Adoptive cell transfer (ACT) immunotherapy represents a promising therapeutic approach for cancer treatment, utilising ex vivo-expanded immune effector cells such as T-lymphocytes, natural killer (NK) cells, and cytokine-induced killer (CIK) cells. CIK cells are characterised by dual T-cell and NK cell-like properties, exhibiting high proliferative capacity, cytolytic activity, and non-MHC-restricted tumour recognition. Canine malignant melanoma (CMM) is an aggressive neoplasm with significant translational relevance to human melanoma, yet the cytotoxic potential of CIK cells against CMM remains unexplored. This study aimed to expand and functionally characterise canine CIK and T-cells from healthy donors, comparing their cytotoxic activity against CMM cell lines in allogeneic and autologous settings. Peripheral blood mononuclear cells were isolated and expanded using standardised protocols with IFN-γ, anti-CD3/CD28 activation, and IL-2 supplementation. Flow cytometry confirmed successful expansion with CIK cells showing 85% NKp46 expression and T-cells reaching 91.96% CD8 expression, with comparable expansion folds (7.94 vs. 7.88). Cytotoxicity assays against seven CMM cell lines demonstrated robust anti-tumour activity for both effector populations, with CIK cells achieving 61.5% mean killing and T-cells 59.8% in allogeneic assays at 10:1 effector-to-target ratios. Pearson's correlation analysis revealed strong negative correlations between effector cell numbers and target viability (r = -0.960 for CIK cells; r = -0.790 for T cells). Cytokine profiling showed distinct secretion patterns, with T-cells producing a broader cytokine repertoire. These findings establish the feasibility and comparable efficacy of both CIK cells and T-cells against CMM, supporting their therapeutic development for canine melanoma immunotherapy.

Programmed Death-Ligand 1 (PD-L1) is an immune-checkpoint molecule involved in tumor-induced immunotolerance. While few studies explored its role in canine B-cell lymphoma (BCL), comprehensive analysis across lymphoma immunophenotypes remains limited. This study investigated associations between time to progression (TTP) and lymphoma-specific survival (LSS) with PD-L1 expression, including surface membrane protein (mPD-L1), mRNA levels in nodal aspirates, and soluble protein (sPD-L1) plasmatic concentrations, in dogs with different nodal lymphoma immunophenotypes at initial presentation. Fifty-eight cases were evaluated: 38 BCL, 11 T-cell lymphomas not otherwise specified (T-NOS), and 9 T-zone lymphomas (TZL). No significant association was found between mPD-L1 expression, mRNA level, sPD-L1 concentration, and TTP or LSS. Although not significant, median TTP was longer in mPD-L1 negative cases in both BCL (306, 95%C.I. 130-482 vs. 136, 95%C.I. 102-170 days) and T-NOS (228, 95%C.I. 40-416 vs. 48, 95%C.I. 0-168 days). The same was true for LSS (273, 95%C.I. 58-386 vs. 191, 95%C.I. 23-236 days for BCL, and 282, 95%C.I. 60-504 vs. 121, 95%C.I. 77-165 days for T-NOS). Notably, the only two TZL cases that progressed were mPD-L1-positive. These findings suggest a potential prognostic role for mPD-L1, warranting further validation in larger cohorts in BCL and T-NOS nodal lymphomas. For TZL, extended follow-up studies are needed due to its indolent behavior. Although sPD-L1 and mRNA levels did not correlate with outcome, their diagnostic and prognostic utility merits further investigation and methodologic refinement.

