Osteosarcoma (OSA) is a primary bone tumour occurring in children but is also prevalent in large breed dogs. Canine OSA (cOSA) has long been viewed as analogous to human OSA (hOSA) with cOSA serving as a surrogate for development of therapeutic approaches to treat the rarer human form. Drug therapy of OSA has remained virtually unchanged over the last four decades and drug testing is challenging due to the genomic heterogeneity of OSA as well as the limited number of patients for clinical trials. Thus, an approach that includes a suitable clinical surrogate at the early stage of therapeutic development may be beneficial. Therefore, to address these challenges, a phenotypic drug screen of 12 targeted anticancer drugs was carried out using 13 cOSA and 6 hOSA cell lines and responses compared at clinically relevant exposures (CRE) estimated from human data. The results identified four drugs (alisertib, crizotinib, onvansertib and sorafenib) with significant responses at the CRE in cOSA and hOSA cell lines and demonstrated that drug responses were indistinguishable across species. Correlations of drug response with genomic biomarkers in the cOSA cell line panel identified Myc and Hedgehog signalling as potential predictors of crizotinib response and Myc, epithelial markers and anti-apoptotic signalling for onvansertib response. The conclusions of these findings are that cOSA and hOSA cell lines show the same range of response to targeted agents and identify potential biomarker pathways for further investigation in OSA tumours for use in future comparative oncology studies including clinical trials in pet dogs.

Tumor-associated macrophages (TAMs) drive cancer progression and metastasis. However, the mechanisms by which tumor cells shape TAM phenotypes in canine cancers remain poorly understood. We investigated correlations between cancer cell gene expression and macrophage polarization to identify potential biomarkers and therapeutic targets. Tumor-conditioned media from 25 canine cancer cell lines were applied to monocyte-derived macrophages from three canine donors for 24 hours. Following washout, supernatants were analyzed for immunomodulatory cytokines and chemokines. Each cell line's polarization capacity was ranked using modified z-scores, then correlated with RNA-sequencing data through Spearman's correlation and differential expression analysis. Cancer cell lines showed marked heterogeneity in macrophage polarization capacity, largely independent of histologic type. MVB12A, a gene involved in exosome biogenesis, strongly correlated with vascular endothelial growth factor (VEGF) stimulation, suggesting exosome-mediated polarization mechanisms. Exosome fractionation experiments confirmed that purified exosomes induced significantly more macrophage VEGF secretion than other conditions, and high-MVB12A cell lines showed greater VEGF enrichment in exosomes. C-C motif chemokine ligand 3 (CCL3) was strongly correlated with tumor necrosis factor-alpha (TNF-α) secretion exclusively in histiocytic sarcoma cells, and recombinant CCL3 induced dose-dependent TNF-α secretion from macrophages. High-polarizing cell lines exhibited upregulation of macrophage activation, epithelial-to-mesenchymal transition (EMT), and metabolic reprogramming genes, and downregulation of immune surveillance and cell adhesion genes. Gene set enrichment analysis confirmed pathways for immune suppression, EMT, and extracellular matrix remodeling. These findings identify exosome-associated VEGF stimulation as a previously uncharacterized mechanism in canine tumors and highlight CCL3 as a potential histiocytic sarcoma-specific driver of macrophage TNF-α secretion. Further validation in canine clinical cohorts will determine whether these pathways can serve as biomarkers or therapeutic targets in veterinary oncology.

