The Escalation of Osteosarcoma Stem Cells Apoptosis After the Co-Cultivation of Peripheral Blood Mononuclear Cells Sensitized with Mesenchymal Stem Cells Secretome and Colony Stimulating Factor-2 in vitro

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.

Untitled

“…There are many extravesicle products secreted by immune cells that can inhibit and stimulate the activity of wild-type-P53 so that it can secrete extravesicle molecules that play a role in inhibiting cancer cells because it acts as an extracellular messenger P53 (Pavlakis et al, 2020). As in previous studies (Mahyudin et al, 2021) in co-cultivation between activated PBMCs with secretome MSCs and OS-SCs, there was an increase in the expression of TNF-alpha, IL-6, which can stimulate apoptosis through extrinsic pathways played by macrophages, NK-cells and as well as other cells such as T-CD8 cells. In this study, we explored the role of extravesicle molecules in inhibiting tumor development through the mechanism of cell senescence, therefore through an exploratory approach to molecules in the microenvironment, which are the result of secretion of immune cells associated with cancer cells to solve problems in the case of osteosarcoma.…”

“…Co-cultivation between sensitized PBMCs and mesenchymal stem cell osteosarcoma (OS-MSCs) at a ratio of 5:1 was then cultured in a 10 cm Petri dish or flash dish, and then given 10 mL of alpha MEM medium, finally, incubated in a 5% CO2 incubator with a temperature of 37°C for 5 days (Mahyudin et al, 2021).…”

“…Finally, it was washed with medium without serum two times. After washing, it was reacted with each anti-CD4, CD8, and CD332 antibodies for 1 hour, finally analyzed using a BD-LSR II FACS Scan (Le Bert et al, 2020;Mahyudin et al, 2021).…”

“…After washing, it was reacted with human conjugate labeled with alkaline phosphatase, added PNpp, incubated in a dark room for 15 minutes, and then stopped with 1 N H2O2 or 1N HCl. Finally, the reading was carried out using an ELISA reader with a wavelength of 450 nm (Pucci et al, 2021;Mahyudin et al, 2021). Meanwhile, for validation was used the ELISA kit (Bioassay Technology Laboratory, Birmingham, UK and Elabscience, Texas, USA).…”

Activation of microvesicle peripheral blood mononuclear cells by mesenchymal stem cells secretome co-cultivated with osteosarcoma stem cell

“…Then wait for 30 minutes. Finally, the debris and osteosarcoma cells were separated by filtration (Mahyudin et al, 2021).…”