Pam3CSK4, a TLR2 ligand, induces differentiation of glioblastoma stem cells and confers susceptibility to temozolomide
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Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and GBM stem cells (GSC) may be responsible for its recurrence and therapeutic resistance. Toll-like receptors (TLRs), which recognize multiple ligands (endogenous and pathogen-associated) and trigger the immune response of mature immune cells, are also expressed by hematopoietic stem and progenitor cells, where their activation results in the differentiation of these cells into myeloid cells. Since TLR expression has been recently described in neural cells, including neural stem cells, we studied TLR expression by GSCs and the effect of stimulation by TLR ligands on promoting GSC differentiation into mature GBM cells. First, our results showed heterogeneous TLR expression by GBM cells from human tumors and, for the first time, by human GSCs defined by their CD133+ and CD44+ phenotypes. Next, the effect of TLR ligands was studied in in vitro cell cultures of neurospheres and CD44+ cells obtained from two GBM cell lines (U-87 and U-118). The expression of GSC markers diminished in the presence of Pam3CSK4 or LPS (TLR2 and TLR4 ligands, respectively), thus indicating TLR-dependent differentiation. Interestingly, simultaneous treatment with Pam3CSK4 plus temozolomide (TMZ), the reference drug in GBM treatment, significantly increased cell death compared to the effect of the ligand alone, which showed no toxicity, or TMZ alone. These results suggest a synergistic effect between Pam3CSK4 and TMZ based on the induction of TLR-dependent GSC differentiation towards mature GBM cells, which exhibited increased sensitivity to chemotherapy, and provide new perspectives in GBM therapy.
KeywordsPam3CSK4 Glioblastoma stem cells Toll-like receptors TLR2 Stem cell differentiation
This work was supported by the following grants: VLC-Bioclínic-TLR-GBMM-GIL-CERDÁ-2015 from INCLIVA and the University of Valencia, FIS-P114/01669 from the Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) and PROMETEOII/2015/007 from the Generalitat Valenciana. We thank Ms. Ana Clari and Guadalupe Herrera for their technical assistance. The funding sources were not involved in the research and/or preparation of the article.
The present work was supported by the following grants: VLC-Bioclínic TLR-GBM-GIL-CERDÁ-2015 from INCLIVA and the University of Valencia, FIS-P114/01669 from the Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) and PROMETEOII/2015/007 from the Generalitat Valenciana. The funding sources were not involved in the research and/or preparation of the article.
Compliance with ethical standards
Conflict of interest
Javier Megías declares that he has no conflicts of interest. Alba Martínez declares that she has no conflicts of interest. Teresa San-Miguel declares that she has no conflicts of interest. Rosario Gil-Benso declares that she has no conflicts of interest. Lisandra Muñoz-Hidalgo declares that she has no conflicts of interest. David Albert-Bellver declares that he has no conflicts of interest. Amara Carratalá declares that she has no conflicts of interest. Daniel Gozalbo declares that he has no conflicts of interest. Concha López-Ginés declares that she has no conflicts of interest. María Luisa Gil declares that she has no conflicts of interest. Miguel Cerdá-Nicolás declares that he has no conflicts of interest.
The study was reviewed and approved by the Institutional Ethics Committee of the University of Valencia and Clinic Hospital of Valencia. All procedures performed in this study were in accordance with the 1964 Declaration of Helsinki.
Informed consent was obtained from all individual participants included in the study.
- 8.Safari M, Khoshnevisan A (2015) Cancer stem cells and Chemoresistance in glioblastoma multiform: a review article. J Stem Cells 10:271–285Google Scholar
- 10.Brown DV, Daniel PM, D'Abaco GM, Gogos A, Ng W, Morokoff AP, Mantamadiotis T (2015) Coexpression analysis of CD133 and CD44 identifies proneural and mesenchymal subtypes of glioblastoma multiforme. Oncotarget 20:6267–6280Google Scholar
