Co-cultivation of PBMCs microvesicle activated by MSCS and OS-OCs

J. Pharm. Pharmacogn. Res., vol. 10, no. 5, pp. 782-790, September-October 2022.

Original Article

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

[Activación de microvesículas de células mononucleares de sangre periférica por secretoma de células madre mesenquimales cocultivadas con células madre de osteosarcoma]

Fachrizal Arfani Prawiragara1,2, Ferdiansyah2*, Mouli Edward2, Dwikora Novembri Utomo2, Mohammad Hardian Basuki2, Alexander Patera Nugraha3, Fedik Abdul Rantam4

1Magister Clinical Medicine Program, Orthopedic and Traumatology Department, General Academic Dr. Soetomo Hospital/Faculty of Medicine, Airlangga, Surabaya, Indonesia.

2Department of Orthopaedic and Traumatology, General Academic Dr. Soetomo Hospital/Teaching Hospital, Faculty of Medicine, Airlangga University, Surabaya, Indonesia.

3Department of Orthodontics, Faculty of Dental Medicine, Airlangga University, Surabaya, Indonesia.

4Stem Cell Research Center and Development, Airlangga University, Surabaya, Indonesia.



Context: Microvesicle is a cell micro molecule that may play a role in the process of osteosarcoma stem cell apoptosis.

Aims: To investigate the activity of peripheral blood mononuclear cells (PBMCs) through the secretion of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), C-X-C Motif Chemokine Ligand 13 (CXCL13) and tissue inhibitor of metalloproteinases-3 (TIMP-3) on co-cultivation of peripheral blood mononuclear cells (PBMCs) sensitized by mesenchymal stem cell secretome (MSCS) co-cultivated with osteosarcoma stem cells (OS-SCs).

Methods: This study was true experimental with a post-test only control group design. This was in vitro study PBMSCs sensitized by MSCS as then samples were divided into 4 treatment groups, respectively: Zero-day treatment (P0) PBMCs were co-cultivated with OS-SCs for 0 hours; First treatment (P1) PBMCs were co-cultivated with OS-SCs for 1 hour; Second treatment (P2) PBMCs were co-cultivated with OS-SCs for 2 days; Third treatment (P3) PBMCs were co-cultivated with OS-SCs for 4 days. The examination method used in this study was flow cytometry and indirect enzyme-linked immunosorbent assay (ELISA). The data were statistically analyzed with analysis of variance (ANOVA) with a p≤0.05 considered a significant difference.

Results: There was a tendency for a significant increase in extravesicular secretion in the secretion of IL-2, IL-6, IL-10, CXCL13, TIMP3, in the microvesicle PBMCs when sensitized by MSCs secretome co-cultivated with OS-SCs environment co-cultivated after the fourth day with significantly different between groups (p≤0.05).

Conclusions: PBMSCs’ microvesicle such as IL-2, IL-6, IL-10, CXCL13, TIMP3 was significantly sensitized by MSCS and co-cultivated with OS-SCs after the fourth day of in vitro.

Keywords: medicine; non-communicable disease; non-infectious disease; osteosarcoma; stem cells.


Contexto: La microvesícula es una micromolécula celular que puede desempeñar un papel en el proceso de apoptosis de las células madre del osteosarcoma.

Objetivos: Investigar la actividad de las células mononucleares de sangre periférica (PBMC) a través de la secreción de interleucina-2 (IL-2), interleucina-6 (IL-6), interleucina-10 (IL-10), C-X-C Motif Chemokine Ligand 13 (CXCL13) e inhibidor tisular de metaloproteinasas-3 (TIMP-3) en el cocultivo de células mononucleares de sangre periférica (PBMC) sensibilizadas por secretoma de células madre mesenquimales (MSCS) cocultivadas con células madre de osteosarcoma (OS-SC).

Métodos: Este estudio fue verdaderamente experimental con un diseño de grupo de control solo posterior a la prueba. Este fue un estudio in vitro de PBMSC sensibilizadas por MSCS, ya que luego las muestras se dividieron en 4 grupos de tratamiento, respectivamente: Tratamiento de día cero (P0) Las PBMC se cocultivaron con OS-SC durante 0 horas; Las PBMC del primer tratamiento (P1) se cocultivaron con OS-SC durante 1 hora; Las PBMC del segundo tratamiento (P2) se cocultivaron con OS-SC durante 2 días; Las PBMC del tercer tratamiento (P3) se cocultivaron con OS-SC durante 4 días. El método de examen utilizado en este estudio fue la citometría de flujo y el ensayo inmunoabsorbente ligado a enzimas indirecto (ELISA). Los datos fueron analizados estadísticamente con análisis de varianza (ANOVA) con p≤0.05 considerado como diferencia significativa.

