The COVID-19 pandemic has highlighted the urgent need for Brazil to establish a robust and cost-effective vaccine platform, implemented on a large scale, utilizing the country's existing industrial infrastructure. The Newcastle disease virus (NDV) is an avian paramyxovirus that elicits strong humoral and cellular immunity, making it a valuable antigen expression vector. Furthermore, NDV can be produced at a low cost in embryonated chicken eggs, leveraging the existing global influenza vaccine manufacturing infrastructure. Here, as proof of concept, we developed a recombinant NDV expressing the N protein of SARS-CoV-2 virus. First, a codon-optimized nucleotide sequence of the N gene was synthesized. The transgene was inserted into an antigenome NDV plasmid, and the recombinant products were transformed into TOP10 competent cells to generate the NDV-N plasmid. Rescue of the NDV-N virus was performed using reverse genetics in BHK-T7/9 cells. The viral particles were rescued from the cells and propagated in embryonated 10-day-old chicken eggs. The concentration, size, and polydispersity of the viral particles were determined using nanoparticle-tracking analysis. The expression of the N gene and protein in NDV was determined using real-time PCR and western blotting. In addition, we infected Canine Mammary Cancer cells with NDV-N, a cell line highly susceptible to NDV infection and propagation, to observe the expression of the N protein using immunofluorescence. Thus, we established a promising platform for recombinant NDV production in Brazil. Other candidates for oncolytic therapy based on genetically modified NDV for human and veterinary medicine are currently under development in our laboratory.

Canine cutaneous mast cell tumour (MCT) is the most common skin neoplasm in dogs, with histopathology serving as both the diagnostic and primary prognostic tool. However, identifying reliable biomarkers is essential for improving clinical decision-making. CD30, a member of the TNF receptor superfamily, is well-characterised in human haematopoietic malignancies. However, its role in canine MCTs remains unclear. This study aimed to evaluate CD30 expression in neoplastic mast cells and tumour-infiltrating lymphocytes (TILs), and to assess its prognostic significance in canine cutaneous MCTs. Immunohistochemical analysis of CD30, CD3, and PAX-5 was performed on 53 samples, and RNA sequencing was conducted to assess CD30 transcript levels in 17 cases. CD30 was detected in 94.6% of neoplastic mast cells, with strong staining observed in 35 cases and weak staining in 18. Strong CD30 expression (p = 0.0051), CD30+ TIL counts (p = 0.0105) and the diffuse infiltrate distribution pattern of CD3+ TILs (p = 0.0497) were associated with shorter post-surgical survival. RNA sequencing confirmed higher CD30 (TNFRSF8) gene expression levels in high-risk tumours (logFC = 2.3182; FDR = 0.0405). These findings suggest that CD30 is a promising biomarker with potential prognostic value in canine cutaneous MCTs.

Canine diffuse large B-cell lymphoma (cDLBCL) is a frequent and highly aggressive hematopoietic malignancy that serves as a robust translational model for human non-Hodgkin lymphomas. However, the molecular determinants underlying disease progression and prognosis remain poorly defined, hindering precise therapeutic decisions. Clarifying these mechanisms is essential to support the development of prognostic tools and targeted interventions in veterinary oncology. This study aimed to identify prognostically relevant genes and immune alterations associated with cDLBCL. RNA-Seq was performed on lymph node samples from 15 dogs with multicentric DLBCL and two healthy controls. Differential gene expression was assessed to identify key molecular alterations. Functional annotation, pathway analysis, and Kaplan–Meier survival correlations were used to characterize the biological and clinical significance of target genes. Additionally, immune cell deconvolution was conducted to explore changes in the tumor microenvironment (TME). Transcriptomic profiling revealed 410 differentially expressed genes (DEGs), comprising 408 downregulated and 2 upregulated genes in tumor samples compared to normal lymph nodes. Among these, STOM, TBC1D8, HMOX1, ABCB1, and CTLA4 emerged as genes of interest due to their involvement in immune regulation, oxidative stress response, vesicular trafficking, drug resistance, and immune checkpoint signaling. Lower expression of these genes was significantly associated with reduced overall survival. Immune infiltration analysis demonstrated a dominance of neoplastic B cells and a marked reduction in cytotoxic T cells and macrophages, consistent with an immunosuppressive TME. This study identifies key molecular and immunological signatures associated with prognosis in cDLBCL, providing insight into mechanisms of immune suppression and therapeutic resistance. The findings reinforce the translational value of canine lymphoma as a model for human disease and support the integration of molecular biomarkers into veterinary oncologic practice to guide individualized treatment strategies.

