Canine Hemangiosarcoma Cell Lines In Vitro Platform Captures Morphological, Functional, and Molecular Diversity

Both canine hemangiosarcoma and human angiosarcoma are aggressive malignancies characterized by clinical and molecular heterogeneity. Current in vitro models rely on individual cell lines, failing to capture this biological complexity. We established a standardized 2D in vitro platform comprising a panel of three canine primary splenic hemangiosarcoma cell lines (EMN009, EMN011, EMN017) and one patient-matched brain metastasis (EMN010) evaluated in parallel. Using gold-standard assays—morphometrics, growth, migration and tube-formation assays—alongside disease-relevant transcriptomic and microRNA profiling, we characterized this multi-line system under harmonized conditions. Neoplastic lines shared a robust core angiogenic signature of 42 overexpressed genes linked to VEGF, AGE-RAGE, and S1P signaling, and a 47-gene cancer stem cell engine enriched in the Hippo/YAP1 pathway. Transcriptomic clustering divided the lines into slower-growing primary tumors (EMN009/EMN011) and highly angiogenic phenotypes (EMN017/EMN010). Isogenic comparison revealed that the metastatic EMN010 uniquely upregulates STAT signaling and peptidyl-tyrosine phosphorylation. MicroRNA profiling demonstrated a global downregulation of tumor-suppressors miR-126-5p and miR-452-5p, unleashing VEGFA-driven angiogenesis, while visceral hemangiosarcoma biomarker miR-214-3p was significantly upregulated. Conversely, miR-10b and miR-133-3p expression varied across lines, reflecting ongoing clonal evolution. This integrated platform successfully recapitulates hemangiosarcoma heterogeneity, providing a reliable, standardized framework for comparative oncology and targeted pathophysiological studies.