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Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and β-catenin expression as well as nuclear β-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and β-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/β-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions. [ABSTRACT FROM AUTHOR]

Simple Summary: In the last few years, the treatment of different types of tumors in humans has benefited from the introduction of new drugs called "immune checkpoint inhibitors" (ICI). These treatments help the patient's immune response in fighting against cancer, therefore limiting tumor growth and aggressiveness. The possibility to use this therapeutical strategy also in pet animals such as horses has been scarcely explored in veterinary medicine. This study aims at investigating the presence and expression of specific checkpoint molecules such as PD-L1 and CTLA-4 in penile tumors of horses, particularly regarding malignant squamous cell carcinomas. In fact, PD-L1 and CTLA-4 presence is pivotal for an effective response to ICI and a successful therapy. Unfortunately, our results seem to indicate that these molecules are not frequently expressed in equine penile tumors. Therefore, horses with penile tumors may not benefit from the therapeutical use of ICI. In horses, penile squamous cell carcinomas (epSCCs) are among the most common cutaneous neoplastic lesions. These tumors usually arise in benign lesions such as viral plaques and papillomas frequently induced by Equus caballus papillomavirus type 2 (EcPV2) infection. In the last decade, the introduction of immune checkpoint inhibitors (ICI) for the treatment of human cancers has demonstrated promising results. Among the most commonly targeted pathways, there is PD-1/PD-L1 and CTLA-4. The aim of this study is to investigate the expression of the PD-1/PD-L1 pathway and CTLA-4 in the tumor microenvironment of epSCCs to assess the feasibility of an immunotherapeutic approach. Twenty equine epithelial tumors were retrospectively selected and submitted to RT-qPCR for PD-1 and PD-L1 genes. After testing antibodies cross-reactivity by western blotting, immunohistochemistry for PD-L1 and CTLA-4 was performed. Results from RT-qPCR demonstrated that 3/20 cases expressed the PD-L1 gene, whereas the PD-1 gene was not detected. Immunohistochemical positivity for PD-L1 was found only in one case. CTLA-4-positive cells were observe in all cases but were few (Mdn = 4.8; IQR = 2.3–7.1 cells/HPF). In this study group, PD-1/PD-L1 and CTLA-4 do not appear to be highly expressed and therefore the use of ICI in epSCCs may not have promising rates of response. [ABSTRACT FROM AUTHOR]

Simple Summary: Squamous cell carcinoma (SCC) is one of the most common cancers in horses, and it can arise at any site on the skin and mucosae. Recent studies associated equine papillomavirus type 2 (EcPV2) infections with this type of cancers of the oral tract and genitals. Larynx and pharynx are frequently recognized as sites of SCC. In humans, squamous cell carcinoma of the larynx (SCCL) is a common cancer associated with papilloma virus (PV) infection and epithelial to mesenchymal transition (EMT). EMT can occur under different biological conditions, upon the same programmed changes: embryogenesis and organ development fibrosis, wound healing, and cancer metastases. This work reports for the first time in a SCCL of a horse a wide immunohistochemical EMT characterization, by analyzing main epithelial markers (E-cadherin, β-catenin, and pan-cytokeratin AE3/AE1), main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1, and HIF-1α). This work illustrates an example of tumor cell adaptation during the metastatic process in the equine SCCL, taking also into consideration the potential influence provided by EcPV2 oncoproteins on the EMT process. Squamous cell carcinoma (SCC) is one of the most frequent tumors of skin and muco-cutaneous junctions in the horse. Equine papillomavirus type 2 (EcPV2) has been detected in equine SCC of the oral tract and genitals, and recently also in the larynx. As human squamous cell carcinoma of the larynx (SCCL), it is strongly etiologically associated with high-risk papillomavirus (h-HPV) infection. This study focuses on tumor cells behavior in a naturally occurring tumor that can undergo the so-called epithelial to mesenchymal transition (EMT). A SCCL in a horse was investigated by immunohistochemistry using antibodies against E-cadherin, pan-cytokeratin AE3/AE1, β-catenin, N-cadherin, vimentin, ZEB-1, TWIST, and HIF-1α. EcPV2 DNA detection and expression of oncogenes in SCC were investigated. A cadherin switch and an intermediate filaments rearrangement within primary site tumor cells together with the expression of the EMT-related transcription factors TWIST-1, ZEB-1, and HIF-1α were observed. DNA obtained from the tumor showed EcPV2 positivity, with E2 gene disruption and E6 gene dysregulation. The results suggest that equine SCCL might be a valuable model for studying EMT and the potential interactions between EcPV2 oncoproteins and the EMT process in SCCL. [ABSTRACT FROM AUTHOR]