Melanoma is a highly aggressive cancer in both humans and dogs with significant biological and clinical similarities. This study aimed to identify therapeutic kinase targets in canine melanoma by screening kinase inhibitors in canine melanoma cell lines. Cell viability assays showed that seven inhibitors reduced viability in both CMGD2 and TLM1 cells, whereas additional compounds showed cell line-specific activity. The most promising kinase targets were AURKA, AURKB, AXL, CLK1/2/4, IGF1R, MAP2K1, and MAP2K2. Gene expression analysis confirmed expression of these targets in the cell lines, except for CLK3. In silico analyses revealed high structural homology (≥90%) between canine and human kinases, supporting the translational relevance of these findings. Molecular docking further demonstrated similar predicted binding profiles between human and canine kinases. Several FDA-approved drugs targeting these kinases were identified as potential repurposing candidates for canine melanoma treatment. These findings highlight the potential of targeted therapies for canine melanoma and reinforce the value of comparative oncology in advancing precision medicine across species. However, these findings are based on an exploratory single-dose in vitro screen, a small tumor cohort, and in silico analyses, and therefore require dose-response, protein-level, and in vivo validation.

Canine splenic hemangiosarcoma (HSA) is a highly aggressive endothelial malignancy and the standard treatment protocol involves splenectomy followed by chemotherapy. To explore a precision oncology approach, we analyzed the expression of 45 cancer-related-genes in formalin-fixed-, paraffin-embedded-splenic HSA specimens from 27 dogs using the Oncomapa™ qPCR panel. Unsupervised principal component analysis and K-means clustering identified three molecular distinct groups, which were further confirmed using Ward’s hierarchical clustering. The clustering results were validated using the within-cluster sum of squares and silhouette scores, supporting an exploratory three-group solution. The first cluster was marked by increased MET, MAP2K1/2, FLT3, SRC, AKT2, TP53, PDGFRA, SETD2, and NR3C1 expression, suggesting activation of the MAPK and PI3K/AKT pathways, specific TK receptor activation (MET, FLT3, and PDGFRA), and stress and immune regulation (NR3C1 and TP53). The second cluster exhibited elevated expression levels of BCL2, NOTCH1, RET, and KIT, indicating mechanisms of apoptosis evasion, enhanced cell survival, and potential targets for tyrosine kinase inhibition. The third cluster, characterized by the overexpression of MYC, TOP2A, RRM2, TYMS, and BRCA1, showed a molecular profile associated with elevated proliferative signaling, DNA synthesis, and DNA repair-related mechanisms. Based on these signatures, we propose cluster-specific candidate therapeutic hypotheses for future validation. This study suggests that gene expression profiling may contribute to categorizing canine hemangiosarcoma into clinically relevant subtypes, providing a foundation for future investigations into personalized therapeutic approaches that require further biological validation.