The hypoxic microenvironment plays a critical role in the progression of canine osteosarcoma (OSA) by promoting different cellular responses, including the release of extracellular vesicles (EVs). Given the clinical aggressiveness of canine OSA, the aim of this study was to evaluate the miRNAome profile in EVs released in vitro by four canine OSA cell lines under hypoxic conditions. In particular, for this study we used two commercial canine osteosarcoma cell lines (D17 and D22) and two primary osteosarcoma cell lines obtained in our laboratory (Penny and Wall). D17, D22, Penny, and Wall cell lines were cultured under normoxic and hypoxic conditions (200 µM CoCl2) for 24 h. EVs were isolated by size-exclusion chromatography and characterized by nanoparticle tracking analysis and Western blotting. miRNAs extracted from EVs were then sequenced and analyzed using bioinformatics approaches. The most representative miRNAs were identified and validated by qPCR using the miRCURY LNA miRNA PCR assay. miRNome profiling identified 233 miRNAs differentially expressed in EVs across all analyzed cell lines. Among these, 94 miRNAs were detected exclusively under hypoxic conditions. From this subset, 43 miRNAs were selected for further validation by qPCR. The qPCR results showed that miR-222-3p and miR-186-5p were significantly downregulated in the Wall cell line under hypoxia (p ≤ 0.05). TargetScan and pathway enrichment analyses demonstrated that miR-186-5p regulates target genes involved in different cellular processes. In human osteosarcoma, low serum levels of miR-222-3p are associated with poor prognosis, while miR-186-5p is recognized as a key hypoxia-responsive miRNA. Collectively, these results suggest the potential of EV-associated miRNAs as biomarkers in canine OSA and support their relevance in translational and comparative oncology.

Advanced-stage sarcomas pose a major challenge in oncology, as they are often resistant to conventional therapies and associated with poor prognosis. CAR-based cellular immunotherapy is emerging as a very promising therapeutic option; however, clinically relevant animal models are urgently needed to accelerate the clinical development of these approaches. B7-H3 molecule, due to its low expression in normal tissue, high prevalence in multiple human cancers, and association with cancer stemness and aggressiveness, represents one of the most attractive targets for CAR-immunotherapy. In this study, we established a preclinical cellular immunotherapy platform based on canine cytokine-induced killer cells (CIK) redirected by an antihuman B7-H3 CAR against canine sarcoma cells and 3D sarcoma spheroids. B7-H3 was consistently detected across all analyzed canine sarcoma subtypes, including osteosarcoma, soft tissue sarcoma, and hemangiosarcoma, although with variable levels of expression intensity. We successfully generated canine B7-H3-CAR.CIK, achieving a mean CAR expression of 39

The most appropriate approach to regional/sentinel lymph nodes (LN) for staging canine oral malignant melanoma (OMM) is still controversial. This study aims to retrospectively evaluate the prognostic impact of neck dissection modality and LN metastasis in a homogeneous cohort of dogs treated by surgery and adjuvant anti-CSPG4 electrovaccination. Seventy-seven dogs were enrolled and divided into two groups based on the presence (Group A, 24 dogs) or absence (Group B, 53 dogs) of histologically confirmed LN metastasis at the time of surgery. The overall LN metastatic rate was 31%; metastasis was found mostly in the mandibular lymph center (83%). Median survival time (MST) and disease-free interval (DFI) in Group A were 406 and 134 days, respectively. Although shorter, these values were not significantly different from MST and DFI in Group B (534 and 219 days, respectively; p = 0.16 and p = 0.11). Stratifying the cases based on the type of lymphadenectomy performed, no statistical differences were observed between Groups 1 (ipsilateral lymphadenectomy) and 2 (bilateral lymphadenectomy) regarding both MST and DFI. Similarly, no significant differences in MST and DFI were observed among subgroups based on ipsilateral (Group 4) and bilateral (Group 6) removal versus ipsilateral (Group 3) and bilateral (Group 5) non-removal of even the medial retropharyngeal LN. No association was found between LN metastasis and recurrence or distant metastasis. Finally, no association was found between lymphadenectomy pattern and progressive disease. The results recorded in this study, i.e., that ipsilateral mandibular lymphadenectomy may be a reasonable surgical option in OMM, apply for this cohort of dogs only, and the translation of this principle to canine OMMs differently treated needs further investigations. Additionally, further efforts should be addressed to studies on sentinel LN identification for canine OMM staging.

