Evaluation of galectin-1 targeting drugs in combination with chemo-immunotherapy in the treatment of high-grade glioma.

Glioblastoma multiforme (GBM) is until today, one of the most aggressive and lethal tumors. Recent advances in surgery, chemo-and radiotherapy were not able to induce a significant shift in prognosis for these patients, and therefore this clearly presents an unmet medical need. Galectin-1 (Gal-1) is a naturally occurring galactose-binding lectin, which is overexpressed in glioblastoma multiforme (GBM). Gal-1 is associated with tumor progression, and is a potent immune suppressor in the tumor micro-environment (TME). Gal-1 in the TME of GBM is known to drive chemo- and immunotherapy resistance. To inhibit Gal-1 in GBM, an effective therapy is required that (selectively) reaches the central nervous system tumor, with limited systemic effects. In this thesis, we report for the first time that concentrated chitosan nanoparticle suspensions can deliver small interfering RNA (siRNA) into the central nervous system tumor within hours after intranasal administration. These nanoparticles are able to complex siRNA targeting Gal-1 to a high percentage, and protect them from RNAse degradation. Moreover, a successful intracellular delivery of anti-Gal-1 siRNA resulted in a decreased expression of Gal-1 in both murine and human GBM cells. Sequence specific RNAinterference, resulted in more than 50 % Gal-1 reduction in tumor bearing mice.

This reduction induces a remarkable switch in the TME contexture where myeloid suppressor cells and regulatory T cells were reduced, while CD4+ helper and CD8+ cytotoxic T cells were increased. gal-1 knock-down seems to reduce macrophages’ polarization switch from M1 to M2 during GBM progression. These changes result in a tumor vasculature normalization and modest increase in survival for tumor bearing mice. The rational combination of intranasal siGal-1 treatment with temozolomide and immunotherapy such as dendritic cell vaccination or PD-1 blocking results in synergistic effects and drastically increases the survival of tumor bearing mice in a prophylactic and established intracranial tumor model respectively. In fact, we could demonstrate an aggravated DNA damage pattern when Gal-1 was reduced and TMZ was administered. Furthermore, we could document an increased infiltration of Th1 and CTL cells in the TME during siGal-1 treatment and PD-1 blocking.

In the presented thesis, we have also developed PEGylated chitosan nanoparticles intended for intranasal transport, to deliver Galectin-1 targeting siRNA molecules from the nasal cavity towards the central nervous system. We explored the potential of PEGylated chitosan nanoparticles in comparison to native chitosan nanoparticles as described before. Successful cytoplasmatic delivery was demonstrated by efficient Gal-1 reduction on GL261. Possible superior nasal epithelial barrier modulation was suggested in comparison to native chitosan. Moreover, we could demonstrate an efficient delivery into the tumor micro-environment of an orthotopic murine glioblastoma model.

This study indicates that the intranasal pathway is an underexplored transport route for delivering siRNA-based therapies targeting Gal-1 in the treatment of GBM. The current observations provide evidence that Gal-1 could be a valuable adjuvant clinical target to further increase the efficiency of checkpoint blockade and chemotherapy.