Resultados: Hubo una tendencia a un aumento significativo en la secreción extravesicular en la secreción de IL-2, IL-6, IL-10, CXCL13, TIMP3, en las microvesículas de PBMC cuando se sensibilizan con el secretoma de MSC cocultivado con el entorno de OS-SC. cocultivados después del cuarto día con diferencias significativas entre grupos (p≤0.05).

Conclusiones: Las microvesículas de PBMSC como IL-2, IL-6, IL-10, CXCL13, TIMP3 fueron significativamente sensibilizadas por MSCS y cocultivadas con OS-SC después del cuarto día de in vitro.

Palabras Clave: células madre; enfermedad no transmisible; enfermedad no infecciosa; medicina; osteosarcoma.

Citation Format: Prawiragara FA, Ferdiansyah, Edward M, Utomo DN, Basuki MH, Nugraha AP, Rantam FA (2022) Activation of microvesicle peripheral blood mononuclear cells by mesenchymal stem cells secretome co-cultivated with osteosarcoma stem cell. J Pharm Pharmacogn Res 10(5): 782–790.

Akdis M, Palomares O, van de Veen W, van Splunter M, Akdis CA (2012) TH17 and TH22 cells: A confusion of antimicrobial response with tissue inflammation versus protection. J Allergy Clin Immunol 129: 1438–1449.

Berraondo, P, Sanmamed, MF, Ochoa MC, ExtreberriaI, Aznar MA, Pérez-Gracia JL, Rodríguez-Ruiz ME, Ponz-Sarvise M, Castañón E and Melero I (2019) Cytokines in clinical cancer immunotherapy. Br J Cancer 120(1): 6–15.

Bian J, Wang Y, Smith MR, Kim H, Jacobs C, Jackman J, Kung HF, Colburn NH, Sun Y (1996) Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3. Carcinogenesis 17: 1805–1811.

Coppé JP, Patil CK, Rodier F, Sun Y, Mun DP, Golstein J, Nelson PS, Desprez PY, Campisi J (2008) Senescence-associated secretory phenotypes reveal cell nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301.

Crotty S (2011) Follicular helper CD4 T cells (TFH). Annu Rev Immunol 29: 621–663.

Cuddihy AR, Farid J, Coackley C, Bristow RG (2008) WTp53 induction does not override MTp53 chemoresistance and radioresistance due to gain-of-function in lung cancer cells. Mol Cancer Ther 7(4): 980–992.

Darnton SJ, Hardie LJ, Muc RS, Wild CP, Casson AG (2005) Tissue inhibitor of metalloproteinase-3 (TIMP-3) gene is methylated in the development of esophageal adenocarcinoma: loss of expression correlates with poor prognosis. Int J Cancer 115: 351358.

Dwi Wibowo RM, Perdanakusuma DS, Tanggo EH (2017) Mechanism of apoptosis inhibition to squamous cell carcinoma of oral cancer in cisplatin treatment. Folia Med Indonesia 55(1): 1–6.

Han XG, Mo HM, Liu XQ, Li Y, Du L, Qiao H, Fan QM, Zhao J, Zhang SH, Tang TT (2018) TIMP3 overexpression improves the sensitivity of osteosarcoma to cisplatin by reducing IL-6 production. Front Genet 9: 135.

Hirahara K, Poholek A, Vahedi G, Laurence A, Kanno Y, Milner JD, O’Shea (2013) Mechanisms underlying helper T-cell plasticity: Implications for immune-mediated disease. J Allergy Clin Immumnol 131(5): 1276-1287

Hitomi K, Okada R, Loo TM, Miyata K, Nakamura AJ, Takahashi A (2020) DNA damage regulates senescence-associated extracellular vesicle release via the ceramide pathway to prevent excessive inflammatory responses. Int J Mol Sci 21: 3720.

Kalluri R (2016) The biology and function of fibroblasts in cancer. Nature Rev 16: 582–598.

Kazanietz MG, Durando M, Cooke M (2019) CXCL13 and its receptor CXCR5 in cancer: Inflammation, immune response, and beyond. Front Endocrinol (Lausanne) 10: 471.