There is poorly understood variability both between and within cancer types in their ability to maintain an immunosuppressive microenvironment and polarize tumor-associated macrophages (TAMs). Identifying genetic biomarkers of cancers that strongly polarize TAMs would enable the discovery of novel therapeutic targets and support the stratification of patients for appropriate immunotherapy. Our aim, therefore, was to correlate gene expression data from cancer cell lines with phenotypic changes in canine macrophages. We harvested standardized tumor-conditioned medium (TCM) from twenty-five canine cancer cell lines with bulk RNA-sequencing data available. Primary monocyte-derived macrophages were obtained from three canine donors and polarized with the twenty-five different TCMs. After a washout period, supernatants were collected, and polarization changes of the macrophages were assessed via secretory products using ELISA and a multiplex cytokine/chemokine assay. For each analyte measured, a modified z-score was used to rank each cell line’s ability to stimulate the secretion of that analyte from macrophages. Multiple regression analysis and pairwise comparison analysis (composed of “strong” and “weak” stimulators) were performed against the full transcriptome. Many significantly differentially expressed genes were identified in the cancer cell lines with the strongest ability to polarize macrophages. For example, cell lines with MVB12A upregulated, a gene involved in selecting cargo for exosomes, had the highest ability to stimulate VEGF production from macrophages. This was subsequently confirmed by depleting the TCM of exosomes which resulted in a significant decrease in VEGF production. Multiple other genes involved in macrophage immunosuppression were upregulated, while those involved in cell adhesion and metastasis prevention were downregulated. In conclusion, canine cancer cell lines have varying abilities to polarize primary macrophages, irrespective of histologic type, as measured by secretory products. The cell lines capable of potent polarization had relevant differentially expressed genes and pathways. These are currently being explored to identify novel biomarkers and therapeutic targets. Rachel V. Brady, Kristen B. Farrell, Eric P. Palmer, Sunetra Das, Dawn L. Duval, Douglas H. Thamm. Genomic determinants of canine tumor-associated macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3979.

Thyroid tumors represent 1-4% of cancers in both dogs and humans. Most canine tumors are follicular (FTC) or medullary carcinomas (MTC), unlike humans, where only 10-15% are FTC and 2% are MTC, with BRAF/NRAS or RET mutations, respectively. Here, we conduct histological and molecular analyses of canine thyroid tumors. Transcriptionally, elevated ERBB2 expression characterizes FTC tumors, whereas MTC tumors show upregulated RET signaling. Elevated HER2 protein-staining and larger tumor size associate with shorter progression-free survival. Recurrent mutations are rarely observed with potential driver variants in MEN1 (10%), KRAS (7%), and TSHR (3%), among others. Notably, mutations in DNA repair pathway genes are the most consistently shared across tumors, occurring in 60% of cases. Thus, the genomic profile of canine FTC differs significantly from that of humans, with limited reliance on RAS/RAF signaling for oncogenic progression. Conversely, RET signaling likely underlies tumorigenesis in both canine and human MTC.

A 7-year-old female spayed Bernese Mountain dog was presented for evaluation of hematuria. Incidentally, a right stifle sarcoma was diagnosed via cytology, which raised concern for histiocytic sarcoma (given the patient's signalment) versus another joint-associated sarcoma. Histopathology and immunohistochemistry revealed a CD18-negative, non-histiocytic origin cell population. Findings were consistent with a joint-associated grade II soft tissue sarcoma (STS). The patient's hematuria was progressive over 5 months, and urinary bladder transitional cell carcinoma (TCC) was diagnosed via cystoscopy and histopathology. An enlarged right medial iliac lymph node was identified on routine restaging via abdominal ultrasound 3 months later. Cytology of the lymph node revealed a markedly pleomorphic cell population, again raising concern for histiocytic sarcoma (HS). Other differentials included an anaplastic metastatic population from the joint-associated STS or the TCC. Immunocytochemistry revealed a cytokeratin-positive, CD18-, CD204-, and vimentin-negative cell population, consistent with a carcinoma. DNA was extracted from cytology slides to sequence cells for BRAF mutation status. Sequencing revealed a homozygous V596E (transcript ENSCAFT00845055173.1) BRAF mutation, consistent with the known biology of TCC. In neither case was HS truly present in this patient, but immunocytochemistry provided information that helped to optimize the patient's chemotherapy recommendations.