Objectives:Dendritic cells (DCs) are pivotal antigen-presenting cells capable of bridging innate and adaptive immunity, making them promising candidates for cancer immunotherapy. While canine mature DCs (cmDCs) have been successfully generated from circulating mononuclear cells (CMCs) in healthy dogs, their derivation and immunomodulatory capacity in tumor-bearing dogs (TbDs) remain poorly characterized. Methods:In this study, we evaluated the efficiency of cmDC generation from peripheral blood of both healthy donors (HDs) and TbDs and investigated their functional responses to Toll-like receptor (TLR) agonists and mRNA-based genetic modification. Results:CD14+ monocytes were successfully isolated from peripheral blood using immunomagnetic sorting and differentiated into cmDCs using recombinant canine GM-CSF and IL-4. The differentiation efficiency was comparable between the two groups. In both cohorts, cmDCs upregulated key maturation markers (CD1a, CD80, CD83) and restored DLA class II expression in TbDs. Stimulation with LPS and R848 significantly increased CD80 and CD83 expression and triggered IL-12/p70 and IL-8 production, confirming the acquisition of a functional immunostimulatory phenotype. To assess their amenability to genetic engineering, cmDCs were transfected using DE-DOPE/mRNA lipoplexes. These lipoplexes exhibited favorable physicochemical properties and achieved robust mRNA delivery, resulting in 100% GFP-positive cells and >60% viability, outperforming electroporation in terms of cytocompatibility. Conclusions:Our findings demonstrate that cmDCs derived from both HDs and TbDs are phenotypically and functionally competent and can be efficiently transfected using a non-viral mRNA delivery system. This strategy offers a viable platform for the development of personalized, DC-based cancer vaccines in canine patients.

Cancer-testis antigens (CTAs) are promising targets for immuno-oncotherapy. They offer the potential to treat cancers for which effective systemic therapies are lacking, including canine malignant melanoma (CMM). In this study, we investigate the suitability of eight canine orthologs of human CTAs as targets for immunotherapy, including cancer-associated gene 1 (CAGE1), CCCTC-binding factor (CTCFL), DEAD-box helicase 53 (DDX53), the melanoma antigen gene (MAGE), 5'-nucleotidase, cytosolic IB (NT5C1B), P antigen family member 3-like (PAGE3-like), preferentially expressed antigen in melanoma (PRAME), and synovial sarcoma X chromosome breakpoint (SSX). MAGE proteins were detected by immunohistochemistry in 12.1 % (4/33) of CMM cases, including digital and oral melanoma, with healthy tissue expression restricted to the testis. CTA mRNA was detected by Real-time PCR in canine testis and validated through gel electrophoresis and Sanger sequencing. MAGE, PAGE3-like, and PRAME mRNA were strongly expressed in canine oral melanoma and metastatic cell lines with restricted expression in normal tissues. CAGE1, CTCFL, DDX53, and SSX6 were only weakly expressed or absent in canine oral melanoma. CTCFL and DDX53 expression in healthy tissues was not restricted to the testis, as moderate expression was found in the kidney. These results suggest that dogs express CTAs, similar to humans, and thus CTAs may serve as a target for immunotherapy in dogs.

Canine osteosarcoma (OSA) is an aggressive and highly malignant tumor of bone with a poor prognosis and it mirrors the disease in humans. Angiogenesis, the formation of new blood vessels, is driven by hypoxia-induced factors such as HIF-1α and VEGF, both of which play a crucial role in tumor growth and metastasis. However, the role of angiogenesis in OSA remains a topic of ongoing debate. This study aimed to investigate the relationship between angiogenesis, measured by intratumoral microvessel density (MVD), hypoxic markers, and clinical outcomes in 28 dogs diagnosed with appendicular OSA. Clinicopathological data such as age, breed distribution, tumor localization, histopathological subtypes, and metastatic behavior were consistent with reported epidemiologic characteristics of canine OSA, though no significant correlation was found among these variables. The results indicated a significant association between higher MVD and high-grade OSA (p = 0.029), suggesting that increased tumor vascularization is linked to more aggressive tumor behavior. Additionally, elevated VEGF expression was strongly correlated with disease-free interval DFI), with a p-value of 0.045. Although HIF-1α positivity showed a trend towards poorer survival, the results did not reach statistical significance (p = 0.07). These findings highlight the potential role of VEGF as a valuable prognostic marker in canine OSA, which could have potentially important implications for therapeutic targeting and clinical management of the disease. This study advances the understanding of angiogenesis in canine OSA, while emphasizing the need for continued research into the complex mechanisms regulating the interplay between hypoxia, angiogenesis and tumor progression.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

