© 2020 Elsevier Inc. read more All rights reserved.Herpes simplex virus type 1 (HSV-1) is a large DNA virus that has been popular for oncolytic virus development in pre-clinical research and clinical trials. An oncolytic HSV-1 encoding granulocyte-macrophage colony stimulating factor (GM-CSF), designated talimogene laherparepvec (T-VEC) was approved for the treatment of patients with advanced melanoma in 2015. There are numerous advantages of HSV-1 for oncolytic development, including the ease of recombinant engineering, presence of non-essential genes allowing attenuation of pathogenicity and space for foreign transgene expression. In addition, most recombinants retain sensitivity to acyclovir providing an additional safety feature. In this chapter, we will focus on the key methods for the development of oncolytic HSV-1 vectors and some of the commonly utilized laboratory protocols used to characterize and assess the structure and oncolytic activity of recombinant HSV-1 viruses. © 2020 Elsevier Inc. All rights reserved.Homing of tumor-specific cytotoxic T lymphocytes (CTLs) to the tumor tissues represents a vital step in procuring an effective anti-tumor immune response. Intratumoral accumulation of tumor-specific CTLs can be supported through local chemokine modulation using immune adjuvants or viral vectors, as well as vaccination, using peptide, protein or cell-based vaccines, including dendritic cell (DC) vaccines. Clinical and pre-clinical studies demonstrate that the current immunotherapy regimens are only effective when high numbers of CTLs are present within the tumor microenvironment (TME). Notably, many types of cancer take advantage of this principle and restrict T cell migration into the tumor, subverting the anti-tumor immune response and allowing uncontrolled tumor growth. This chapter discusses the mechanisms involved in the migration of CTLs into tumors and describes the feasible method of evaluating treatment-induced changes in the numbers of polyclonal tumor-specific CTLs in the TME and lymphoid tissues. The described method is widely applicable to multiple tumor models with wild-type antigen expression patterns, without the need for genetically-manipulated cancer cells or animals expressing defined T cell receptors. © 2020 Elsevier Inc. All rights reserved.Tumor infiltration of conventional dendritic cells has been shown to be essential for triggering efficient antitumor immune responses. These findings have generated an increasing demand for reliable methods to accurately identify and quantify specific DC-subpopulations, both in immune monitoring of clinical trial samples as well as in preclinical mouse tumor models. Here, we describe a flow cytometric approach to assess percentages and absolute counts of conventional dendritic cells in solid mouse tumors. © 2020 Elsevier Inc. All rights reserved.Urothelial bladder cancer is the most common malignancy of the urinary tract resulting in over 165,000 deaths worldwide. Immunotherapies targeting the programmed cell death protein-1 (PD-1) checkpoint pathway were recently approved for the treatment of bladder cancer, but favorable responses to this treatment are still limited to a minority of patients. This resistance to therapy has driven a need to optimize syngeneic models of bladder cancer that enable evaluation of the tumor immune microenvironment under varying conditions. Several models have been in place for many years, and we discuss in this chapter the optimization of an orthotopic model of bladder cancer that can be employed to study the anti-tumor immune response. © 2020 Elsevier Inc. All rights reserved.Focal radiation therapy has the potential to generate systemic tumor-targeting immune responses so potent as to eradicate anatomically distant, non-irradiated malignant lesions, a phenomenon commonly referred to as “the abscopal response.” In cancer patients, bona fide abscopal responses are rare, although the recent introduction of immune checkpoint blockers into the clinical practice has significantly increased their incidence. In rodents, abscopal responses can be conveniently modeled by establishing two, slightly asynchronous and anatomically distant subcutaneous tumors in syngeneic immunocompetent hosts, provided that the therapeutic partners of radiation potentially included in the regimen of choice do not mediate systemic anticancer effects per se. Here, we describe such method to monitor abscopal responses based on mammary carcinoma TSA cells implanted in syngeneic immunocompetent BALB/c mice. With minor variations, the same technique can be conveniently applied to a variety of transplantable mouse tumors. © 2020 Elsevier Inc. All rights reserved.Biomarker assessments of tumor specimens is widely used in cancer research to audit tumor cell intrinsic as well as tumor cell extrinsic features including the diversity of immune, stromal, and mesenchymal cells. To comprehensively and quantitatively audit the tumor-immune microenvironment (TiME), we developed a novel multiplex immunohistochemistry (mIHC) platform and computational image processing workflow using a single formalin-fixed paraffin-embedded (FFPE) tissue section. Herein, we validated this platform using nine matched primary newly diagnosed and recurrent head and neck squamous cell carcinoma (HNSCC) sections sequentially subjected to immunodetection with a panel of 29 antibodies identifying malignant tumor cells, and 17 distinct leukocyte lineages and their functional states. Image cytometric analysis was applied to interpret chromogenic signals from digitally scanned and coregistered light microscopy-based images enabling identification and quantification of individual tumor cells, structural features, immune cell phenotypes and their functional state. In agreement with our previous study via a 12-plex imaging mIHC platform, myeloid-inflamed status in newly diagnosed primary tumors associated with significantly short progression free survival, independent of lymphoid-inflamed status. Spatial distribution of tumor and immune cell lineages in TiME was also examined and revealed statistically significant CD8+ T cell exclusion from tumor nests, whereas regulatory T cells and myeloid cells, when present in close proximity to tumor cells, highly associated with rapid cancer recurrence. These findings indicate presence of differential immune-spatial profiles in newly diagnosed and recurrent HNSCC, and establish the robustness of the 29-plex mIHC platform and associated analytics for quantitative analysis of single tissue sections revealing longitudinal TiME changes. © 2020 Elsevier Inc. All rights reserved.