Osteosarcoma (OS) is the most common primary bone tumor in both humans and dogs, occurring approximately 10 times more frequently in dogs. OS is a highly aggressive neoplasm with evidence of metastasis often found at the time of diagnosis. Long-term survival in afflicted dogs continues to remain low despite aggressive therapy. This highlights the need for improved diagnostics and development of more effective therapeutics in OS. Ongoing research has been investigating mesenchymal stem cells (MSCs) and their role in the tumor microenvironment with various cancers. Mesenchymal stem cells are a multipotential nonhematopoietic progenitor with probable immunomodulatory effects, including possible suppression of anti-tumor immunity, in OS. The precise mechanism and overall effect are not defined and little information is known regarding effects in human patients. There are no current publications in canine OS regarding this topic. The objective of our study is to investigate the communication of MSCs with OS cells and how this interaction may affect the anti-tumor response (particularly that of CD8 T cells). Additionally, we will compare serum cytokine levels in canine OS patients to those in healthy canines to investigate trends in immune dysregulation due to OS. Our hypotheses are: 1) Immune dysregulation reflected in systemic cytokine alterations occurs in canine OS; 2) Peripheral blood mononuclear cell (PBMC) function including cytotoxic lymphocyte function will be downregulated by MSC-derived exosomes and will allow for increased OS proliferation.
Cytokine levels in harvested healthy and OS donors were measured by commercial 11-plex immune cytokine kit and transforming growth factor-β magnetic bead 3-plex kit. Serum levels of interleukin-8 and interleukin-12p40 in OS patients were significantly increased over those of healthy controls. Additional analytes including monocyte chemoattractant protein-1 and transforming growth factor-β1 appeared to have potential diagnostic utility. Exosomes were isolated from healthy canine adipose-derived MSCs, and exosome concentrations determined by BCA analysis. PBMCs were isolated from samples from healthy dogs using density gradient centrifugation. MSC-derived exosomes were exposed to PBMCs and caused increased proliferation of PBMCs. Exosome-treated PBMCs were co-incubated with OS cells, but no significant change in OS cell proliferation and/or apoptosis was identified. Flow cytometry was used to evaluate the effects of MSC-derived exosomes on the relative percentages of CD4 and CD8 T cell populations. No significant differences between exosome-treated PBMCs and untreated PBMCs were identified via preliminary flow cytometric analysis.
To summarize, our findings have provided initial insight into cytokine alterations in OS patients that may contribute to pathogenesis and progression of disease. Our research also demonstrates effects MSCs may have in shifting host lymphocyte populations via exosome signaling. Based on our findings, further research will evaluate the direct communication between OS cells and MSCs, with investigation of alterations in exosomes after interaction, and will continue to assess the potential impact of host immune alterations due to MSC-derived exosomes on anti-tumor immunity. For the latter goal, next steps include continued flow cytometric analysis with additional samples, and isolation of CD8 T-cells and evaluation of their functional status after incubation with MSC-derived exosomes. This planned work has potential to promote improved future diagnostics and targeted therapy for canine OS.