Equine sarcoids are the most common skin tumors in horses and are characterized by local invasiveness, high recurrence rates, and inconsistent responses to current therapies. Oncolytic virotherapy represents a promising alternative approach that exploits defects in antiviral signaling pathways in tumor cells. Newcastle disease virus (NDV), an avian paramyxovirus that is nonpathogenic in mammals, has demonstrated tumor-selective activity in several species; however, its effects on equine sarcoids have not been previously investigated. In this study, primary cell cultures derived from equine sarcoid tumors were established to evaluate the selective cytotoxicity of NDV. Sarcoid tissues from six horses generated thirteen primary sarcoid cultures, while three primary equine fibroblast cultures served as non-tumor controls. Bovine papillomavirus (BPV) DNA was assessed by quantitative polymerase chain reaction. Cells were infected with a green fluorescent protein-expressing Newcastle disease virus (NDV-GFP), and cell viability was measured after seventy-two hours using a metabolic viability assay. Sarcoid cultures exhibited significantly greater sensitivity to viral infection than normal fibroblasts, with substantially lower half-maximal inhibitory concentrations. Viral infectivity, quantified by fluorescence intensity, correlated with increased susceptibility to virus-induced cytotoxicity. Notably, the oncolytic activity of Newcastle disease virus was observed in both bovine papillomavirus-positive and bovine papillomavirus–negative sarcoid cultures. These findings demonstrate that NDV-GFP induces stronger cytotoxic effects in equine sarcoid cells than in normal fibroblasts, supporting its potential as a novel therapeutic strategy in equine oncology.

Lymphoma is one of the most prevalent types of feline cancer. It is characterized as a group of diseases that can affect various organs, such as the gastrointestinal tract, kidneys, thymus, and skin. In feline medicine, the search for alternative treatments is of utmost importance, given the significant number of animals that relapse or are unresponsive to conventional chemotherapy treatment. As an alternative to existing modalities of treatment for a variety of cancers, oncolytic viruses have been studied in the last few years. Those viruses possess a unique ability to target and eliminate cancer cells while simultaneously stimulating an immune response against malignant cells, acting as an immunotherapy. Newcastle Disease Virus (NDV) is an avian paramyxovirus that affects both domestic and wild birds, causing symptoms that range from severe to asymptomatic, depending on the viral strain. Less virulent strains are considered safe for use as a vaccine against Newcastle Disease. In the Oncology field, those strains are also being studied to be used as oncolytic virotherapy for mammals, and several results demonstrate their efficacy in vitro and in vivo. The present study aimed to explore the oncolytic potential of Newcastle Disease Virus expressing green fluorescent protein (NDV-GFP) in feline lymphoma cells isolated from a FeLV-positive patient with thymic lymphoma. The NDV-GFP infected, replicated, and induced apoptosis in feline lymphoma cells. Therefore, these results provide preliminary evidence of the oncolytic activity of NDV in feline leukemia virus-induced lymphoma.

Background Multiple correspondence analysis (MCA) is an unsupervised data science methodology that aims to identify and represent associations between categorical variables. Gliomas are an aggressive type of cancer characterized by diverse molecular and clinical features that serve as key prognostic factors. Thus, advanced computational approaches are essential to enhance the analysis and interpretation of the associations between clinical and molecular features in gliomas. Objective This study aims to apply MCA to identify associations between glioma prognostic factors and also explore their associations with stemness phenotype. Methods Clinical and molecular data from 448 patients with brain tumors were obtained from the Cancer Genome Atlas. The DNA methylation stemness index, derived from DNA methylation patterns, was built using a one-class logistic regression. Associations between variables were evaluated using the χ² test with k degrees of freedom, followed by analysis of the adjusted standardized residuals (ASRs >1.96 indicate a significant association between variables). MCA was used to uncover associations between glioma prognostic factors and stemness. Results Our analysis revealed significant associations among molecular and clinical characteristics in gliomas. Additionally, we demonstrated the capability of MCA to identify associations between stemness and these prognostic factors. Our results exhibited a strong association between higher DNA methylation stemness index and features related to poorer prognosis such as glioblastoma cancer type (ASR: 8.507), grade 4 (ASR: 8.507), isocitrate dehydrogenase wild type (ASR:15.904), unmethylated MGMT (methylguanine methyltransferase) Promoter (ASR: 9.983), and telomerase reverse transcriptase expression (ASR: 3.351), demonstrating the utility of MCA as an analytical tool for elucidating potential prognostic factors. Conclusions MCA is a valuable tool for understanding the complex interdependence of prognostic markers in gliomas. MCA facilitates the exploration of large-scale datasets and enhances the identification of significant associations.

