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Among the main aberrations occurring in GBM, those in MEK/ERK and PI3K/akt/mTOR pathways predominate and confer GBM Stem-like Cells (GSCs) sustained proliferation and resistance to therapy. A panel of eight patient-derived primary GSCs lines have been screened for their sensitivity to a small kinase MEK inhibitor (MEKi) with AnnexinV/PI staining. Among these, five display a sensitive phenotype with at least 50% reduction on cell viability after 72 hours of treatment. Then, four cell lines, two MEKi-sensitive (ICH001 and ICH003) and two MEKi-resistant (ICH013 and ICH027) were selected for a deeper molecular characterization based on MGMT methylation status, mesenchymal index and main hotspot mutations associated with GBM pathology MEKi incubation on GSC caused a prompt phospho-ERK reduction already after 3 hours. Of note, we report a concomitant activation of AKT and downstream molecules pointing to an ERK-mTOR redundant activity. To this end, we combined MEKi to PI3K/akt/mTOR inhibitor and we observed an increased cell death even in GSCs displaying moderate sensitivity to MEKi as single-agent (MEKi: 90% vs MEKi-PI3K/akt/mTOR inhibitor: 30% cell viability). Then, MEKi ability to cross the Blood Brain Barrier (BBB) and target GBM cells was investigated using a transwell BBB in vitro model. The PI3K/akt/mTOR pathway inhibitor, known from the literature to readily cross the BBB, was included as positive control. Obtained results showed MEKi inability to efficiently cross the BBB, thus limiting its utility as GBM therapy. These results suggest the need for a specific drug delivery strategy in the brain that might be therapeutically effective. Recently our laboratory has provided proof-of-concept of a combination strategy based on radiation and adjuvant drug-loaded liposomes (LPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate BBB crossing. To strengthen therapeutic efficacy and to lower off-target effects, we implemented mApoE-LPs with a matrix metalloproteinases 2 and 9 sensitive lipopeptide (M2-9SLP) that allows controlled payload release only in the tumor microenvironment rich in MMPs. To this end, MEKi was encapsulated into the M2-9SLP/mApoE-LPs, and its capacity to promote cell death was evaluated. M2-9SLP/mApoE-MEKi-LPs caused in all the sensitive cell lines GSCs proliferation inhibition and induction of apoptosis upon 72h in vitro treatment indicating that the encapsulation process did not alter drug efficacy. In conclusion, our in vitro results support MEKi encapsulation into M2P/mApoE-LP as nanotherapeutic strategy that could guarantee specific delivery of MEKi in a MMP2-enriched tumor microenvironment without altering its capacity to inhibit GSC proliferation and survival. Funding by FRRB grant NEVERMIND (CP2_16/2018) Citation Format: Milena Mattioli, Elisabetta Stanzani, Valentino Ribecco, Marco Pizzocri, Eliana Lauranzano, Margherita Maria Ravanelli, Simone Olei, Maria Pia Tropeano, Pierfausto Seneci, Francesca Re, Federico Pessina, Michela Matteoli, Lorena Passoni. Smart encapsulation of a MEK inhibitor into M2-9SLP/mApoE-liposomes for specific GBM targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2711.

Background The radio- and chemo- resistance of Glioblastoma Stem-like Cells (GSCs), together with their innate tumor-initiating aptitude, make this cell population a crucial target for effective therapies. However, targeting GSCs is hardly difficult and complex, due to the presence of the Blood Brain Barrier (BBB) and the infiltrative nature of GSCs arousing their dispersion within the brain parenchyma. Methods Liposomes (LIPs), surface-decorated with an Apolipoprotein E-derived peptide (mApoE) to enable BBB crossing, were loaded with doxorubicin (DOXO), as paradigm of cytotoxic drug triggering immunogenic cell death (ICD). Patient-derived xenografts (PDXs) obtained by GSC intracranial injection were treated with mApoE-DOXO-LIPs alone or concomitantly with radiation. Results Our results indicated that mApoE, through the engagement of the Low-Density Lipoprotein Receptor (LDLR), promotes mApoE-DOXO-LIPs transcytosis across the BBB and confers target specificity towards GSCs. Irradiation enhanced LDLR expression on both BBB and GSCs, thus further promoting LIP diffusion and specificity. When administered in combination with radiations, mApoE-DOXO-LIPs caused significant reduction of in vivo tumor growth due to GSC apoptosis. GSC apoptosis prompted microglia/macrophage phagocytic activity, together with the activation of the antigen-presenting machinery crucially required for anti-tumor adaptive immune response. Conclusions Our results advocate for radiotherapy and adjuvant administration of drug-loaded, mApoE-targeted nanovectors as an effective strategy to deliver cytotoxic molecules to GSCs at the surgical tumor margins, the forefront of GBM recurrence, circumventing BBB hurdles. DOXO encapsulation proved in situ immune response activation within GBM microenvironment.