AbstractCanine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features. Histologically, canine STS sub-types are difficult to discern at the light microscopic level due to their overlapping features. Thus, genomic, and transcriptomic profiling of canine STS may prove valuable in differentiating the diverse sub-types of mesenchymal neoplasms within this group. To this purpose we sought to characterize the transcript expression and genomic mutation profiles of canine STS. To delineate transcriptomic sub-types, hierarchical clustering was used to identify 4 groups with district expression profiles. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. Sensitivity to imatinib was evaluated in a canine STS cell line also bearing aPDGFBfusion. Using whole exome sequencing, recurrent driver variants were identified in the cancer genesKMT2D(21% of the samples) andTP53(21%) along with copy number losses of RB1 and CDKN2A. Gene amplifications and resulting transcript increases were identified in genes on chromosomes 13, 14, and 36. A subset of STS was identified with high T-cell infiltration. This multi-omics approach has defined canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for therapy.

Supplementary Table S1A. Metadata associated with canine cancer cell line panel. Supplementary Table S1B. Metadata associated with normal samples. Supplementary Table S2. Certificate of Analysis - Cell Line Validation. Supplementary Table S5. List of protein coding somatic variants identified in 33 canine cancer cell lines. Supplementary Table S7. Functional clustering of genes into selected categories.

Supplementary Fig. S1. Pipeline for post-processing of somatic variants. Supplementary Fig. S2. Distribution of genes and variants. Supplementary Fig. S3. MDM2 and TP53 transcript levels. Supplementary Fig. S4. KEGG MAPK pathway. Supplementary Fig. S5. KEGG PI3K-AKT pathway. Supplementary Fig. S6 Western blot analyses for ERK/pERK and AKT/pAKT.

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 μM and 2.1-5.1 μM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.

Section 1: Supplementary Materials and Methods Section 2: Supplementary Tables Supplementary Table S3. Tools and databases used in this study. Supplementary Table S4. Primers used for validating variants. Supplementary Table S6. Putative driver gene mutations. Supplementary Table S8. Mutated MAPK genes mapped to KEGG pathway (cfa:04010). Supplementary Table S9. Mutated PI3K-AKT genes mapped to KEGG pathway (cfa:04151).

Soft tissue sarcomas (STS) are a heterogenous group of mesenchymal tumors representing over 50 distinct types with overlapping histological features and non-specific anatomical locations. Currently, localized sarcomas are treated with surgery + / − radiation in both humans and dogs with few molecularly targeted therapeutic options. However, to improve precision-based cancer therapy through trials in pet dogs with naturally occurring STS tumors, knowledge of genomic profiling and molecular drivers in both species is essential. To this purpose, we sought to characterize the transcriptomic and genomic mutation profiles of canine STS subtypes (fibrosarcoma, undifferentiated pleomorphic sarcoma, and peripheral nerve sheath tumors), by leveraging RNAseq, whole exome sequencing, immunohistochemistry, and drug assays. The most common driver mutations were in cell cycle/DNA repair (31%, TP53 -21%) and chromatin organization/binding (41%, KMT2D -21%) genes. Similar to a subset of human sarcomas, we identified fusion transcripts of platelet derived growth factor B and collagen genes that predict sensitivity to PDGFR inhibitors. Transcriptomic profiling grouped these canine STS tumors into 4 clusters, one PNST group (H1), and 3 FSA groups selectively enriched for extracellular matrix interactions and PDFGB fusions (H2), homeobox transcription factors (H3), and elevated T-cell infiltration (H4). This multi-omics approach provides insights into canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for chemo- and immuno-therapy.

Pet dogs develop spontaneous cancers at a rate estimated to be five times higher than that of humans, providing a unique opportunity to study disease biology and evaluate novel therapeutic strategies in a model system that possesses an intact immune system and mirrors key aspects of human cancer biology. Despite decades of interest, effective utilization of pet dog cancers has been hindered by a limited repertoire of necessary cellular and molecular reagents for both in vitro and in vivo studies, as well as a dearth of information regarding the genomic landscape of these cancers. Recently, many of these critical gaps have been addressed through the generation of a highly annotated canine reference genome, the creation of several tools necessary for multi-omic analysis of canine tumours, and the development of a centralized repository for key genomic and associated clinical information from canine cancer patients, the Integrated Canine Data Commons. Together, these advances have catalysed multidisciplinary efforts designed to integrate the study of pet dog cancers more effectively into the translational continuum, with the ultimate goal of improving human outcomes. The current review summarizes this recent progress and provides a guide to resources and tools available for comparative study of pet dog cancers.