- 12.Anido J, Sáez-Borderías A, Gonzàlez-Juncà A, Rodón L, Folch G, Carmona MA, Prieto-Sánchez RM, Barba I, Martínez-Sáez E, Prudkin L, Cuartas I, Raventós C, Martínez-Ricarte F, Poca MA, García-Dorado D, Lahn MM, Yingling JM, Rodón J, Sahuquillo J, Baselga J, Seoane J (2010) TGF-β receptor inhibitors target the CD44(high)/Id1(high) glioma-initiating cell population in human glioblastoma. Cancer Cell 18:655–668CrossRefGoogle Scholar
- 15.Bradshaw A, Wickremsekera A, Tan ST, Peng L, Davis PF, Itinteang T (2016) Cancer stem cell hierarchy in glioblastoma Multiforme. Front Surg 15:21Google Scholar
- 16.Wei KC, Huang CY, Chen PY, Feng LY, Wu TW, Chen SM, Tsai HC, Lu YJ, Tsang NM, Tseng CK, Pai PC, Shin JW (2010) Evaluation of the prognostic value of CD44 in glioblastoma multiforme. Anticancer Res 30:253–259Google Scholar
- 17.Denysenko T, Gennero L, Roos MA, Melcarne A, Juenemann C, Faccani G, Morra I, Cavallo G, Reguzzi S, Pescarmona G, Ponzetto A (2010) Glioblastoma cancer stem cells: heterogeneity, microenvironment and related therapeutic strategies. Cell Biochem Funct 28:343–351. https://doi.org/10.1002/cbf.1666 CrossRefGoogle Scholar
- 28.Alvarado AG, Thiagarajan PS, Mulkearns-Hubert EE, Silver DJ, Hale JS, Alban TJ, Turaga SM, Jarrar A, Reizes O, Longworth MS, Vogelbaum MA, Lathia JD (2017) Glioblastoma Cancer stem cells evade innate immune suppression of self-renewal through reduced TLR4 expression. Cell Stem Cell 20:450–461.e4. https://doi.org/10.1016/j.stem.2016.12.001 CrossRefGoogle Scholar
- 33.Pellegatta S, Poliani PL, Corno D, Menghi F, Ghielmetti F, Suarez-Merino B, Caldera V, Nava S, Ravanini M, Facchetti F, Bruzzone MG, Finocchiaro G (2006) Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas. Cancer Res 66:10247–10252CrossRefGoogle Scholar
- 36.Binder ZA, Wilson KM, Salmasi V, Orr BA, Eberhart CG, Siu IM, Lim M, Weingart JD, Quinones-Hinojosa A, Bettegowda C, Kassam AB, Olivi A, Brem H, Riggins GJ, Gallia GL (2016) Establishment and biological characterization of a panel of glioblastoma Multiforme (GBM) and GBM variant Oncosphere cell lines. PLoS One 11:e0150271. https://doi.org/10.1371/journal.pone.0150271 CrossRefGoogle Scholar
- 43.Campos B, Wan F, Farhadi M, Ernst A, Zeppernick F, Tagscherer KE, Ahmadi R, Lohr J, Dictus C, Gdynia G, Combs SE, Goidts V, Helmke BM, Eckstein V, Roth W, Beckhove P, Lichter P, Unterberg A, Radlwimmer B, Herold-Mende C (2010) Differentiation therapy exerts antitumor effects on stem-like glioma cells. Clin Cancer Res 15:2715–2728. https://doi.org/10.1158/1078-0432.CCR-09-1800 CrossRefGoogle Scholar
- 44.Dong Y, Han Q, Zou Y, Deng Z, Lu X, Wang X, Zhang W, Jin H, Su J, Jiang T, Ren H (2012) Long-term exposure to imatinib reduced cancer stem cell ability through induction of cell differentiation via activation of MAPK signaling in glioblastoma cells. Mol Cell Biochem 370:89–102. https://doi.org/10.1007/s11010-012-1401-0 CrossRefGoogle Scholar
- 45.Daniele S, Costa B, Zappelli E, Da Pozzo E, Sestito S, Nesi G, Campiglia P, Marinelli L, Novellino E, Rapposelli S, Martini C (2015) Combined inhibition of AKT/mTOR and MDM2 enhances glioblastoma Multiforme cell apoptosis and differentiation of cancer stem cells. Sci Rep 5:9956. https://doi.org/10.1038/srep09956 CrossRefGoogle Scholar
- 48.Megías J, Martínez A, Yáñez A, Goodridge HS, Gozalbo D, Gil ML (2016) TLR2, TLR4 and Dectin-1 signalling in hematopoietic stem and progenitor cells determines the antifungal phenotype of the macrophages they produce. Microbes Infect 18:354–363. https://doi.org/10.1016/j.micinf.2016.01.005 CrossRefGoogle Scholar
- 49.Alexander BM, Galanis E, Yung WK, Ballman KV, Boyett JM, Cloughesy TF, Degroot JF, Huse JT, Mann B, Mason W, Mellinghoff IK, Mikkelsen T, Mischel PS, O'Neill BP, Prados MD, Sarkaria JN, Tawab-Amiri A, Trippa L, Ye X, Ligon KL, Berry DA, Wen PY (2015) Brain malignancy steering committee clinical trials planning workshop: report from the targeted therapies working group. Neuro-Oncology 17:180–188. https://doi.org/10.1093/neuonc/nou154 CrossRefGoogle Scholar
- 50.Prados MD, Byron SA, Tran NL, Phillips JJ, Molinaro AM, Ligon KL, Wen PY, Kuhn JG, Mellinghoff IK, de Groot JF, Colman H, Cloughesy TF, Chang SM, Ryken TC, Tembe WD, Kiefer JA, Berens ME, Craig DW, Carpten JD, Trent JM (2015) Toward precision medicine in glioblastoma: the promise and the challenges. Neuro-Oncology 17:1051–1063. https://doi.org/10.1093/neuonc/nov031 CrossRefGoogle Scholar