Pam₃CSK₄, a TLR2 ligand, induces differentiation of glioblastoma stem cells and confers susceptibility to temozolomide

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.