The anti-tumor peptide tat-cx43(266-283) modulates the interplay between glioblastoma cells and tumor-associated astrocytes

Resumen

Glioblastoma is the most aggressive and frequent form of primary brain tumor and, despite continuous effort to find an effective treatment, is considered as one of the deadliest types of cancer, with a median survival of only 16 months. Glioblastomas are characterized by fast and aggressive growth, high infiltrative capacity, and resistance to current treatments. These tumors are composed of a heterogeneous population of cells, including some with stem cell properties (glioblastoma stem cells, GSCs), which are highly tumorigenic and have high oncogenic Src activity. The presence of tumor cells in the brain triggers the activation of the astrocytes in the tumor microenvironment, which ultimately cooperate with glioblastoma cells to promote tumor progression. Previous results from our laboratory show that a cell-penetrating peptide that mimics the inhibitory effect of connexin43 on Src (Tat-Cx43266-283) exerts potent anti-tumoral effects in glioblastoma cells in vitro and in vivo, without deleterious effects in healthy brain cells. In this PhD thesis, we explore the mechanisms of astrocyte activation in the context of glioblastoma and analyze the effect of Tat-Cx43266-283 in the crosstalk between tumor cells and astrocytes. First, we use ex vivo and in vitro co-culture models to study astrocyte activation. We show that the presence of glioblastoma cells promotes a change in the phenotype of astrocytes that is characterized by an increase in the phosphorylation and nuclear translocation of the transcription factor STAT3. We find that laminin activates astrocytes, increasing the activity of STAT3 and the expression of its target genes N-cadherin and b-catenin. Importantly, we find that the treatment with Tat-Cx43266-283 reverts these effects. Second, we set up an in vivo model consisting in the intracranial injection of GL261 GSCs in immunocompetent syngeneic mice. We show that the treatment of GSCs derived from GL261 with Tat-Cx43266-283 reduces Src levels and activity, diminishes proliferation, and promotes differentiation towards an oligodendrocyte-like state that favors temozolomide (TMZ) anti-tumor effect. We generate drug-resistant GL261 GSCs and characterize their response to Tat-Cx43266-283 and TMZ treatments. Third, we use the in vivo model to confirm the effect of Tat-Cx43266-283 on astrocyte activation. We find that tumor progression induces a vast response of astrocytes, including STAT3 activation, and that Tat-Cx43266-283 reverts this effect. Finally, we take advantage of high-throughput single-cell technologies to confirm the main findings of this PhD Thesis and obtain a global picture that helps understanding glioblastoma biology and, ultimately, provide a cure for this deadly malignancy. In summary, the results presented in this PhD Thesis confirm the anti-tumor effect of Tat-Cx43266-283 in glioblastoma cells and uncover a new role for this peptide in the modulation of astrocyte activation. The combination of these effects disrupts the cross-communication between glioblastoma cells and astrocytes, contributing to the reduction of tumor growth and invasion.