Le Bert N, Tan AT, Kunasegaran K, Tham CYL, Hafezi M, Chia A, Chng MHY, Lin M, Tan N, Linster M, Chia WN, Chen MI, Wang LF, Ooi EE, Kalimuddin S, Tambyah PA, Low JG, Tan YJ, Bertoletti A (2020) SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584(7821): 457–462.

Legler DF, Loetscler M, Ross RS, Clark-Lewis I, Baggiolini M, Moser B (1998) B Cell–attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5. J Exp Med 187(4): 655–660.

Mahyudin F, Prawiragara FA, Edward M, Utomo DN, Basuki MH, Bari YA, Nugraha AP, Rantam FA (2021) 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. J Blood Med 12: 601–611.

Mahyudin F, Yazid H, Edward M, Basuki MH, Bari YA, Rantam FA (2020) The enhancement apoptosis of osteosarcoma mesenchymal stem cells co-cultivation with peripheral blood mononuclear cells sensitized by secretome and granulocyte macrophage colony-stimulating factor. J Adv Pharm Res 11(4): 213–219.

Mastutik G, Rahniayu A, Kurniasari N, Rahaju AS, Alia R, Mustokoweni S (2021) The expression of E6 HPV, P53 and P16ink4a at well, moderately, and poorly differentiated cervical adenocarcinoma. Folia Med Indonesia 55(4): 295–300.

Monleón I, Martínez-Lorenzo MJ, Monteagudo L, Lasierra P, Taulés M, Iturralde M, Piñeiro A, Larrad L, Alava MA, Naval J, Anel A (2001) Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells. J Immunol 167: 6736–6744.

Oft M (2014) IL-10: Master switch from tumor-promoting inflammation to anti-tumor immunity. Cancer Immunol Res 2(3): 194-199.

Pavlakis E, Neuman M, Stiewe T (2020) Extracellular vesicles: Messengers of p53 in tumor–stroma communication and cancer metastasis. Int J Mol Sci 21: 9648.

Pavlakis E, Stiewe T (2020) p53’s extended reach: The mutant p53 secretome. Biomolecules 10(2): 307.

Pucci M, Raimondo S, Urzi O, Moschetti M, Bella MAD, Conigliaro A, Caccamo N, LaManna MP, Fontana S, Allessandro B (2021) Tumor-derived small extracellular vesicles induce pro-Inflammatory cytokine expression and PD-L1 regulation in M0 macrophages via IL-6/STAT3 and TLR4 signaling pathways. Int J Mol Sci 22: 12118.

Rallis K, Corrigan AE, Dadah H, George AM, Keshwara SM, Sideris M, Sabados (2021) Cytokine-based cancer immunotherapy: Challenges and opportunities for IL-10. Anticancer Res 41: 3247–3252.

Rantam FA, Setiawan B, Wibisono S (2015) Induced monocytes-derived HSCs (CD34 + ) with LPS accelerated homing rat bone marrow-mesenchymal stem cell (BM-MSCs, CD105) in injured pancreas. J Biomed Sci Eng 8: 333–344.

Sahai E, Astsaturov I, Cukierman E, DeNardo DG, Egeblad M, Evans RM, Fearon D, Greten FR, Hingorani SR, Hunter T, Hynes RO, Jain RK, Janowitz T, Jorgensen C, Kimmelman AC, Kolonin MG, Maki RG, Powers RS, Puré E, Ramirez DC, Scherz-Shouval R, Sherman MH, Stewart S, Tlsty TD, Tuveson DA, Watt FM, Weaver V, Weeraratna AT, Werb Z (2020) A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer 20: 174–186.

Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133: 775–787.

Schuldner M, Dörsam B, Shatnyeva O, Reiners KS, Kubarenko A, Hansen HP, Finkernagel F, Roth K, Theurich S, Nist A, Stiewe T, Paschen A, Knittel G, Reinhardt HC, Müller R, Hallek M, von Strandmann EP (2019) Exosome-dependent immune surveillance at the metastatic niche requires BAG6 and CBP/p300-dependent acetylation of p53. Theranostics 9: 6047–6062.

Su C-W, LinC_W, Yang W-E, Yang S-F (2019) TIMP-3 as a therapeutic target for cancer. Ther Adv Med Oncol 11: 1758835919864247.

Tan C, Gery I (2012) The unique features of Th9 cells and their products. Crit Rev Immunol 32: 1–10.

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