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Tumor cells acquire distinct genetic characteristics as a means to survive and proliferate indefinitely. Changes in the genetic code can also translate in changes at the protein level, therefore creating a distinguishable signature unique for tumor cells, and absent in normal tissues. The presence of discernable moieties in tumors is particularly attractive because it represents a therapeutic opportunity to target tumor cells with specificity, while sparing non-transformed cells. In this sense neoantigens, short peptides containing a mutated sequence, are seen attractive therapeutic targets because of their confinement within tumor cells. Neoantigens can be recognized with high affinity and specificity by tumor-targeting T cells, which consequently can initiate a potent anti-tumor immune response. While this is feasible and it has been tested in numerous cancer types including melanoma, colon and lung cancer, to mention a few, there are technical challenges in identifying immunogenic neoantigens. In this manuscript we address the topic of neoantigen identification from tumor samples, offering a technical overview of the bioinformatic methods utilized to profile the neoantigenic load of tumor samples obtained from clinical specimens. This is meant to guide readers through the steps of neoantigen identification using genomic data, by suggesting tools and methods that can provide, with a high degree of confidence, reliable results for downstream in vitro and in vivo applications. © 2020 Elsevier Inc. All rights reserved.Emergency of the CRISPR technology, a new genome editing tool, revolutionized the biomedical research field in the past 6 years. At the same time, recent advances in cancer immunotherapy reinvigorated our hope to cure most if not all cancer patients with further development of various treatment options. A combination of the CRISPR technology with immuno-oncology research will undoubtedly accelerate the development of new cancer therapies. This review will focus on the CRISPR system and its applications in immune-oncology including identification of immune-oncology gene targets, generation of cancer animal models, and enabling better cell design and manufacture for adoptive cellular therapies. © 2020 Elsevier Inc. All rights reserved.Among the many immunotherapies being developed and tested both preclinically and clinically, oncolytic viruses (OVs) are gaining traction as a forerunner in the search for potent new therapeutic agents, with a genetically engineered herpes simplex virus type 1 (HSV-1) recently approved by the FDA for the treatment of melanoma. The great potential of OVs to fight cancer is driving different approaches to improve OV-based therapy, with genetic modification of OVs to enhance host antitumor immunity being one of the most promising approaches. In this chapter we describe possible modifications in the OV genome that could increase its antitumor activity and immunostimulatory capacity, together with different methods to achieve these goals. Finally, we present different analyses to verify the desired genetic modification and evaluate its impact on host antitumor immunity in preliminary stages. © 2020 Elsevier Inc. All rights reserved.The tumor microenvironment (TME) is composed of a set of cellular compartments comprising vascular, neuroendocrine, stromal, epithelial and immune cells. These compartments constitute a heterogeneous and dynamic set, where intercellular communication is driven by a complex network of cytokines, chemokines, growth factors, and inflammatory and matrix remodeling enzymes. Based on this complexity, an increasing number of assays may be required to identify and locate specific proteins in the tissue section and the standard procedure is to perform one stain at a time on serial sections. Recently, interest in performing multiple assays on formalin-fixed, paraffin embedded (FFPE) specimens has gained ground, and is referred to as multiplexing, i.e., multiple staining of the same section at the same time. Multiple staining is a promising approach that may help to improve understanding of the interactions between the different cellular components of the TME, stratify cancer patients, and help clinicians in their patient management. In this chapter, we detail a simple methodological approach to perform multiple staining on the same section using tissue obtained from patients with melanoma. This procedure evaluates the presence and location of three different proteins, human leukocyte antigen (HLA), forkhead box protein 3 (FoxP3) and Granzyme B (GRZB). © 2020 Elsevier Inc. All rights reserved.Despite exciting proof-of-concept data mediated by adoptive T-cell transfer and checkpoint blockade, major challenges imposed by the tumor microenvironment restrict clinical benefits to a minority of patients with advanced or metastatic solid malignancies. read more While employment of toxic pre- and postconditioning regimens to circumvent the inefficacy of T-cell transfer presents a fundamental problem for heavily pretreated cancer patients, for checkpoint blockade, the main issue relates to low single-agent response rates. To overcome these hurdles, combination therapy with oncolytic adenovirus is becoming an attractive solution given multiple intrinsic modulatory effects on the intratumoral immune compartment, engineering capabilities and safety profile. Here, we provide a short overview on the tumor microenvironmental challenges in solid tumors, and how oncolytic adenoviruses can counteract these barriers. Finally, the immunotherapeutic potential of oncolytic adenoviruses will be discussed in the context of clinical experience with adoptive T-cell therapy and immune checkpoint inhibitors. © 2020 Elsevier Inc. All rights reserved.Recombinant adeno-associated viruses (rAAVs) are attractive tools for research in cancer immunotherapy. A single administration of an AAV vector in tumor mouse models induces a progressive increase in transgene expression which reaches a plateau 1 or 2 weeks after administration. The rAAV is then able to maintain the expression of the immunostimulatory transgene. Thus, the use of these vectors obviates the need for frequent administrations of the therapeutic protein to achieve the antitumor effect. The long-term expression of AAV vectors can be exploited for the evaluation of the antitumor activity of immune-enhancing proteins. Most preclinical studies have focused on the expression of cytokines and on the induction of immune responses elicited by tumor-associated antigens expressed by rAAVs. Notwithstanding, rAAVs may not be suitable for immunostimulatory proteins that require high and/or immediate expression. In this chapter, we review a feasible, reliable and detailed protocol to produce and purify AAV vectors as a tool for cancer immunotherapy strategies.