IntroductionProgrammed Death-Ligand 1 is a well-known immune checkpoint molecule. Recent studies evaluated its expression in different canine cancer types through different laboratory techniques. The present study aims to evaluate the surface membrane protein expression (mPD-L1) by means of flow cytometry (FC) in different canine lymphoma immunophenotypes. Furthermore, in a subset of cases, mRNA and plasmatic soluble protein (sPD-L1) have been assessed in the same patient, and correlations among results from the three analyses investigated.MethodsSamples obtained for diagnostic purpose from untreated dogs with a confirmed lymphoma immunophenotype were included: surface protein was assessed via FC and quantified with median fluorescence index ratio (MFI ratio), gene expression was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and plasmatic concentration of soluble protein (sPD-L1) measured with ELISA. Statistical analyses were performed to investigate any difference among FC immunophenotypes, updated Kiel cytological classes, and in the presence of blood infiltration.ResultsConsidering FC, most B-cell lymphomas (BCL) were positive, with higher MFI ratios than other subtypes (81%, median MFI ratio among positive samples = 1.50, IQR 1.21–2.03, range 1.01–3.47). Aggressive T-cell lymphomas had a lower percentage of positive samples (56%) and showed low expression (median MFI ratio in positive samples = 1.14, IQR 1.07–1.32, range 1.02–2.19), while T-zone lymphomas (TZL) were frequently positive (80%) but with low expression (median MFI ratio in positive samples = 1.19, IQR 1.03–1.46, range 1.02–6.03). Cellular transcript and sPD-L1 were detected in all samples, without differences among immunophenotypes. No correlation between results from different techniques was detected, but sPD-L1 resulted significantly increased in FC-negative lymphomas (p = 0.023).DiscussionPD-L1 molecule is involved in canine lymphoma pathogenesis, with differences among immunophenotypes detected by FC. Specifically, BCL have the highest expression and aggressive T-cell lymphomas the lowest, whereas TZL need further investigations.

B-cell lymphomas (BCL) is the most frequent hematological cancer in dogs. Treatment typically consists of chemotherapy, with CHOP-based protocols. However, outcome remains generally poor, urging the exploration of new therapeutic strategies with a targeted approach. Myc tran-scription factor plays a crucial role in regulating cellular processes and its dysregulation is im-plicated in numerous human and canine malignancies, including canine BCL (cBCL). This study aims to evaluate the efficacy of indirectly inhibiting Myc in cBCL using BI2536 and MZ1 com-pounds in two in vitro models (CLBL-1 and KLR-1201). Both BI2536 and MZ1, alone and com-bined, affected cell viability in a significant concentration and time-dependent manner. Western Blot revealed an upregulation of PLK1 expression in both cell lines upon treatment with BI2536, in association with a reduction in c-Myc protein levels. Conversely, MZ1 led to a decrease in its primary target, BRD4, along with a reduction in c-Myc. Furthermore, BI2536, both alone and in combination with MZ1, induced larger transcriptomic changes in cells compared to MZ1 alone, primarily affecting MYC target genes and genes involved in cell cycle regulation. These data un-derscore the potential role of Myc as therapeutic target in cBCL, providing a novel approach to indirectly modulate this molecule.

Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80–90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.

