ABSTRACT
INTRODUCTION: 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.
ABSTRACT
The advanced, metastasis, and reccurent of osteosarcoma (OS) patients have a poor prognosis postaggresive surgery and chemotherapy. Peripheral blood mononuclear cells (PBMCs) as cell-based immunotherapy may successful in the OS treatment. To investigate the enhancement apoptosis of OS-mesenchymal stem cells (OS-MSCs) co-cultivated with PBMCs sensitized using the secretome and granulocyte macrophage colony-stimulating factor (GMCSF). This true experimental study with posttest only control group design and in vitro study. The sample was cultured OS-MSCs which confirmed by Cluster of Differentiation-133 using immunocytochemistry (ICC) and histopathology analysis. The sample divided into six groups accordingly: OS-MSC, OS-MSC + PMBC, OS-MSC + PMBC + Secretome, OS-MSC + PMBC + GMCSF, OS-MSC + PBMC + Secretome + GMCSF (n = 5/N = 30). The enhancement of OS-MSCs apoptosis was analyzed through Interleukin-2 (IL-2) level through the Enyzme-Linked Immunosorbent Assay examination, expression of Signal Transducers and Activators of Transcription (STAT)-3 and caspase-3 by ICC. One-way analysis of variance test and Tukey Honestly Significant Difference to analyze the difference between the groups (P < 0.05). The highest of IL-2 level was found in the PBMC + Secretome + GMCSF group. The highest expression of caspase-3 was found in OS-MSC + PBMC + Secretome + GMCSF group with significant different between groups (P < 0.05). There was insignificant difference of STAT-3 epxression and IL-2 level between groups (P > 0.05). The co-cultivation of OS-MSCs and PBMSCs activated using secretome and GMCSF has a great ability to enhance OS-MSCs apoptosis.
