Cancers represent one of the most significant public health challenges worldwide. Immunotherapy has demonstrated promising potential in cancer treatment; however, it still faces challenges, including limited response rates, drug resistance, and immune-related adverse events (irAEs) (1, 2). The immunosuppressive tumor microenvironment (TME) constitutes a major barrier to the therapeutic efficacy of immunotherapy. Therefore, developing novel therapeutic modalities that can specifically target tumors while simultaneously reversing the immunosuppressive microenvironment has become an urgent priority and a frontier direction in cancer research.

Oncolytic virus (OV) therapy, a highly promising platform for tumor treatment, has attracted significant research interest. OVs have dual mechanisms of action: first, they selectively replicate within tumor cells and directly lyse them, producing the “oncolytic” effect (3); second, and more importantly, the viral replication and tumor cell lysis stimulate robust systemic anti-tumor immune responses in the host (4–8).

Newcastle disease virus (NDV) is an oncolytic virus known for its high safety profile. It primarily infects avian species and exhibits extremely low pathogenicity in humans, providing a strong basis for its clinical safety. In most tumor cells, the antiviral interferon signaling pathway is suppressed, allowing NDV to evade immune clearance and replicate extensively within these cells (9–12). In addition to directly lysing tumor cells, NDV activates the host’s innate and adaptive immune responses, demonstrating significant antitumor activity (13).

Several wild-type NDV strains, such as PV701 and 73-T, have entered clinical trials, demonstrating favorable safety profiles and antitumor efficacy. Although wild-type NDV inherently targets tumors and exhibits immunogenicity, its oncolytic effectiveness remains limited. This limitation primarily results from factors such as neutralization by pre-existing antibodies, insufficient replication efficiency, and impaired immune responses. The development of reverse genetics systems allows researchers to edit the NDV genome precisely, generating rNDV strains with significantly enhanced oncolytic efficacy. Current strategies primarily focus on modifying viral F or HN proteins to improve targeting, optimizing F protein cleavage sites to increase replication capacity within tumor cells, and utilizing NDV as a vector to express various exogenous genes. These recombinant viruses not only retain NDV’s inherent advantages but also exert localized effects within the TME through the expression of functional proteins, synergistically amplifying antitumor immune responses to ultimately achieve a “1 + 1>2” therapeutic effect.

Therefore, this article aims to provide a systematic review of research progress on rNDV in tumor therapy. By examining its modification strategies, mechanisms of action, preclinical and clinical research outcomes, and analyzing current challenges and future directions, it seeks to offer guidance for further research and clinical translation in this field.

Genetic engineering has generated a new generation of highly effective and safe recombinant NDV by removing non-essential viral genes or inserting exogenous genes. This genetic modification primarily relies on reverse genetics techniques, which involve manipulating the viral genome at the DNA level, followed by rescuing viruses with specific characteristics. Genetic modification strategies for NDV include enhancing its targeting ability by modifying viral proteins, increasing its oncolytic capacity through the insertion of apoptosis-related genes or by antagonizing antiviral pathways in tumor cells, and promoting NDV’s antitumor immune response by incorporating immunomodulatory genes (Figure 1, Tables 1, 2).

Recombinant NDV exhibits vastly enhanced antitumor potency. Its action transcends mere oncolysis, evolving into a multidimensional, synergistic process. The basis of rNDV’s attack on tumors is its direct oncolytic activity. This process begins with the specific binding of viral envelope proteins—such as the engineered, enhanced-targeting HN protein—to receptors on tumor cells. Subsequently, the virus enters the cell via membrane fusion or endocytosis, releasing viral ribonucleoprotein complexes (vRNPs) into the cytoplasm. The virus exploits the host cell’s transcriptional and translational machinery to synthesize substantial amounts of viral RNA and proteins, assembling them into new viral particles. Ultimately, the exocytosis and release of numerous viruses rupture the tumor cell membrane, leading to complete lysis and cell death. This process not only directly reduces tumor burden but, more importantly, induces immunogenic cell death, thereby laying the groundwork for subsequent activation of the immune system.

rNDV is capable of infecting normal cells within the tumor microenvironment, which have preserved type I IFN response. After rNDV infection, PAMPs inherent to the virus and DAMPs released by dying cancer cells are recognized by PRRs within immune cells. This activates a cascade of downstream signaling pathways involving nuclear factor kappa-B (NF-κB) and interferon regulatory factors (IRFs), leading to the rapid secretion of type I interferons and pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β (13). These cytokines recruit innate immune cells—including neutrophils, natural killer (NK) cells, and macrophages—to rapidly infiltrate the tumor. Concurrently, NK cells activated by cytokines such as IFNs directly kill tumor cells and secrete IFN-γ, further amplifying the immune response (66). High concentrations of inflammatory factors also suppress pre-existing immunosuppressive molecules within the TME, such as transforming growth factor-beta (TGF-β), and interfere with the immunosuppressive functions of Tregs and myeloid-derived suppressor cells (MDSCs). This process reprograms the TME, converting “cold” tumors into “hot” tumors and creating favorable conditions for initiating adaptive immune responses.

