Colorectal cancer (CRC) is one of the most common cancers in the world and ranks third in terms of incidence, but second in terms of mortality [1]. The three dominant conventional cancer therapies, i.e., surgery, chemotherapy, and radiotherapy, are powerful but show some limitations such as being non-specific, toxic to normal cells, developing chemo-resistance sub-clones, and inability to create long-lasting immunity [2], [3]. Novel treatment approaches are constantly required to enhance the safety and efficacy of cancer therapy to overcome these drawbacks. An encouraging new approach to fighting cancer is cancer immunotherapy, which involves activating the immune system against tumors. Various immunotherapy modalities include oncolytic virus therapy (OVT), checkpoint blockades, adoptive T-cell therapy, etc. [2]. Combination treatments with immunotherapies and other approaches can increase treatment efficacy, enhance immune responses, reduce immunosuppression, and generate long-lasting immunity against cancer cells compared to monotherapy [4]. Reovirus (RV), a double-stranded benign human RNA virus, possesses oncolytic properties and is a leading candidate for therapeutic development [5]. Both apoptotic and apoptosis-independent cell death mechanisms are considered prominent mechanisms of RV-mediated cancer cell killing [6]. In addition to their main effect, OVs can induce an immune system response. Tumors create an immunosuppressive environment to prevent the immune system from recognizing cancer cells. Delivering OVs into tumors enables a potent and durable response against tumor antigens and supports immunological memory. Since the immune system recognizes viruses as pathogens, the consequent antiviral response may be a significant barrier for OVs [2]. Evidence suggests that optimal antitumor responses may be achieved by combining oncolytic virotherapy with other cancer immunotherapy approaches [7]. Nowadays, two types of cancer vaccines typically utilize tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) to activate the patient's immune system. TAAs are widely distributed on the surface of cancer cells compared to normal cells [8]. T cells recognize TAA-derived peptides presented by human leukocyte antigen (HLA), initiating an immune response against cancer [9]. Identifying suitable TAAs and selecting optimal adjuvants remain crucial for developing effective vaccines [10]. The carcinoembryonic antigen (CEA) is one of the first TAAs ever identified which is expressed in 98.8% of CRC cases compared with matched healthy tissues [11] and it has been employed in numerous studies to treat cancer [12], [13], [14], [15], [16]. Targeting antigens to dendritic cell (DC)–specific receptors along with immune-stimulatory adjuvants elicits a strong T-cell response against the antigen [17]. The most widely used molecule for delivering antigens to DCs is DEC-205, which requires co-administration of adjuvants to optimally induce anti-tumor cytotoxic T lymphocytes (CTLs) and T helper cells [18]. In this study, programmed cell death protein 1 (PD1/PDL1) inhibitor, lenalidomide (L), and lysate of necrotic cancer cells (N) were used as adjuvants to potentiate DC-targeted CEA vaccination. A growing body of research in DC therapy suggests that blocking PD-1 enhances the ability of the immune system to combat cancer cells [19], [20]. Lenalidomide is a synthetic immunomodulatory drug with potent antineoplastic, anti-angiogenic, and anti-inflammatory properties [21], [22]. Combining lenalidomide as an adjuvant with tumor antigen–loaded DC vaccination significantly inhibits tumor growth and enhances antitumor immunity in mouse cancer models [23], [24]. Lysate of necrotic cancer cells contains a mixture of proteins that induce DC maturation and stimulate CD4+ and CD8+ T cells, triggering a rapid immune response at the tumor site [25], [26].

In this study, the effects of combining immunotherapy and oncolytic virotherapy for CRC treatment were investigated in a mouse model. For immunotherapy, adenoviral vectors expressing the CEA antigen targeted to DEC205 on DCs were used along with PD1/PDL1 inhibitor, lenalidomide, and lysate of necrotic CEA-expressing CRC cells.