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features including hemangiosarcoma, lymphangiosarcoma, histiocytic sarcoma, synovial sarcoma, leiomyosarcoma, and rhabdomyosarcoma. Histologically, canine STS sub‐types are difficult to discern at the light microscopic level due to their overlapping microscopic features. Thus, genomic, and transcriptomic profiling of canine STS may prove more valuable in differentiating the diverse sub‐types of mesenchymal neoplasms within this grouping. To this purpose we sought to characterize the mutation and expression profiles of canine STS. In this study we used whole exome capture (Agilent Sure Select Canine V2) to sequence 29 tumors, grade I‐III, from non‐visceral locations, and matched normal samples, along with RNAseq of tumor samples. For the WES data, 40‐170 million 150 bp paired‐end reads were obtained from Illumina sequencing which were mapped against CanFam3.1 genome via BWA and short variants were called and annotated Mutect2 and VEP tools, respectively. The median depth of sequencing for normals and tumors were 97X (range: 35X–128X) and 110X (range: 22X–133X), respectively. Additionally, RNAseq reads were sequenced from 29 tumors and mapped against CanFam3.1 genome via STAR and gene count data was generated by HTSeq‐count tool. The total number of somatic variants identified across 29 samples ranged from 275 to 4,196, of which, 6% to 31% were annotated as cDNA variants. The protein coding mutations per callable megabase ranged from 0.3 to 37.3. Known cancer genes (COSMIC database) with recurrent mutations were KMT2D(21% of the samples), TP53 (21%), CNTNAP2(14%) genes. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. These gene fusions were confirmed through Sanger sequencing of amplified cDNA. To delineate the transcriptomic sub‐types, we used hierarchical and K‐means clustering methods. This resulted in 4 groups with similar expression profiles comprising of 11, 4, 8 and 6 samples in each. Further, genes set variation analysis (GSVA) was used to profile the distinct pathways across all 4 clusters. Preliminary data indicates that the 11‐sample and 6‐sample clusters had up‐regulated non‐homologous end joining pathway and immune response pathways, respectively. Further study of genes and pathways that are enriched in sample clusters will help in categorizing the canine STS tumors for diagnosis and treatment.

Activating BRAF mutations are drivers of oncogenesis in several human cancers and in over 70% of canine urothelial carcinomas (cUC). Selective BRAF inhibitors are effective alone and in combination with MEK inhibitors in approximately half of BRAF mutant melanoma cases, but the majority of these patients ultimately develop resistance. Thus, intrinsic and acquired resistance to MAP kinase pathway targeted agents remains a challenge requiring the development of novel therapeutic strategies. Using canine urothelial carcinoma as a spontaneous cancer model to explore treatment of BRAF mutant cancers and acquired drug resistance, we sought to identify determinants of MEK1/2 inhibitor sensitivity and resistance. We applied the human‐derived MAPK Pathway Activity Score (MPAS), a predictor of sensitivity to MEK inhibition in human cancers based on expression levels of 10 downstream targets, to a panel of 32 canine cancer cell lines and found that MPAS correlates with trametinib sensitivity in this canine dataset. MPAS genes with significant (p<0.05) Pearson correlations to sensitivity were CCND1, DUSP6, ETV4, ETV5, and SPRY2. We also identified 30 non‐MPAS genes whose expression levels are significantly correlated with trametinib sensitivity, suggesting their role in de novo sensitivity to trametinib. Each of the 5 urothelial carcinoma lines had high MPAS scores and trametinib IC50 values <10nM. CRISPR‐Cas9 knockouts in two canine urothelial carcinoma lines also identified MEK1 as a critical dependency. To investigate mechanisms of acquired resistance, we generated trametinib‐resistant (TramR) clonal derivatives of the BRAF mutant Tyler1 canine urothelial carcinoma cell line (Tyler1‐TramR), with trametinib IC50 values greater than 500 nM versus 1 nM in parental Tyler1. Tyler1‐TramR cell lines exhibited sustained inhibition of ERK1/2 phosphorylation when treated with trametinib and were also resistant to pharmacologic inhibition of ERK1/2. Collectively, these data suggest an ERK‐independent mechanism of resistance. Gene expression analysis of two Tyler1‐TramR clones identified dramatic downregulation of epithelial markers accompanied by an increase in expression of mesenchymal genes and transcription factors that regulate the epithelial‐to‐mesenchymal transition (EMT). One Tyler1‐TramR clone exhibited metabolic alterations including decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and reduced glycolytic capacity. Overall, the findings in this study suggest that the mechanisms of acquired and intrinsic MEK inhibitor resistance are conserved between man and dog, further elucidating the translational value of cUC as a model to optimize therapies for Ras/Raf/MAP kinase driven cancers.