Inducing specific anti-tumor immunity is the primary mechanism through which rNDV exerts its long-lasting antitumor effects and prevents distant metastasis and recurrence. Upon lysis, tumor cells infected with rNDV release substantial quantities of DAMPs, such as calreticulin (CRT), ATP, and high-mobility group box 1 (HMGB1) (12). The uptake of these antigens triggers activation and maturation in cross-presenting DCs. Subsequently, activated DCs present processed antigen peptides via MHC-I and MHC-II molecules to naive CD8⁺ and CD4⁺ T cells, thereby activating and expanding large numbers of antigen-specific cytotoxic T lymphocytes (CTLs) and helper T (Th) cells. These activated T cells subsequently circulate through the bloodstream, infiltrate tumor tissues, and specifically recognize and eliminate tumor cells expressing the corresponding antigens via the perforin-granzyme pathway or the Fas/FasL pathway. The exogenous genes carried by rNDVs act as boosters in this process. GM-CSF enhances the recruitment and maturation of DCs and strengthens their antigen-presenting capabilities (28). IL-12 promotes the activation and proliferation of CTLs and NK cells (30–33). Meanwhile, immune checkpoint inhibitor single-chain variable fragments (scFv), such as anti-PD-L1, can relieve the suppression of infiltrating T cells by tumor cells (45), reversing T cell exhaustion and rendering the immune response more potent and enduring.

NDV selectively infects and lyses tumor vascular endothelial cells, playing a crucial role in tumor therapy (67). At the same time, it inhibits tumor angiogenesis by stimulating the production of TNF-α and IFN-γ (68). IFN-γ not only inhibits angiogenesis by downregulating the DLL4/Notch signaling pathway but also promotes vascular normalization through its interactions with endothelial cells, thereby enhancing antitumor immune responses (69, 70). Furthermore, TNF-α enhances immunotherapy efficacy by altering vascular endothelial barrier function and increasing T lymphocyte infiltration (71). These findings provide novel insights into NDV’s applications in tumor therapy and offer theoretical support for the development of inflammation-based antitumor strategies (72). A study demonstrates that VEGF-Trap-expressing rNDV reduces the growth rate of vascular endothelial cells by 85.37% (73). This rNDV exhibits an enhanced inhibition of colon cancer through amplified anti-angiogenesis.

The anti-tumor mechanism of rNDV involves an interconnected, stepwise, and synergistic process. NDV not only directly lyses cells but also induces innate and adaptive immune responses in the TME (Figure 2). This process is further enhanced by the expression of immunoregulatory genes, ultimately resulting in potent, long-lasting anti-tumor immunity.

Recombinant NDV has demonstrated therapeutic potential in multiple cancer types, including melanoma, primary hepatocellular carcinoma, colorectal cancer, breast cancer, and glioma. Recombinant NDV LX/IL (15 + 7), which co-expresses IL-7 and IL-15, enhanced the infiltration of CD8+ and CD4+ T cells, significantly inhibiting tumor growth in melanoma (59). rNDV-MIP3α induced cellular immune responses in melanoma, as demonstrated by a sevenfold increase in IFN-γ–secreting CD8+ T cells compared to the control group (36). Recombinant NDV with a modified F cleavage site prolonged survival in primary hepatocellular carcinoma, extending it to 35 days compared to 18 days in the control group (49). rNDV-IL12 suppressed the growth of colon cancer, resulting in a smaller tumor volume (537.90 ± 12.99 mm³) compared to that of the AF2240-i-treated group (1113.00 ± 32.16 mm³) (57). rNDV-IL12 modulated immune responses by increasing the levels of CD4+ T cells, CD8+ T cells, IL-2, IL-12, and IFN-γ (57). Recombinant NDV expressing IL-12 (rAF-IL12) resulted in 52% inhibition of breast cancer growth, whereas AF2240 caused 34.5% inhibition (74). Recombinant NDV expressing the human p53 gene has been shown to induce apoptosis in glioma cells, enhance cytotoxic T cell infiltration, and prolong survival in glioma-bearing mice (26). These studies provide evidence for the further application of rNDV in solid tumor therapy.

