Live-attenuated Japanese encephalitis virus inhibits glioblastoma growth and elicits potent antitumor immunity.

Glioblastomas (GBMs) are highly aggressive brain tumors that have developed resistance to currently available conventional therapies, including surgery, radiation, and systemic chemotherapy. In this study, we investigated the safety of a live attenuated Japanese encephalitis vaccine strain (JEV-LAV) virus as an oncolytic virus for intracerebral injection in mice. We infected different GBM cell lines with JEV-LAV to investigate whether it had growth inhibitory effects on GBM cell lines in vitro. We used two models for evaluating the effect of JEV-LAV on GBM growth in mice. We investigated the antitumor immune mechanism of JEV-LAV through flow cytometry and immunohistochemistry. We explored the possibility of combining JEV-LAV with PD-L1 blocking therapy. This work suggested that JEV-LAV had oncolytic activity against GBM tumor cells in vitro and inhibited their growth in vivo. Mechanistically, JEV-LAV increased CD8+ T cell infiltration into tumor tissues and remodeled the immunosuppressive GBM microenvironment that is non-conducive to immunotherapy. Consequently, the results of combining JEV-LAV with immune checkpoint inhibitors indicated that JEV-LAV therapy improved the response of aPD-L1 blockade therapy against GBM. The safety of intracerebrally injected JEV-LAV in animals further supported the clinical use of JEV-LAV for GBM treatment.

In Situ Tumor Vaccine Expressing Anti-CD47 Antibody Enhances Antitumor Immunity.

In situ tumor vaccine is a potential cancer therapy due to their advantages in induction of antitumor immune responses. Oncolytic virotherapy utilizes natural or engineered oncolytic viruses to kill tumors selectively, representing a promising in situ tumor vaccine for cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in "don't eat me" signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As potential tumor vaccine vectors, oncolytic viruses can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Therefore, we developed an adenovirus-based tumor vaccine loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer models in immunocompetent mice were established to evaluated in vivo antitumor efficacy of in situ tumor vaccination. The tumor vaccine armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice, and also elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in immunocompetent mice following in situ vaccination with the anti-CD47 tumor vaccines; tumorigenesis was completely inhibited in these mice after tumor re-challenge. The recombinant anti-CD47 tumor vaccine has an effectual antitumor activity and may be a promising antitumor agent.

Long noncoding RNA RP11-241J12.3 targeting pyruvate carboxylase promotes hepatocellular carcinoma aggressiveness by disrupting pyruvate metabolism and the DNA mismatch repair system.

Accumulating evidence indicates that hepatitis B virus X protein (HBx) plays a key role in HBV-related hepatocellular carcinoma (HCC) aggressiveness; however, the underlying mechanisms are not entirely clear. Long non-coding RNAs (lncRNAs), which participate in the regulation of diverse biological processes, may be critical for the function of HBx. Our research indicated that HBx induced changes in the expression of numerous lncRNAs and implicated the novel lncRNA RP11-241J12.3 in HBx-mediated HCC aggressiveness. Although RP11-241J12.3 expression was downregulated in transient HBx-expressing HCC cells (similar to the early stage of HBV infection), its oncogenic properties remained. The results showed that RP11-241J12.3 not only accelerated DNA synthesis and upregulated the expression of pyruvate carboxylase (PC) and MSH3, which is a key protein in pyruvate metabolism and DNA mismatch repair (MMR), but also promoted tumor growth in vitro and in vivo, thus promoting HCC aggressiveness. More importantly, we revealed that RP11-241J12.3 may interact with PC and identified its location in the cytoplasm close to the nucleus using fluorescence in situ hybridization (FISH). We also observed RP11-241J12.3 expression was upregulated in HCC tissues compared with the paracarcinomatous tissues. Furthermore, RP11-241J12.3 expression levels showed a close relationship with clinical stage and tumor size and that low RP11-241J12.3 expression was significantly correlated with longer HCC patient survival. These results further our understanding of the lncRNAs regulated by HBx in HCC, and provide evidence that dysregulation of RP11-241J12.3 contributes to HCC aggressiveness.

High-dose VitC plus oncolytic adenoviruses enhance immunogenic tumor cell death and reprogram tumor immune microenvironment.

Preclinical and clinical studies have validated the antitumor effects of several oncolytic viruses (OVs). However, the efficacy of OVs is limited when they are administered as monotherapies. Combination therapy is a promising direction for oncolytic virotherapy in the future. A high dose of vitamin C (VitC) exerts anticancer effects by triggering the accretion of substantial amounts of reactive oxygen species (ROS). OVs can induce immunogenic tumor cell death and elicit an antitumor immune response. ROS play an important role in immunogenic cell death (ICD). This study aimed to explore whether high-dose VitC in combination with oncolytic adenoviruses (oAds) exhibited a synergistic antitumor effect. High-dose VitC synergized with oAds against tumor by enhancing immunogenic tumor cell death. Combination therapy with high-dose VitC and oAds significantly increased the number of T cells in the tumor microenvironment (TME) and promoted the activation of T cells. Furthermore, the antitumor effect of the combination therapy was CD8+ T cell dependent. In addition, combination therapy with high-dose VitC and oAds reprogramed the immunosuppressive TME. Our study provides a new strategy for combination therapy of OVs.

Antifibrotic Therapy Augments the Antitumor Effects of Vesicular Stomatitis Virus Via Reprogramming Tumor Microenvironment.