Despite major gains with immunotherapy, many tumors remain non-responsive because of poor antigen release and a suppressive tumor microenvironment (TME). Oncolytic virotherapy (OVT) directly lyses cancer cells and secondarily inflames the TME via immunogenic cell death and type I interferon (IFN-I) signaling. Here we present primary clinical and preclinical evidence on (i) mechanisms that govern OV selectivity and immune priming, (ii) genetic engineering strategies linked to clinical signals, and (iii) translational lessons across species, with emphasis on companion-animal oncology as a bridge to human trials. We highlight trials where OVs prime checkpoint response (e.g., DNX-2401→pembrolizumab in recurrent glioblastoma) and where vector design (e.g., TK-deleted vaccinia, CG0070) or payloads (e.g., IFNβ, NIS) drive measurable benefit. We conclude with actionable priorities, patient selection by IFN-pathway competence, receptor-tropism panels, and rational OV-ICI sequencing, to accelerate durable responses.

Simple and complex carcinomas are the most common type of malignant Canine Mammary Tumors (CMTs), with simple carcinomas exhibiting aggressive behavior and poorer prognostic. Stemness is an ability associated with cancer initiation, malignancy, and therapeutic resistance, but is still few elucidated in canine mammary tumor subtypes. Here, we first validated, using CMT samples, a previously published canine one-class logistic regression machine learning algorithm (OCLR) to predict stemness (mRNAsi) in canine cancer cells. Then, using the canine mRNAsi, we observed that simple carcinomas exhibit higher stemness than complex carcinomas and other histological subtypes. Also, we confirmed that stemness is higher and associated with basal-like CMTs and with NMF2 metagene signature, a tumor-specific DNA-repair metagene signature. Using correlation analysis, we selected the top 50 genes correlated with higher stemness, and the top 50 genes correlated with lower stemness and further performed a gene set enrichment analysis to observe the biological processes enriched for these genes. Finally, we suggested two promise stemness-associated targets in CMTs, POLA2 and APEX1, especially in simple carcinomas. Thus, our work elucidates stemness as a potential mechanism behind the aggressiveness and development of canine mammary tumors, especially in simple carcinomas, describing evidence of a promising strategy to target this disease.

ABSTRACT: There are limited publications about canine subcutaneous mast cell tumors (MCT). International studies have shown that subcutaneous MCT has longer survival times than cutaneous MCT, with lower recurrence and metastasis rates. In addition, subcutaneous MCT has a specific histopathological classification (circumscribed, combined, or infiltrative pattern). Our study evaluated 162 cases of subcutaneous MCT diagnosed from 2014 to 2017 in Brazil. The mean age of the animals was 8.6 years, with a predominance of females and higher incidence in dogs with mixed breed (n=40), followed by Boxer (n=20), Labrador Retriever (n=14), Golden Retriever (n=11) and Pug (n=10). Regarding histopathological characterization, the most common infiltrative pattern represented 54.3% of cases, followed by circumscribed (34.8%) and combined (11%) patterns. The mean mitotic index (MI) was 1.04, with 93.9% of cases presenting MI≤4 and 53.1% MI=0. The data found in this Brazilian study regarding subcutaneous MCT does not differ from those described in American studies, suggesting similar genetic and epidemiological factors. The evaluated proliferation indices suggest that subcutaneous MCT presents slow progression and should be evaluated as a distinct form of cutaneous MCT.

Lymphoma is the most prevalent type of cancer in cats, and the search for alternative treatments is of utmost importance given the significant number of animals that exhibit tumor recurrence or are unresponsive to conventional chemotherapy treatment. Oncolytic viruses possess a unique ability to target and eliminate cancer cells while simultaneously stimulating an immune response against these malignant cells. The present study aimed to explore the oncolytic potential of Newcastle Disease Virus expressing green fluorescent protein (NDV-GFP) in a feline leukemia virus (FeLV)-positive lymphoma cell line. Our research suggests that NDV-GFP has a propensity to selectively target, infiltrate, and replicate with-in feline lymphoma cells, resulting in the induction of apoptosis in these cells, as opposed to non-tumor cells. Therefore, our research provides evidence supporting the development of novel oncolytic therapies based on NDV for the treatment of feline lymphomas.