Clinical trials of NDV have demonstrated its efficacy and safety (Table 3). MEDI5395, a recombinant NDV expressing the human GM-CSF gene, demonstrated antitumor activity in Phase I clinical trials. When combined with durvalumab, it exhibited favorable safety and efficacy profiles in patients with advanced-stage tumors (75). NDV-GT, a recombinant NDV expressing porcine α1,3GT gene, induced surface expression of the αGal antigen on tumor cells. Pre-existing anti-αGal antibodies in the human body rapidly recognized this antigen, triggering a hyperacute immune rejection response that causes tumor vascular embolism and extensive tumor cell death. Results from a Phase I clinical trial reported in 2025 indicated that 90% of advanced cancer patients treated with NDV-GT achieved disease control without experiencing severe adverse effects (48). The clinical safety of NDV has been extensively demonstrated by data from multiple trials. No serious adverse events were reported in the clinical trials of NDV therapies. While current preclinical studies and preliminary clinical outcomes are encouraging, most rNDV therapies remain in preclinical or early clinical stages. Their safety, efficacy, and optimal application strategies require validation through further large-scale clinical trials.

Due to tumor heterogeneity, mutation burden, and variations in the TME, monotherapy-based tumor immunotherapy often leads to resistance, limiting the ability of OVs to achieve optimal antitumor efficacy. Combining OV therapy with other treatment modalities—such as chemotherapy, ICIs, adoptive cell therapy (ACT), and radiotherapy—may be essential for improving therapeutic outcomes.

The strategy of combining oncolytic viruses with chemotherapy remains a topic of controversy. Some clinical studies suggest that the combination of oncolytic viruses and chemotherapy can achieve satisfactory antitumor effects (86–90), but a large-scale clinical trial has produced conflicting results (91). Eigl et al. reported that metastatic prostate cancer patients treated with the oncolytic virus Pelareorep plus docetaxel had a median survival of 19.1 months, compared to 21.1 months for patients receiving docetaxel monotherapy (91). Another clinical trial found that Pelareorep combined with FOLFOX/Bevacizumab resulted in worse progression-free survival (PFS) compared to monotherapy (92). These conflicting results do not demonstrate the superiority of combining oncolytic viruses with chemotherapy over chemotherapy alone, suggesting that the combination may exhibit a “1 + 1 < 1” effect.

The combination of NDV with immunotherapy currently represents the most promising strategy. Possible reasons for the limited effectiveness of ICIs include a relatively low mutation burden, minimal T-cell infiltration, and an immunosuppressive TME. Studies have demonstrated that following NDV treatment, immune checkpoints such as CTLA-4 and PD-1 are significantly upregulated on infiltrating T cells in both the injected tumors and distant tumors (43, 44). Furthermore, the expression of programmed cell death ligand 1 (PD-L1) is increased in tumor cells, myeloid cells, and stromal cells following NDV infection (44). These findings suggest that combining NDV with ICIs may enhance antitumor efficacy (Figure 3). Co-administration of NDV with PD-1 or PD-L1 antibodies significantly improves the therapeutic response in melanoma, resulting in 90–100% tumor-free rates compared to 70% in the NDV monotherapy group (44). Another study demonstrates the superior efficacy of NDV combined with CTLA-4 antibodies compared to monotherapy in treating melanoma, colorectal cancer, and prostate cancer (43). Recombinant NDV expressing inducible co-stimulator ligand (NDV-ICOSL) increases infiltration of GrB+ICOS+CD8+ T cells. When combined with CTLA-4 antibodies, it demonstrated a 74% tumor-free rate in distant tumors, which was superior to the combination of wild-type NDV with anti-CTLA-4 (46). Although the combination strategy of rNDV and ICIs has shown promising results in preclinical studies, further prospective clinical trials are required to validate the efficacy.

ACT has demonstrated significant efficacy in treating leukemia, melanoma, and lymphoma. However, its application to epithelial tumors is limited due to challenges related to cell infiltration and tumor heterogeneity (93). Combining OVs with ACT could potentially modify the TME of solid tumors, thereby overcoming current limitations. Chen et al. compared the efficacy of OV monotherapy with combination therapies involving chimeric antigen receptor T (CAR-T) cells and innate-like natural killer T (iNKT) cells. The results demonstrated the superiority of combination therapy over monotherapy (94). An oncolytic virus expressing truncated CD19 (CD19t) enhanced CAR-T cell targeting of solid tumors and further stimulated local antitumor immunity (95). On the other hand, CAR-T cells can serve as vectors for oncolytic viruses, delivering these viruses to tumor cells that express specific antigens. Delivering the myxoma virus to tumor cells expressing homologous antigens via CAR-T cells induces targeted tumor cell death and autophagy, resulting in tumor regression (96). These findings suggest that oncolytic viruses could be powerful tools to overcome limitations in adoptive cell therapy.

