Spatial resolution of the metastatic osteosarcoma tumor microenvironment using immunolabeling across murine, canine and human lung.

BACKGROUND: Animal models are crucial resources for studying cancer, metastasis, and the tumor microenvironment (TME). Spatial resolution improves our ability to decipher the functions and interactions of distinct components within the TME. Traditionally, this is accomplished through mapping of protein immunolabeling within tissue sections using immunohistochemistry (IHC). Emerging techniques in this field, including multiplex imaging and spatial transcriptomics, continue to rely on IHC to facilitate cell type and tissue compartment identification. Therefore, appropriate antibody validation remains part of the foundation on which sound and reproducible scientific findings are built. This is important for studies investigating tissue-based hypotheses in murine, canine, or human tissues. METHODS: In this work, we aimed to develop a panel of antibodies to label TME components of metastatic osteosarcoma across murine, canine, and human lung. Candidate antibodies were evaluated across species based on sequence homology, immunolabeling in control samples (positive, negative, isotype), and finally, by western blot. RESULTS: Herein we present a robust panel of antibodies that label murine, canine, and human immune cells (CD20, CD204, CD3, FOXP3, Iba1), osteosarcoma tumor cells (ALPL, RUNX2, SATB2), and other components of the TME (CD31, cytokeratin, FAPα, PROX1, TTF-1, vimentin), and outline methods in which IHC can be pursued within the context of comparative oncology. CONCLUSION: The identification of antibodies that label murine, canine, and human tissues supports the investigation of cancer biology across models and patient populations, highlighting a critical strength of comparative oncology research. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-025-07367-5.