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Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.