Despite advances in diagnostic and therapeutic approaches for lung cancer, new therapies targeting metastasis by the specific regulation of cancer genes are needed. In this study, we screened a small library of epigenetic inhibitors in non-small-cell lung cancer (NSCLC) cell lines and evaluated 38 epigenetic targets for their potential role in metastatic NSCLC. The potential candidates were ranked by a streamlined approach using in silico and in vitro experiments based on publicly available databases and evaluated by real-time qPCR target gene expression, cell viability and invasion assays, and transcriptomic analysis. The survival rate of patients with lung adenocarcinoma is inversely correlated with the gene expression of eight epigenetic targets, and a systematic review of the literature confirmed that four of them have already been identified as targets for the treatment of NSCLC. Using nontoxic doses of the remaining inhibitors, KDM6B and PADI4 were identified as potential targets affecting the invasion and migration of metastatic lung cancer cell lines. Transcriptomic analysis of KDM6B and PADI4 treated cells showed altered expression of important genes related to the metastatic process. In conclusion, we showed that KDM6B and PADI4 are promising targets for inhibiting the metastasis of lung adenocarcinoma cancer cells.

ABSTRACT: LIN28 is a RNA-binding protein including two highly conserved homologous, LIN28A and LIN28B. Proto-oncogenes such as LIN28A and LIN28B are generally targeted by the let-7 miRNAs in different types of human cancers. Here, we determined the expression of LIN28A in canine mammary tumor samples and the LIN28/let-7 pathway in canine mammary cell lines. In those cell lines, we identified a functional LIN28/let-7 pathway which exhibited high expression of let-7 members and low expression of its targets, including LIN28A and LIN28B. However, the mammary carcinoma tissue samples showed a frequent expression of LIN28A being expressed mainly in the epithelial cells. No association was observed between LIN28A expression and histopathological classification and grade, TNM and survival time. Our results suggested a possible role of the LIN28A protein in the development of canine mammary carcinomas due to the high frequency observed in the tumor samples (28 of 32). The in vitro experiments suggested that the LIN28/let-7 pathway is active in the tumor cells evaluated. However, more studies are necessary to elucidate the exact role of LIN28/let-7 pathway in canine mammary carcinomas.

This study assessed differential gene expression and identified expression quantitative trait loci (eQTLs) from samples of Longissimus lumborum muscle from bulls at 15 months of age submitted to different prenatal nutrition. Upon confirmation of pregnancy, 126 dams were separated into three diet treatments varying the period of inclusion of energy protein supplementation (NP, PP, and FP). At calving, 63 males were genotyped with GGP LD BeadChip. The skeletal muscle of 15 bulls was sequenced (RNA-seq) at 15 months of age. The EdgeR package was used for differential gene expression and principal component analysis (PCA), and the Matrix eQTL package was used for the eQTLs analysis (R statistical). The functional enrichment analysis was performed using the MetaCore® software. No genes differentially expressed were found between treatments (FDR > 0.05); nevertheless, we found 179 cis-tag-eQTLs and 20,762 trans-tag-eQTLs (FDR < 0.05) after linkage disequilibrium analysis. The functional enrichment analysis identified terms from gene ontology related to genes associated to trans-eQTLs (FDR < 0.05) as well as metabolic pathways (> gScore). Most biological pathways and genes found had been previously associated to fetal programming. The different prenatal supplementation strategies did not impact on muscle transcriptome of bulls. Additionally, there is a link between genotype and gene expression levels related to developmental traits in Nellore cattle.

Mast cell tumours (MCTs) are the most frequent malignant skin neoplasm in dogs. Due to the difficulty in purifying large numbers of canine neoplastic mast cells, relatively little is known about their properties. A reproducible in vitro model is needed to increase the understanding about the phenotype and functional properties of neoplastic mast cells. In the present study, we describe the establishment of primary cocultures of neoplastic mast cells from canine cutaneous MCTs and cancer-associated fibroblasts. We confirmed the inability of canine neoplastic mast cells to remain viable for long periods in vitro without the addition of growth factors or in vivo passages in mice. Using a transwell system, we observed that mast cell viability was significantly higher when there is cell-to-cell contact in comparison to non-physical contact conditions and that mast cell viability was significantly higher in high-grade than in low-grade derived primary cultures. Moreover, the use of conditioned medium from co-cultured cells led to a significantly higher tumoral mast cell viability when in monoculture. Signalling mechanisms involved in these interactions might be attractive therapeutic targets to block canine MCT progression and deserve more in-depth investigations.