Recent evidence from our laboratory provided proof-of-concept for therapeutic potential for glioblastoma (GBM) of a combination strategy based on radiation and adjuvant doxorubicin-loaded liposomes (LIPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate Blood Brain Barrier (BBB)-crossing. Significant glioma stem cell (GSC) apoptosis, tumor growth inhibition and increased overall survival were observed in vivo upon combined treatment offering attractive and innovative therapeutic possibilities for GBM. To strengthen therapeutic efficacy and lower off-target effects, we implemented mApoE-LIPs with a matrix metalloproteinases (MMP)-activable element that allows controlled payload release only in the MMPs rich tumor microenvironment, thus concurring to reduce unspecific interaction in healthy tissue where MMPs are low or absent Given the MMP2 overexpression in GBM, a MMP2-activable block (M2AB) was included in the phospholipid bilayer of mApoE-LIPs. The M2AB efficacy was evaluated on patient-derived GSCs displaying different MMP2 enzymatic activities by means of calcein-loaded M2AB/mApoE-LIPs. Human endothelial cells (hCMEC/d3), not expressing MMP2, were included to validate the targeted strategy as well as to assay the cytotoxicity on non-tumoral cells. Intracellular calcein quantification showed that: 1) calcein uptake correlates with MMP2 activity level; 2) M2AB/mApoE functionalization augmented calcein internalization into GSCs compared to mApoE alone. The MEK/ERK pathway, known to supports GBM cell survival, migration, and radio-resistance was considered as target strategy against GBM. A survey of the MEK/ERK inhibitors Trametinib (TRAM) and Pimasertib (PIMA) in several GSC lines indicated significant induction of GSC apoptosis associated to reduction of ERK phosphorylation. TRAM and PIMA were then encapsulated into mApoE-LIPs and their anti-GSC activity was investigated. Indeed, a dose dependent inhibition of GSC survival and induction of apoptosis combined to a significant lower level of phospho-ERK was observed upon 72h treatment. In conclusion, M2AB/mApoE-LIPs demonstrate: 1) a MMP2-dependent payload release; 2) stability when MMP2 lacks in the cellular milieu; 3) a synergic effect of the double M2AB/mApoE functionalization. TRAM and PIMA encapsulation into LIPs does not alter their anti-GSC activity. Funding by FRRB grant NEVERMIND (CP2_16/2018) Citation Format: Milena Mattioli, Marco Pizzocri, Elisabetta Stanzani, Valentino Ribecco, Simone Olei, Maria Pia Tropeano, Sabrina Giofrè, Antonio Renda, Sara Pellegrino, Pierfausto Seneci, Francesca Re, Federico Pessina, Michela Matteoli, Lorena Passoni. Improving glioblastoma treatment specificity and efficacy of mApoE-targeted liposome by MMP2-controlled drug releasee [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 368.

