pt517CS) inhibited the proliferation, colony formation, and migration ability of CRC cells; induced cell apoptosis; and altered cell cycle distribution. Daily intragastric administration of Pt517 significantly inhibited tumor growth in mice; increased the expression levels of TNF-α, INF-γ, and CD8 in tumor tissues; and decreased the levels of IL-6, IL-10, and TGF-β. Pt517 intervention significantly modulated the gut microbiota composition with increased relative abundance of Parabacteroides goldsteinii, Lachnospiraceae, and Enterorhabdus caecimuris B7. The long-chain fatty acids (LCFAs), stearic acid and palmitic acid, were increased in the serum of treatment group mice and detected in Pt517CS. Functional verification indicated that stearic acid and palmitic acid directly inhibited the proliferation of CT26 cells in a dose-dependent manner, suggesting that Pt517 might exert its anti-CRC effect by secreting LCFAs. These findings indicate that P. tenue Pt517 is a potential new candidate for the microbial treatment of CRC, which warrants further validation for its safety and efficacy before clinical translation. Full article

Telmisartan, an angiotensin II type 1 receptor blocker with established anti-inflammatory and antihypertensive properties, has been reported to inhibit tumor cell proliferation, yet its impact on the tumor immune microenvironment remains poorly understood. In this study, we evaluated the immunomodulatory effects of telmisartan using a syngeneic MC38 colorectal cancer model in C57BL/6 mice. Daily intragastric administration of telmisartan significantly suppressed tumor growth and reduced endpoint tumor weight compared with controls. To elucidate the underlying mechanisms, we performed single-cell RNA sequencing on tumor-infiltrating CD45⁺ immune cells and revealed a macrophage-dominated immune landscape comprising multiple transcriptionally distinct subclusters. Telmisartan broadly downregulated pro-tumoral and M2-associated macrophage programs, including decreased expression of genes such as Mrc1 and Spp1, while also suppressing cell proliferation-related pathways. In contrast to its overall suppressive impact on macrophages, telmisartan increased the proportion of cytotoxic CD8⁺ T cells, reduced regulatory T cell counts, and enhanced major histocompatibility complex class I antigen presentation, consistent with an immune-activating effect. These results indicate that telmisartan reshapes the colorectal tumor immune microenvironment by simultaneously attenuating tumor-promoting macrophage activity and augmenting cytotoxic T cell responses. Overall, this study provides a single-cell framework to understand how angiotensin receptor blockade reshapes tumor-infiltrating immune programs, highlighting the translational potential of repurposing telmisartan for novel cancer immunotherapy strategies. Full article

Background/Objectives: Glioblastoma remains the most lethal primary brain tumor in adults, and progress in oncolytic virotherapy is limited by the lack of immunocompetent models permissive to human-tropic viruses. Methods: Here, murine CT-2A and GL261 glioma and B16 melanoma cell lines were engineered to express human Coxsackievirus and Adenovirus Receptor (CXADR) fused to tagBFP, generating “humanized” tumors that preserve parental growth characteristics while acquiring high susceptibility to group B Coxsackieviruses (CVBs) and adenovirus serotype 5. Results: CXADR expression in CT-2A, GL261, and B16 cells markedly enhanced binding, internalization, and replication of CVBs in vitro, with the strongest effect observed for LEV14 (attenuated CVB5), which reached up to 10⁵-fold higher viral titers in humanized cells compared with parental cells. Unchanged sensitivity to vesicular stomatitis virus indicated receptor-specific effects. Humanized CT-2A-CXADR-BFP and GL261-CXADR-BFP cells initiated aggressive subcutaneous and intracranial tumors in syngeneic C57BL/6 mice without signs of immune rejection, and histology and MRI confirmed invasive high-grade glioma phenotypes. In intracranial CT-2A-CXADR-BFP tumors, repeated intratumoral LEV14 administration induced extensive tumor necrosis and prolonged survival despite the rapid development of neutralizing antibodies. Systemic intravenous LEV14 dosing produced strong oncolytic activity against subcutaneous CT-2A-CXADR-BFP tumors, as demonstrated by pronounced tumor growth inhibition, long-lasting regression in a subset of animals with gliomas, and improved overall survival. Conclusions: Collectively, these data establish CXADR-humanized models as versatile, immunocompetent platforms for evaluation of CXADR-dependent oncolytic enteroviruses. Full article