Other combination strategies include pairing with radiotherapy and targeted therapy. Results from a Phase I clinical trial of OPB301 combined with radiotherapy in esophageal cancer patients demonstrated an objective response rate (ORR) of 91.7% in 13 patients, with a favorable safety profile (97). The Phase III clinical trial of JX-594 combined with sorafenib for advanced hepatocellular carcinoma (NCT02562755) reported an ORR of only 19.2%, which was lower than the 20.9% observed in the sorafenib monotherapy group (98). Despite promising preclinical data suggesting that JX-594 could sensitize tumors to subsequent therapy with vascular endothelial growth factor (VEGF) inhibitors or vascular endothelial growth factor receptor (VEGFR) inhibitors, the trial results indicated that the combination did not demonstrate greater clinical benefit compared to sorafenib monotherapy (98, 99). Therefore, additional prospective clinical trials are necessary to validate the efficacy of NDV in combination with radiotherapy or targeted therapies.

Compared to other oncolytic viruses, NDV has unique features: (1) NDV is a single-stranded negative-sense RNA virus that replicates in the cytoplasm, independently of the host cell's DNA replication machinery. It cannot integrate with the host genome. (2) While NDV replicates efficiently within tumors, it does not replicate in the normal cells of mammalian hosts. (3) NDV is cost-effective to produce, can be administered through various methods, and has minimal side effects.

NDV has demonstrated promising antitumor properties and a high safety profile in both preclinical and clinical studies. Despite its promising potential, studies of NDV therapy face significant challenges related to heterogeneity and methodological inconsistency, which complicate interpretation and hinder progress in the field. The primary issue is the profound heterogeneity of viral platforms and study designs across different investigations. Researchers have employed diverse reverse genetics systems, parental viral strains, and transgene payloads, which offer technical advantages during exploratory phases. However, this diversity has become a critical drawback—variations in viral backbones, production methods, and genetic modifications, making meaningful comparisons across trials nearly impossible. Moreover, early clinical trials frequently suffer from small sample sizes, single-arm designs, and a lack of standardized efficacy endpoints. Consequently, observed clinical outcomes are susceptible to confounding factors, preventing clear attribution to the oncolytic NDV itself. A third critical issue is the absence of reliable biomarker data to validate the NDV’s mechanism of action in patients. In most trials, it remains unclear whether limited efficacy results from the virus failing to infect and replicate within tumors, premature clearance by the host immune system, or an inability to overcome the immunosuppressive TME. The inconsistencies and limitations discussed above highlight the most urgent challenges currently facing the NDV field. Overcoming these hurdles is essential to translating preclinical promise into clinical reality. Therefore, future research must strategically focus on addressing the following key issues:

Although NDV is of low pathogenicity in humans, it is highly contagious among poultry, which is a persistent threat to the poultry industry. The manufacture and use of NDV may result in its release into the environment, thereby necessitating regulatory oversight. Evaluating an oncolytic NDV for environmental safety is also important. However, there are few studies on the environmental shedding of NDV. Further research should be conducted and thoroughly documented.

Current delivery approaches include local delivery and systemic delivery. Most oncolytic virotherapies are administered through intratumoral injection. However, this method has specific drawbacks, including (1) Intratumoral injection may cause bleeding and unwanted metastasis at the lesion site. (2) Difficulties in accessing deep tumor tissues significantly limit the number of applicable cases. Thus, intravenous administration is essential for treating deep-seated or metastatic tumors. However, following intravenous injection, viral particles encounter multiple barriers. First, complement proteins and neutralizing antibodies in the bloodstream can neutralize and eliminate NDV before it reaches the tumor site. Second, the elevated pressure within the tumor stroma and the dense extracellular matrix hinders effective viral penetration and diffusion. Consequently, only a small fraction of the virus successfully reaches and infiltrates the tumor, thereby limiting its therapeutic efficacy. Altering the capsid, applying polymer coatings (100), or enhancing the extracellular envelope of NDVs can improve their shielding (101). Another effective strategy for intravenous injection involves encapsulating NDVs within cellular vectors or loading them onto these cells. Mesenchymal stem cells (MSCs) are promising candidates for delivering oncolytic viruses because they can transport viral particles, serve as factories for viral replication, and modulate the immune system (102–106).