Solid tumors are characterized by abundant extracellular matrix originating from cancer-associated fibroblasts (CAFs). High collagen content can trigger the collapse of vascular system in the tumor and form physical barrier that eventually impedes the penetration of drug particles and cytotoxic immune cells. Moreover, CAFs is able to promote the enrichment of tumor-associated macrophages (TAMs) and differentiation of myeloid-derived suppressor cells (MDSCs) that work in concert to develop a highly immunosuppressive tumor microenvironment (TME). In this study, we investigated if halofuginone, an antifibrotic drug, can augment the therapeutic effects of oncolytic vesicular stomatitis virus (VSV). The results revealed that halofuginone significantly disrupts the collagen network in tumors and promotes the distribution of VSV and infiltration of CD8+ T cells (p < 0.0001). Combined treatment of VSV and halofuginone also modulates the immunosuppressive TME via deletion of TAM, MDSCs, and regulatory T cells (Tregs). Collectively, the combination therapy remarkably inhibits the tumor growth in multiple murine models and prolongs survival of mice. The results demonstrate the clinical potential of halofuginone in combination with oncolytic virus.

Engineered exosome-like nanovesicles suppress tumor growth by reprogramming tumor microenvironment and promoting tumor ferroptosis.

Tumor vaccines that induce effective and sustained antitumor immunity are highly promising for cancer therapy. However, the antitumor potential of these vaccines is weakened due to the immunosuppressive characteristics of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells within the TME; they play an important role in tumor growth, metastasis, immunosuppression, and drug resistance. Fibroblast activation protein-α (FAP) is overexpressed in CAFs in more than 90% of human tumor tissues. Further, FAP+CAFs are an ideal interstitial target for the immunotherapy of solid tumors. Exosomes derived from tumor cells contain many tumor antigens, which can be used as the basis of tumor vaccines that elicit strong antitumor immunity. Almost all exosome-based cancer vaccines have been designed and developed for tumor parenchymal cells. Moreover, the exosome production is very low and the purification is very difficult, limiting their clinical application as tumor vaccines. In this study, we developed FAP gene-engineered tumor cell-derived exosome-like nanovesicles (eNVs-FAP) as a tumor vaccine that can be prepared easily and in large quantities. The eNVs-FAP vaccine inhibited tumor growth by inducing strong and specific cytotoxic T lymphocyte (CTL) immune responses against tumor cells and FAP+CAFs and reprogramming the immunosuppressive TME in the colon, melanoma, lung, and breast cancer models. Moreover, eNVs-FAP vaccine-activated cellular immune responses could promote tumor ferroptosis by releasing interferon-gamma (IFN-γ) from CTLs and depleting FAP+CAFs. Thus, eNVs-FAP is a candidate tumor vaccine targeting both the tumor parenchyma and the stroma. STATEMENT OF SIGNIFICANCE: Nanovaccines can activate immune cells and promote an antitumor immune response. In this study, we developed the fibroblast activation protein-α (FAP) gene-engineered tumor cell-derived exosome-like vesicle vaccines (eNVs-FAP). A large number of eNVs-FAP were obtained by continuously squeezing FAP gene-engineered tumor cells. eNVs-FAP showed excellent antitumor effects in a variety of tumor-bearing mouse models. The mechanistic analysis showed that eNVs-FAP promoted the maturation of dendritic cells (DCs), increased the infiltration of effector T cells into target tumor cells and FAP-positive cancer-associated fibroblasts (FAP+CAFs), and reduced the proportion of immunosuppressive cells, including M2-like tumor-associated macrophages (M2-TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), in the tumor microenvironment (TME). Moreover, the clearance of FAP+CAFs helped enhance interferon-gamma-induced tumor cell ferroptosis.

The Effect of Residual Triton X-100 on Structural Stability and Infection Activity of Adenovirus Particles.

To ensure the high purity and biological activity of the adenovirus vector to be used for clinical applications, a stable and linearly scalable preparation method is highly imperative. During the adenovirus-harvesting process, the Triton X-100-based lysis method possesses the advantages of higher efficiency as well as easier linearization and amplification. Most Triton X-100 can be removed from the adenovirus sample by chromatographic purification. However, there is no report that a small amount of residual Triton X-100, present in adenovirus sample, can affect the particle integrity, infectivity, and structure of adenoviruses. Here, we found that although residual Triton X-100 affected the short-term stability, purity, infectivity, and structure of adenoviruses at 37°C, it did not hamper these properties of adenoviruses at 4°C. This study suggests that although the Triton X-100-based lysis method is a simple, efficient, and easy-to-scale process for lysing host cells to release the adenovirus, the storage conditions of adenovirus products must be taken into consideration.

A novel method to purify adenovirus based on increasing salt concentrations in buffer.

With the rapid development of gene therapy, gene-based medicine with adenovirus as vectors has become a new method for disease treatment. However, there are still enormous challenges in the large-scale production of adenoviruses for clinical use. Recent reports show that ion-exchange chromatography (IEC) is an effective tool for the isolation and purification of adenovirus. However, during the separation and purification, host cell protein and DNA, as well as serum from the culture medium, can non-specifically occupy numerous binding sites of the chromatography packings, thereby reducing the binding between the adenovirus and packing media. We here report a novel method for highly efficient purification of adenoviruses by increasing the salt concentrations of the samples to be ultrafiltrated by tangential flow filtration, the diafiltration buffer, and the samples for IEC purification. This method could significantly remove a large amount of serum proteins and host cell proteins, increase the amount of sample loaded on the IEC column, and improve the binding of the adenovirus samples to the packing media. A purity of > 95% could be obtained after one chromatography operation, and the number of purification steps and the amount of used packing media were reduced. The method is simple, economical, and efficient, and has excellent applications.