Background: Thyroid tumors represent 1–3% of canine cancers with most tumors classified as follicular carcinomas. Medullary carcinomas arising from c-cells are less frequently diagnosed in both dogs and humans. In comparison, papillary thyroid carcinomas are the most common type of human thyroid cancer (70–80%) with follicular thyroid cancer diagnosed in 10–15% of cases. Medullary carcinomas are 2% of human cases and 20% of these are associated with inherited gene mutations in RET. Human papillary and follicular carcinomas frequently bear activating mutations in BRAF and NRAS. Materials and Methods: To determine if canine and human thyroid tumors share molecular characteristics, we conducted whole exome (WES) and RNA sequence analysis of canine thyroid tumors. We used whole exome capture (Agilent Sure Select V2) to sequence 27 thyroid tumors and matched normal genomic samples and ribosomal RNA depleted total RNA was sequenced from 30 tumors. The tumors were histologically typed as solid, follicular compact, or follicular, diagnosed as carcinomas or adenocarcinomas, and contained >70% tumor tissue. Results: For the WES, 150 bp paired end reads (Illumina) were mapped against CanFam3.1 using BWA and short variants were called and annotated with Mutect2 and VEP tools. Average depth of sequencing was 212 ± 27. RNAseq reads were mapped against CanFam3.1 with STAR and normalized counts were generated with DESeq2. Protein coding somatic variants per tumor ranged from 17 to 346 (total 2181). Known cancer genes with somatic mutations in 2 or more tumors were SALL4, HSP90AA1, MEN1, SFPQ, LEPROTL1, CDK4, RAD17, MGAM, NOTCH4, and RANBP2. No somatic variants were identified in BRAF or NRAS, although individual variants of unknown impact were identified in KRAS, ARAF, and RASA1. Individual tumors also had variants in TSHR and THRAP3. Hierarchical clustering of gene expression data separated the tumors into 2 groups: C1 and C2. Differential gene expression analysis between these groups identified high expression (>1000-fold change) of calcitonin transcripts in C1, suggesting that the C1 cluster is comprised of medullary thyroid carcinomas. Notable among the upregulated genes in C1 relative to C2 were: FOXA1, SCG5, RET, ERBB4, NTRK1, WNK2, and MUC1. Upregulated genes in C2 included: FGFR2, MECOM, PAX8, ERBB2, GRM3, AR, SMO, IGF2BP2, and SOCS1. Pre-ranked gene set enrichment analysis (GSEA) identified the “Hallmark KRAS signaling UP” and “GOBP Regulation of Membrane Potential” pathways enriched in C1, while enrichment for C2 was limited, with “GOBP thyroid hormone generation” being the most significant. Conclusions: These data suggest that medullary thyroid carcinomas in the dog, like their human counterparts, may be driven by RET signaling. In contrast, follicular tumors in dogs show limited reliance on RAS/RAF signaling for oncogenic progression. No conflict of interest.