NDV itself, as a foreign antigen, rapidly induces a strong antiviral immune response. Complement and neutralizing antibodies in the bloodstream can neutralize and eliminate NDV before it reaches the tumor site, severely compromising therapeutic efficacy. This may render subsequent doses ineffective, hindering the implementation of repeated, multi-course intensive therapy. Overcoming or circumventing host innate immunity represents the primary challenge in enhancing NDV efficacy. Employing cellular vector systems for NDV delivery represents a highly promising strategy. Cells such as mesenchymal stem cells (MSCs), cytokine-induced killer cells (CIK), or CD8+ T cells, which exhibit natural chemotaxis towards tumors, can serve as viral carriers. These cells carry and protect the virus, effectively evading neutralizing antibodies and complement attacks until reaching the tumor, where they release progeny viruses, achieving tumor-targeted delivery and efficient infection.

Regarding safety, the most frequently reported side effects in OV clinical trials include fever, fatigue, nausea, flu-like symptoms, and pain at the injection site. There is an overall incidence of adverse events of 26.6% associated with OV therapy (107). Compared to other immunotherapy products, OV has a more favorable safety profile. However, due to the heterogeneity both between and within tumors, the absence of specific receptors or promoters in tumor cells may lead to off-target effects, resulting in immune-related adverse events (108). The risks increase significantly when NDV is used in combination with immune checkpoint inhibitors or is modified to express immune modulators (109, 110). Cytokine release syndrome (CRS), a serious adverse effect, can also be triggered by OV therapy (111–113). Essential strategies to minimize side effects include enhancing tumor targeting through genetic engineering, dosing guided by pharmacokinetic principles, intratumoral administration to reduce systemic toxicity, and a low-dose fractionated schedule optimized for efficacy (114–116).

Cancer cells exhibit high heterogeneity in receptor expression, integrity of interferon pathways, and proliferative states. One the one hand, NDV infection requires attachment to cell surface receptors, primarily sialic acid-containing glycoproteins. There is considerable variability in the expression levels of these receptors both between different types of cancer and among patients with the same type of cancer. The absence or low expression of receptors in tumors renders them resistant to infection, posing a significant barrier to oncolysis. On the other hand, most cancer cells have defects in their IFN signaling pathways, which allows NDVs to replicate efficiently within them. However, the impairment is typically not absolute. Cancer cells with an intact IFN pathway can mount an effective antiviral response, clearing the virus and resulting in treatment resistance. In addition, the highly immunosuppressed TME lacks T-cell infiltration. Even when NDV lyses tumor cells and releases antigens, it fails to effectively initiate specific anti-tumor immune responses, thus limiting therapeutic efficacy. Hence, developing strategies that combine therapies for synergistic effects is a major research objective. Chemotherapy and radiotherapy can induce ICD, further promoting tumor antigen release and complementing NDV’s immune-activating effects. Combination with immune checkpoint inhibitors can relieve T-cell suppression, facilitating the successful removal of PD-L1-positive tumor cell subpopulations. This synergy addresses tumor heterogeneity.

Biomarkers play a crucial role in advancing individualized precision oncology by enabling clinicians to provide care that is more effective, less toxic, and more cost-efficient. However, research on biomarkers for oncolytic viruses remains limited. It is necessary to search for biomarkers that can predict the efficacy of NDV, such as pre-existing antibody titers, tumor viral receptor expression levels, IFN signaling pathway status, immune cell infiltration, post-treatment viral replication in peripheral blood, and cytokine dynamics. This will enable the identification of populations likely to benefit, facilitate real-time efficacy monitoring, and allow timely adjustment of treatment strategies.

In summary, this review has outlined the modification strategies, antitumor mechanisms, clinical translation, combination strategies, and challenges of rNDV immunotherapy. Table 4 provides a quick reference summary of all that this review entails.

In conclusion, advancements in genetic engineering technology have enabled the enhancement of NDV’s targeting potential through the modification of viral proteins, as well as the improvement of its immune-stimulating capabilities by inserting exogenous genes. However, numerous unresolved challenges remain in NDV-based cancer therapy, including determining the most effective delivery route, developing strategies to evade neutralizing antibodies, overcoming tumor heterogeneity, and identifying relevant biomarkers. Future research priorities will focus on four key areas: the rational design of a new generation of rNDVs armed with potent immunomodulatory transgenes; the use of cellular vehicles for targeted delivery; the development of bispecific NDVs capable of redirecting immune effector cells; and the creation of synergistic therapeutic regimens that combine NDV with immune checkpoint inhibitors.