Supplementary Table 1 from Spontaneous Feline Mammary Carcinoma Is a Model of HER2 Overexpressing Poor Prognosis Human Breast Cancer

EDITORIAL article Front. Vet. Sci., 31 January 2023Sec. Comparative and Clinical Medicine Volume 10 - 2023 | https://doi.org/10.3389/fvets.2023.1141666

Supplementary Table S1. Breed, sex and age of canine patient population Supplementary Table S2. Age and body weight characteristics of canine patient population

Canine diffuse large B-cell lymphoma (cDLBCL) is characterized by high mortality and clinical heterogeneity. Although chemo-immunotherapy improves outcome, treatment response remains mainly unpredictable. To identify a set of immune-related genes aberrantly regulated and impacting the prognosis, we explored the immune landscape of cDLBCL by NanoString. The immune gene expression profile of 48 fully clinically characterized cDLBCLs treated with chemo-immunotherapy was analyzed with the NanoString nCounter Canine IO Panel using RNA extracted from tumor tissue paraffin blocks. A Cox proportional-hazards model was used to design a prognostic gene signature. The Cox model identified a 6-gene signature ( IL2RB, BCL6, TXK, C2, CDKN2B, ITK) strongly associated with lymphoma-specific survival, from which a risk score was calculated. Dogs were assigned to high-risk or low-risk groups according to the median score. Thirty-nine genes were differentially expressed between the 2 groups. Gene set analysis highlighted an upregulation of genes involved in complement activation, cytotoxicity, and antigen processing in low-risk dogs compared with high-risk dogs, whereas genes associated with cell cycle were downregulated in dogs with a lower risk. In line with these results, cell type profiling suggested the abundance of natural killer and CD8+ cells in low-risk dogs compared with high-risk dogs. Furthermore, the prognostic power of the risk score was validated in an independent cohort of cDLBCL. In conclusion, the 6-gene-derived risk score represents a robust biomarker in predicting the prognosis in cDLBCL. Moreover, our results suggest that enhanced tumor antigen recognition and cytotoxic activity are crucial in achieving a more effective response to chemo-immunotherapy.

Supplementary Figure S1. T cell-mediated IFN-γ response induced by hCSPG4 DNA vaccination. Supplementary Figure S2. CSPG4 specific antibody response from each vaccinated dog. Supplementary Figure S3. Effect of CSPG4 specific antibodies induced by vaccination on SK-MEL-28 cell proliferation. Supplementary Figure S4. Anti cCSPG4 specific antibody response induced by vaccination.

Supplementary Materials and Methods and Figure Legends

Osteosarcoma is the most common primary malignant bone tumour in dogs, characterized by a locally aggressive and highly metastatic behaviour. Despite the current standards of care, most dogs succumb to the disease, indicating the need for novel treatment strategies. Polo-like kinase 1 (PLK1) is dysregulated in a variety of human cancer types, including osteosarcoma, and induces c-Myc accumulation. The crosstalk between the two molecules coordinates cell proliferation, differentiation, self-renewal and apoptosis. Therefore, PLK1 has recently emerged as a potential therapeutic target, mainly in tumours overexpressing c-Myc. BI 2536 is a selective PLK1 inhibitor promoting mitotic arrest and apoptosis in a variety of cancer cells. This research aimed at evaluating PLK1 and c-Myc protein expression in 53 appendicular canine osteosarcoma (cOSA) samples and the in vitro effects of BI 2536 on a c-Myc and PLK1-overexpressing cOSA cell line (D17). PLK1 and c-Myc expression in cOSA samples showed no correlation with clinicopathological data. However, c-Myc overexpression was associated with a significantly reduced overall survival (p = .003). Western Blot and RT-qPCR assays revealed that D17 expressed high protein and transcript levels of both PLK1 and MYC. When treated with BI 2536 (range 2.5-15 nM) for 24 h, D17 showed a substantial decrease in cell growth, inducing apoptosis and G2 /M cell cycle arrest. Interestingly, under BI 2536 treatment, D17 showed decreased c-Myc protein levels. Consistent with human OSA, these preliminary data outline the prognostic value of c-Myc expression in cOSA and highlight the potential role of PLK1 as an antiproliferative therapeutic target for tumours overexpressing c-Myc.