Canine mammary carcinoma (CMC) is one of the major health threats in dogs. The oncolytic virotherapy is a promising strategy to treat canine as well as human cancer patients with non-pathogenic replicating viruses. Here, we evaluated the antitumor activity of one lentogenic, non-lytic Newcastle disease virus (NDV) LaSota strain expressing GFP (NDV-GFP) on five different CMCs and one non-tumorigenic cell line, regarding cell viability, cell death, selectivity index, morphology, global and target gene expression analysis. As evidenced by the selectivity index, all CMC cell lines were more susceptible to NDV-GFP in comparison with the non-tumorigenic cells (~3.1× to ~78.7×). In addition, the oncolytic effect of NDV-GFP was more evident in more malignant CMC cells. Also, we observed an inverse association of the IFN pathway expression and the susceptibility to NDV. The downregulated genes in NDV-GFP-sensitive cells were functionally enriched for antiviral mechanisms by interferon and immune system pathways, demonstrating that these mechanisms are the most prominent for oncolysis by NDV. To our knowledge, this is the first description of oncolysis by an NDV strain in canine mammary cancer cells. We also demonstrated specific molecular pathways related to NDV susceptibility in these cancer cells, opening the possibility to use NDV as a therapeutic-targeted option for more malignant CMCs. Therefore, these results urge for more studies using oncolytic NDVs, especially considering genetic editing to improve efficacy in dogs.

Abstract Despite advances in diagnostic and therapeutic approaches, the majority of lung cancer patients still progress to advanced stages with metastatic lesions. Abnormalities in epigenetic mechanisms are related to the most malignant cancer phenotypes. Thus, there is an increasing interest in the development and validation of drugs that targets epigenetic proteins. Trimethylation of H3K27 is an important post-translational modification and some studies have shown that KDM6B (lysine (K)-specific demethylase 6B), an H3K27 demethylase, is a crucial regulator involved in tumorigenesis and play important roles in various types of cancers. In this study, our goal was to determine potential epigenetic targets to control invasion and migration of NSCLC cancer cells. Initially, KDM6B was found a promise target through in silico analysis for the inverse correlation between gene expression and overall survival on 2438 NSCLC cases from GEO, EGA and TCGA using KMplotter tool and selecting the genes according to HR and p-values (HR > 1 and p < 0.05). Next, NSCLC cell lines (A549, H23, H2126 and H1568) were evaluated for the expression of the KDM6B using CellExpress tool and next by real-time PCR and one cell was chosen for cytotoxicity of the KDM6B inhibitor (GSK-J4) for 72h by MTT assay. The IC50 value and the regression curve were calculated with 6.0 Prism (GraphPad Software, USA). KDM6B gene expression was inversely correlated with patient survival rate for pulmonary adenocarcinoma (HR= 2.81; p= 6,3E-09). All pulmonary adenocarcinoma cell lines expressed KDM6B gene and low expression in healthy lung tissue (Z-score= 2.8). The KDM6B IC50 for A549 cells was 2.78±0.09μM. These early findings report that KDM6B as a promising target for epigenetic therapy for pulmonary adenocarcinoma. Therefore, our next step will be to evaluate the potential of KDM6B inhibition on the phenotype of cancer cell migration and invasion and global analysis of gene expression to understand genes and mechanisms associated with possible inhibitory effects of KDM6B. Citation Format: Jessika C. Lesbon, Pedro L. Xavier, Taismara K. Garnica, Arina L. Rochetti, Rui M. Reis, Susanne Müller, Heidge Fukumasu. Inhibition of histone demethylase KDM6B as a promising target for non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1752.