To promote cell growth, invasion and therapy resistance, glioblastoma (GBM) makes use of different communication routes with the neighbor environment which include Extracellular Vesicles (EVs). EVs are a heterogeneous group of cell-released membranous structures, which contain a wide mixture of active molecules. Each cell type secretes a unique combination of different EV subpopulations that vary in size, content and function. In GBM, the subfraction of small-EVs derived from multivesicular bodies, also referred as exosomes (EXOs), have received considerable attention for their capacity to create a tumor-supportive microenvironment through their actions on immune cells, vasculature and glial cells. Only recently it has been observed that large-EVs formed by the budding of the cell membrane, classically called microvesicles (MVs), are more abundant than EXOs in the plasma of GBM patients. Large-MVs have been associated with disease progression in the context of prostate cancer, but their functional significance remain largely uncharacterized in GBM. To explore EV migratory potential in the GBM context, an in vitro migration test performed on spheroids of patient-derived Glioma Stem-like Cells (GSC) has been set up. EXOs from GSC culture supernatants exert no migratory effects, whereas MVs triggered remarkable cell migration. Differently from GSC culture supernatants, both EXOs and MVs isolated from surgical washing exerted a remarkable migratory effect suggesting that not only EVs from tumoral cells are actively implicated in GSC mobility but also EVs from the non-tumoral microenvironment act synergistically to sustain GBM invasion. To better understand the contribution of the cell sub-population of the tumor environment, an EV separation based on CD45(leukocyte common antigen) expression was performed. CD45-positive EVs (released by immune cells) and CD45-negative EVs (released by tumor and stroma cells) were employed in the spheroid migration assay. Results showed the absence of any migratory effect of CD45-positive subsets, both EXOs and MVs, indicating that EVs selectively generated by tumor and stroma cells play a key role in tumoral invasion. Furthermore, a multiplex bead-based flow-cytometry analysis performed on EVs from six GBM patients revealed the presence of specific antigen clusters related to migration and could also be investigated as tumoral biomarkers in a liquid biopsy context. Citation Format: Valentino Ribecco, Matteo Tamborini, Elisabetta Stanzani, Marco Pizzocri, Milena Mattioli, Simone Olei, Maria Pia Tropeano, Federico Pessina, Michela Matteoli, Lorena Passoni. Deciphering the role of tumor-released microvesicles in glioblastoma mobility and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 274.

The neurovascular unit (NVU) is the most important biological barrier between vascular districts and central nervous system (CNS) parenchyma, which maintains brain homeostasis, protects the CNS from pathogens penetration, and mediates neuroimmune communication. T lymphocytes migration across the blood-brain barrier is heavily affected in different brain diseases, representing a major target for novel drug development. In vitro models of NVU could represent a primary tool to investigate the molecular events occurring at this interface. To move toward the establishment of personalized therapies, a patient-related NVU-model is set, incorporating human primary astrocytes integrated into a microfluidic platform. The model is morphologically and functionally characterized, proving to be an advantageous tool to investigate human T lymphocytes transmigration and thus the efficacy of potential novel drugs affecting this process.

Nerve Growth Factor (NGF) plays a key role in development and function of specific neuronal populations of the CNS. Decreased NGF availability is also responsible for neuronal vulnerability in neurodegenerative disorders, such as Alzheimer’s disease (AD). We used PC12 cells and primary neurons to investigate the potential role of NGF in modulating neuronal autophagy, a cellular process whose deregulation has been linked to the loss of neuronal. The mammalian target of rapamycin has been recently proposed as a therapeutic target for AD due to the role of the autophagy pathway in improving cognitive function by reducing Aβ and Tau pathology. We here show that NGF treatment induces LC3 conversion (LC3-I to LC3-II), a marker of autophagy, through the activation of AMP-activated protein kinase (AMPK). We show that NGF increases the autophagic flux (the dynamic process of autophagosome synthesis, delivery to the lysosome and degradation) as indicated by western blot and fluorescence microscopy analyses in the presence of autophagy inhibitors. These data were confirmed by RT-PCR array analysis and RNA interference-mediated knockdown of autophagy-related genes: Atg9b, Atg12 and AMBRA1, a positive regulator of the BECLIN 1-dependent program of autophagy. In addition, flow cytometry analysis by DCF-DA showed that treatment with autophagy inhibitors determined a strong increase of reactive oxygen species (ROS) followed by decreased cell survival. These changes were fully reversed by NGF treatment, suggesting its potential role in clearing dysfunctional mitochondria. This hypothesis was further confirmed by fluorescence microscopy studies using LC3 and Cox-IV antibodies, showing co-localization of autophagosomes and mitochondria. Overall these data identify a novel aspect of the neuroprotective function of NGF in promoting the clearance of dysfunctional mitochondria (or mitophagy), thus further supporting its therapeutic potential in neurodegenerative pathologies.