Development and characterization of two novel nanomedicine-based approaches to restore the anti-tumor activity of the immune system in glioblastoma patients

Glioblastoma multiforme (GBM) is a malignant primary brain tumors, that, despite aggressive treatments, remains one of the deadliest cancer types with an overall survival of less than 15 months. Immunotherapy has emerged as a promising approach for cancer treatment, showing the possibility of long-term effects. However, in the case of GBM, approaches with immune checkpoint inhibitors have failed to demonstrate a significant therapeutic activity. Moreover, delivering drugs to the CNS, is challenging due to the to the presence of the BBB, which hinders a homogeneous diffusion of the drugs to cancer cells. Consequently, there is an urgent demand for the development of novel targeted therapies. In this context nanomedicine presents a potential solution to address the poor solubility challenges of certain drugs and offers selective targeting of specific cell populations. Another obstacle to immunotherapy in GBM is the fact that tumor is endowed with an immunosuppressive microenvironment that impedes the efficacy of standard and immune therapies, and which is mainly driven by bone marrow-derived macrophages (BMDMs). We previously showed that BMDMs are the main leukocytes infiltrating GBM. Of note, compared to resident microglial cells (MG), BMDMs have a significant immunosuppressive activity, that is associated with their sustained iron metabolism. In fact, our studies indicated that inhibiting HO-1, an enzyme with a key role in iron metabolism and immunosuppressive activity of BMDMs, could be a promising therapeutic strategy for their reprogramming towards a more pro-inflammatory and anti-tumour activity. In my doctoral project, we combined both the field of nanomedicine and tumor immunology to capitalize on two novel nano-based approaches. Our objectives include: 1) inducing a re-programming of macrophages through targeting of immune metabolism of BMDMs using a nanosystem; and 2) inducing immunogenic cell death (ICD) and depleting myeloid suppressive cells within the tumor microenvironment of GBM using another nanosystem. We developed an Oil in Water (O/W) nanoemulsion containing a Zinc protoporphyrin IX (ZnPPIX), a potent inhibitor of heme-oxygenase HO-1. Since ZnPPIX is a highly hydrophobic drug, the aim was to improve its drug delivery. In line with our previous work, results demonstrate that also targeting HO-1 by ZnPPIX encapsulation, reduced the immunosuppressive activity of in vitro-derived macrophages. In addition, the nanoemulsions containing ZnPPIX showed better efficacy in targeting macrophages and modulating the expression of CD163, a marker involved in the iron recycling phenotype of pro-tumoral and immunosuppressive macrophages, as compared to the treatment with the free ZnPPIX. We also developed and studied a polymeric nanosystem loaded with an oxaliplatin derivate, diaminocyclohexane-platinum II (DACHPt-NPs). Oxaliplatin derivatives activate immunogenic cell death (ICD), a phenomenon that can be detected through the measurement of specific danger-associated-molecular-patterns (DAMPs). Our studies demonstrate that NPs can target both myeloid immunosuppressive and tumour cells. This provides a potential novel nanomedicine-based approach capable of both depleting suppressive cells and inducing ICD. Of note, in vitro cytotoxicity studies revealed that DACHPt-loaded nanoparticles had a superior anticancer activity compared with free oxaliplatin. Moreover, evaluation of specific DAMPs, showed the higher efficacy in ICD induction of DACHPt-NPs compared to free oxaliplatin treatment. Finally, uptake studies showed the potential of both nanosytems to be internalized by immunosuppressive myeloid derived cells (particularly BMDMs), malignant cells in the TME and circulating monocytes in the blood while maintaining a low level of incorporation by T cells. This suggests the potential of these nanosystems for both local and systemic treatments.