**3.1 Targeting glioma stem cells (GSCs)**

Glioma stem cells (GSCs) are a subpopulation of glioma cells with stem-like properties. These cells are thought to promote tumor initiation, chemo- and radioresistance, and tumor invasiveness. GSCs were first defined by their expression of prominin 1 or CD133, however, it was later discovered that CD133-negative cells were also capable of causing tumor initiation. In addition, several different models of GSC initiation have been proposed.

Vora *et al.* generated three different therapeutic modalities to target CD133<sup>+</sup> GSCs and tested their efficacy using human GBM models. The first modality, a CD133-binding IgG, was found to be ineffective at causing a significant reduction in proliferation *in vitro* and *in vivo* tumor burden. The second modality, a dual-antigen T cell engager or DATE, specific for CD133 and CD3, caused significant tumorkilling both *in vitro* and *in vivo*. Finally, the CD133-specific CAR T cells provided profound T cell proliferation and secretion of the anti-tumor cytokines IFNγ and TNFα upon co-culture with various human GBM cells. In addition, when mice were intracranially injected with human GBM cells followed by subsequent intracranial injection of CD133-specific CAR T cells, a significant reduction in tumor burden and prolonged survival was observed relative to control-treated mice. Importantly, they found administration of CD133-specific CAR T cells did not significantly impair hematopoiesis [131].

An additional novel method of targeting GSCs is through the use of NK cells. These cells are cytotoxic lymphocytes capable of killing target tumor cells. GSCs have been shown to express activating ligands of NK cells, such as CD155 and B7-H6. In addition, NK cells were shown to be able to lyse GSCs *in vitro* upon co-culture with target GSCs. Contrarily, GSCs were found to promote NK cell

dysfunction that was determined to be contact-dependent. Mechanistically, the NK cell dysfunction was found to be mediated via TGFβ-1 released by GSCs and upon treatment with a TGFβ inhibitor, the dysfunction could be significantly diminished. When evaluated in an *in vivo* model of human GSC, the combination of allogeneic NK cells and a TGFβ inhibitor provided superior survival relative to any control groups. These results suggest combinatorial NK cell therapy and TGFβ inhibitor may provide promising anti-tumor responses [132].
