Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

Primary malignant brain tumors are the most common solid neoplasm in childhood. Despite recent advances, many children affected by aggressive or metastatic brain tumors still present poor prognosis, therefore the development of more effective therapies is urgent. Cancer stem cells (CSCs) have been discovered and isolated in both pediatric and adult patients with brain tumors (e.g., medulloblastoma, gliomas and ependymoma). CSCs are a small clonal population of cancer cells responsible for brain tumor initiation, maintenance and progression, displaying resistance to conventional anticancer therapies. CSCs are characterized by a specific repertoire of surface markers and intracellular specific pathways. These unique features of CSCs biology offer the opportunity to build therapeutic approaches to specifically target these cells in the complex tumor bulk. Treatment of pediatric brain tumors with classical chemotherapeutic regimen poses challenges both for tumor location and for the presence of the blood–brain barrier (BBB). Lastly, the application of chemotherapy to a developing brain is followed by long-term sequelae, especially on cognitive abilities. Novel avenues are emerging in the therapeutic panorama taking advantage of nanomedicine. In this review we will summarize nanoparticle-based approaches and the efficacy that NPs have intrinsically demonstrated and how they are also decorated by biomolecules. Furthermore, we propose novel cargoes together with recent advances in nanoparticle design/synthesis with the final aim to specifically target the insidious CSCs population in the tumor bulk.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults’ molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the K _D value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection. IMPORTANCE: Bacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.

The structure and conformation adopted by protein vaccine antigens significantly influence the exposure of their antigenic determinants. Structural knowledge of antigens in native state could drive the design of recombinant vaccines that resemble their cognate native forms, although such information is often difficult to obtain, particularly for membrane proteins. Here, we assessed the structural and functional features of the native Neisseria Adhesin A (NadA), a meningococcal trimeric outer membrane protein included as soluble recombinant antigen in the 4CMenB vaccine. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to generate a structural model of NadA and to compare the fold and structural dynamics of the recombinant NadA as soluble vaccine form, and the native NadA in situ , as embedded in meningococcal outer membrane vesicles (OMVs), complementing the HDX data with electron microscopy imaging. While their overall structures are similar, conformational differences between the two forms were observed. Especially, OMV- embedded NadA appears more susceptible to trimer opening than its cognate soluble antigen, suggesting that NadA in its native membrane could display a larger antigenic surface. Accordingly, we show that mice immunized with OMV-embedded NadA elicited antibodies with superior bactericidal activity and capable of better preventing bacterial adhesion compared to the soluble antigen. Collectively, these data support the hypothesis that protein vaccine antigens presented in native-like environments can elicit a more potent immune response than recombinant forms.