Modulation of Reoviral Cytolysis (I): Combination Therapeutics.

Patients with stage IV gastric cancer suffer from dismal outcomes, a challenge especially in many Asian populations and for which new therapeutic options are needed. To explore this issue, we used oncolytic reovirus in combination with currently used chemotherapeutic drugs (irinotecan, paclitaxel, and docetaxel) for the treatment of gastric and other gastrointestinal cancer cells in vitro and in a mouse model. Cell viability in vitro was quantified by WST-1 assays in human cancer cell lines treated with reovirus and/or chemotherapeutic agents. The expression of reovirus protein and caspase activity was determined by flow cytometry. For in vivo studies, athymic mice received intratumoral injections of reovirus in combination with irinotecan or paclitaxel, after which tumor size was monitored. In contrast to expectations, we found that reoviral oncolysis was only poorly correlated with Ras pathway activation. Even so, the combination of reovirus with chemotherapeutic agents showed synergistic cytopathic effects in vitro, plus enhanced reovirus replication and apoptosis. In vivo experiments showed that reovirus alone can reduce tumor size and that the combination of reovirus with chemotherapeutic agents enhances this effect. Thus, we find that oncolytic reovirus therapy is effective against gastric cancer. Moreover, the combination of reovirus and chemotherapeutic agents synergistically enhanced cytotoxicity in human gastric cancer cell lines in vitro and in vivo. Our data support the use of reovirus in combination with chemotherapy in further clinical trials, and highlight the need for better biomarkers for reoviral oncolytic responsiveness.

Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome.

Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21 de novo. The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses. IMPORTANCE As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.

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.

Oncolytic Virotherapy Treatment of Breast Cancer: Barriers and Recent Advances.

Oncolytic virotherapy (OV) is an emerging class of immunotherapeutic drugs. Their mechanism of action is two-fold: direct cell lysis and unmasking of the cancer through immunogenic cell death, which allows the immune system to recognize and eradicate tumours. Breast cancer is the most common cancer in women and is challenging to treat with immunotherapy modalities because it is classically an immunogenically "cold" tumour type. This provides an attractive niche for OV, given viruses have been shown to turn "cold" tumours "hot," thereby opening a plethora of treatment opportunities. There has been a number of pre-clinical attempts to explore the use of OV in breast cancer; however, these have not led to any meaningful clinical trials. This review considers both the potential and the barriers to OV in breast cancer, namely, the limitations of monotherapy and the scope for combination therapy, improving viral delivery and challenges specific to the breast cancer population (e.g., tumour subtype, menopausal status, age).

Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges.

Oncolytic viruses have emerged as a promising strategy for cancer therapy due to their dual ability to selectively infect and lyse tumor cells and to induce systemic anti-tumor immunity. Among various candidate viruses, coxsackievirus group B (CVBs) have attracted increasing attention in recent years. CVBs are a group of small, non-enveloped, single-stranded, positive-sense RNA viruses, belonging to species human Enterovirus B in the genus Enterovirus of the family Picornaviridae. Preclinical studies have demonstrated potent anti-tumor activities for CVBs, particularly type 3, against multiple cancer types, including lung, breast, and colorectal cancer. Various approaches have been proposed or applied to enhance the safety and specificity of CVBs towards tumor cells and to further increase their anti-tumor efficacy. This review summarizes current knowledge and strategies for developing CVBs as oncolytic viruses for cancer virotherapy. The challenges arising from these studies and future prospects are also discussed in this review.

Role of Myeloid Cells in Oncolytic Reovirus-Based Cancer Therapy.

Oncolytic reovirus preferentially targets and kills cancer cells via the process of oncolysis, and additionally drives clinically favorable antitumor T cell responses that form protective immunological memory against cancer relapse. This two-prong attack by reovirus on cancers constitutes the foundation of its use as an anticancer oncolytic agent. Unfortunately, the efficacy of these reovirus-driven antitumor effects is influenced by the highly suppressive tumor microenvironment (TME). In particular, the myeloid cell populations (e.g., myeloid-derived suppressive cells and tumor-associated macrophages) of highly immunosuppressive capacities within the TME not only affect oncolysis but also actively impair the functioning of reovirus-driven antitumor T cell immunity. Thus, myeloid cells within the TME play a critical role during the virotherapy, which, if properly understood, can identify novel therapeutic combination strategies potentiating the therapeutic efficacy of reovirus-based cancer therapy.

