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High-grade osteosarcoma, a primary malignant bone tumour, is experiencing a global increase in reported incidence with varied prevalence. Despite advances in management, which include surgery and neoadjuvant chemotherapy often an unsatisfactory outcome is found due to poor or heterogeneous response to chemotherapy. Our study delved into chemotherapy responses in osteosarcoma patients and associated molecular expressions, focusing on CD95 receptor (CD95R), interferon (IFN)-γ, catalase, heat-shock protein (Hsp)70, and vascular endothelial growth factor (VEGF). Employing immunohistochemistry and Huvos grading of post-chemo specimens, we analysed formalin-fixed paraffin-embedded (FFPE) osteosarcoma tissue of resected post-chemotherapy specimens from Dr. Soetomo General Academic Hospital in Surabaya, Indonesia (DSGAH), spanning from 2016 to 2020. Results revealed varied responses (poor 40.38%, moderate 48.08%, good 11.54%) and distinct patterns in CD95R, IFN-γ, catalase, Hsp70, and VEGF expression. Significant differences among response groups were observed in CD95R and IFN-γ expression in tumour-infiltrating lymphocytes. The trend of diminishing CD95R expression from poor to good responses, accompanied by an increase in IFN-γ, implied a reduction in the count of viable osteosarcoma cells with the progression of Huvos grading. Catalase expression in osteosarcoma cells was consistently elevated in the poor response group, while Hsp70 expression was highest. VEGF expression in macrophages was significantly higher in the good response group. In conclusion, this study enhances our understanding of immune-chemotherapy interactions in osteosarcoma and identifies potential biomarkers for targeted interventions., (© 2024. The Author(s).)

Ferdiansyah Mahyudin,1 Fachrizal Arfani Prawiragara,1 Mouli Edward,1 Dwikora Novembri Utomo,1 Mohammad Hardian Basuki,1 Yunus Abdul Bari,1 Alexander Patera Nugraha,2 Fedik Abdul Rantam3 1Orthopedic and Traumatology Department, Faculty of Medicine, Dr Soetomo General Hospital, Airlangga University, Surabaya, Indonesia; 2Orthodontics Department, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia; 3Laboratory of Virology and Immunology, Microbiology Department, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, IndonesiaCorrespondence: Ferdiansyah Mahyudin Email ferdiansyah@fk.unair.ac.idIntroduction: Peripheral blood mononuclear cells (PBMCs) sensitized with mesenchymal stem cells (MSCs) secretome and/or colony stimulating factor-2 (CSF-2) as an immunotherapy candidate may escalate osteosarcoma stem cells (OS-SCs) apoptosis. This study aimed to investigate the escalation of osteosarcoma stem cells’ apoptosis after the co-cultivation with PBMCs sensitized by MSCs secretome with/or CSF-2 and it was completed by analyzing the level of serum tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) and tumor necrosis factor-α (TNF-α) level, annexin V binding, caspase-3 and caspase-8 expression in vitro.Methods: OS-SCs were derived from a single human osteosarcoma sample with its high grade and osteoblastic essential clinical characteristics obtained from a biopsy before the chemotherapy treatment. They were then isolated and cultured confirmed by the cluster of differentiation-133 (FITC) by applying immunofluorescence analysis with fluorescein isothiocyanate (FITC) labeled. MSCs secretome was obtained with cells extracted from the bone marrow of a healthy patient. Furthermore, enzyme linked immunosorbent assay (ELISA) was utilized to analyze sTRAIL and TNF-α level in each group. The expression of caspase-3, caspase-8, and annexin V assay in each group was examined by applying the immunofluorescence labeled with FITC. The comparison analysis between treatment groups and the control group was performed by utilizing the analysis of variance (ANOVA) and continued with Tukey Honest Significant Difference (HSD) (p< 0.05).Results: There was a significant difference in the upregulation of sTRAIL and TNF-α level indicated by the increased annexin V, caspase-3, and caspase-8 expression binding between groups (p< 0.05).Conclusion: MSCs Secretome and CSF-2 could significantly increase the activity of PBMCs through the improvement of sTRAIL and TNF-α levels which could lead to the escalation of OS-SCs apoptosis through an enhanced expression of caspase 3, caspase 8 and annexin V binding in vitro.Keywords: cancer, caspase, osteosarcoma, peripheral blood mononuclear cells, mesenchymal stem cells, tumor necrosis factor