The role of telomerase and viruses interaction in cancer development, and telomerase-dependent therapeutic approaches.

Human telomerase reverse transcriptase (hTERT) is an enzyme that is critically involved in elongating and maintaining telomeres length to control cell life span and replicative potential. Telomerase activity is continuously expressed in human germ-line cells and most cancer cells, whereas it is suppressed in most somatic cells. In normal cells, by reducing telomerase activity and progressively shortening the telomeres, the cells progress to the senescence or apoptosis process. However, in cancer cells, telomere lengths remain constant due to telomerase's reactivation, and cells continue to proliferate and inhibit apoptosis, and ultimately lead to cancer development and human death due to metastasis. Studies demonstrated that several DNA and RNA oncoviruses could interact with telomerase by integrating their genome sequence within the host cell telomeres specifically. Through the activation of the hTERT promoter and lengthening the telomere, these cells contributes to cancer development. Since oncoviruses can activate telomerase and increase hTERT expression, there are several therapeutic strategies based on targeting the telomerase of cancer cells like telomerase-targeted peptide vaccines, hTERT-targeting dendritic cells (DCs), hTERT-targeting gene therapy, and hTERT-targeting CRISPR/Cas9 system that can overcome tumor-mediated toleration mechanisms and specifically apoptosis in cancer cells. This study reviews available data on the molecular structure of telomerase and the role of oncoviruses and telomerase interaction in cancer development and telomerase-dependent therapeutic approaches to conquest the cancer cells.

Oncolytic Viruses as a Platform for the Treatment of Malignant Brain Tumors.

Malignant brain tumors remain incurable diseases. Although much effort has been devoted to improving patient outcome, multiple factors such as the high tumor heterogeneity, the strong tumor-induced immunosuppressive microenvironment, and the low mutational burden make the treatment of these tumors especially challenging. Thus, novel therapeutic strategies are urgent. Oncolytic viruses (OVs) are biotherapeutics that have been selected or engineered to infect and selectively kill cancer cells. Increasingly, preclinical and clinical studies demonstrate the ability of OVs to recruit T cells and induce durable immune responses against both virus and tumor, transforming a "cold" tumor microenvironment into a "hot" environment. Besides promising clinical results as a monotherapy, OVs can be powerfully combined with other cancer therapies, helping to overcome critical barriers through the creation of synergistic effects in the fight against brain cancer. Although many questions remain to be answered to fully exploit the therapeutic potential of OVs, oncolytic virotherapy will clearly be part of future treatments for patients with malignant brain tumors.

The limiting factors of oncolytic virus immunotherapy and the approaches to overcome them.

Oncolytic virus (OV) immunotherapy is characterized by viruses which specifically target cancer cells and cause their cytolysis. They provide a unique and promising new tool for the eradication of cancer as they interact with and affect the tumor microenvironment (TME), vasculature, and immune system. Advancements of genetic engineering have allowed for these viruses to be armed in such a way to have enhanced targeting, strong immunomodulation properties, and an ability to modify the TME. However, there are still major limitations in their use, mostly due to difficulties in delivering the viral particles to the tumors and in ensuring that the immunomodulatory properties are able to stimulate the host immune response to mount a complete response. Using novel delivery systems and using OVs as a complementary therapy in a combinatorial treatment have shown some significant successes. In this review, we discuss the major issues and difficulties in using OVs as anti-tumor agents and some of the strategies put in place so far to overcome these limitations. KEY POINTS: • Oncolytic viruses (OVs) infect cancer cells and cause their cytolysis. • The major limitations in using OVs as anti-tumor therapy were discussed. • The potential strategies to overcome these limitations were summarized.

Immunomodulatory effects of ablation therapy on tumors: Potentials for combination with immunotherapy.

As a promising area of tumor treatment, immunotherapies, such as immune checkpoint inhibitors, have been applied to various types of cancer. However, many patients do not respond to such therapies. Increasing application of tumor ablation therapy, a minimally invasive treatment, has been observed in the clinic. Although it can boost the anti-tumor immune response of patients in many ways, ablation alone is not sufficient to remove the tumor completely or stop tumor recurrence in the long term. Currently, there is emerging research focusing on ablation in combination with immunotherapy, aiming to confirm the therapeutic value of this treatment for cancer patients. Hence, in this article, we review the classification, guideline recommendations, and immunomodulatory effects of ablation therapy, as well as the pre-clinical and clinical research of this combination therapy.