Glioblastoma (GBM) still remains an incurable disease being radiotherapy (RT) the mainstay treatment. Glioblastoma intra-tumoral heterogeneity and Glioblastoma-Initiating Cells (GICs) challenge the design of effective therapies. We investigated GICs and non-GICs response to RT in a paired in-vitro model and addressed molecular programs activated in GICs after RT. Established GICs heterogeneously expressed several GICs markers and displayed a mesenchymal signature. Upon fractionated RT, GICs reported higher radioresistance compared to non-GICs and showed lower α- and β-values, according to the Linear Quadratic Model interpretation of the survival curves. Moreover, a significant correlation was observed between GICs radiosensitivity and patient disease-free survival. Transcriptome analysis of GICs after acquisition of a radioresistant phenotype reported significant activation of Proneural-to-Mesenchymal transition (PMT) and pro-inflammatory pathways, being STAT3 and IL6 the major players. Our findings support a leading role of mesenchymal GICs in defining patient response to RT and provide the grounds for targeted therapies based on the blockade of inflammatory pathways to overcome GBM radioresistance.

Background: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to ameliorate short long-term progression-free survival. We examined 5-aminolevulinic acid (5-ALA)-mediated PDT effects as therapeutical treatment and determined optimal conditions for PDT efficacy without causing phototoxic injury to the normal brain tissue. Methods: We used a platform of Glioma Initiation Cells (GICs) infiltrating cerebral organoids with two different glioblastoma cells, GIC7 and PG88. We measured GICs-5-ALA uptake and PDT/5-ALA activity in dose-response curves and the efficacy of the treatment by measuring proliferative activity and apoptosis. Results: 5-ALA (50 and 100 µg/mL) was applied, and the release of protoporphyrin IX (PpIX) fluorescence measures demonstrated that the emission of PpIX increases progressively until its stabilization at 24 h. Moreover, decreased proliferation and increased apoptosis corroborated the effect of 5-ALA/PDT on cancer cells without altering normal cells. Conclusions: We provide evidence about the effectiveness of PDT to treat high proliferative GB cells in a complex in vitro system, which combines normal and cancer cells and is a useful tool to standardize new strategic therapies.

Therapeutic resistance after multimodal therapy is the most relevant cause of glioblastoma (GBM) recurrence. Extensive cellular heterogeneity, mainly driven by the presence of GBM stem-like cells (GSCs), strongly correlates with patients’ prognosis and limited response to therapies. Defining the mechanisms that drive stemness and control responsiveness to therapy in a GSC-specific manner is therefore essential. Here we investigated the role of integrin a6 (ITGA6) in controlling stemness and resistance to radiotherapy in proneural and mesenchymal GSCs subtypes. Using cell sorting, gene silencing, RNA-Seq, and in vitro assays, we verified that ITGA6 expression seems crucial for proliferation and stemness of proneural GSCs, while it appears not to be relevant in mesenchymal GSCs under basal conditions. However, when challenged with a fractionated protocol of radiation therapy, comparable to that used in the clinical setting, mesenchymal GSCs were dependent on integrin a6 for survival. Specifically, GSCs with reduced levels of ITGA6 displayed a clear reduction of DNA damage response and perturbation of cell cycle pathways. These data indicate that ITGA6 inhibition is able to overcome the radioresistance of mesenchymal GSCs, while it reduces proliferation and stemness in proneural GSCs. Therefore, integrin a6 controls crucial characteristics across GBM subtypes in GBM heterogeneous biology and thus may represent a promising target to improve patient outcomes.

The anionic cobaltabis (dicarbollide) [3,3′-Co(1,2-C₂B₉H₁₁)₂]⁻, [o-COSAN]⁻, is the most studied icosahedral metallacarborane. The sodium salts of [o-COSAN]⁻ could be an ideal candidate for the anti-cancer treatment Boron Neutron Capture Therapy (BNCT) as it possesses the ability to readily cross biological membranes thereby producing cell cycle arrest in cancer cells. BNCT is a cancer therapy based on the potential of ¹⁰B atoms to produce α particles that cross tissues in which the ¹⁰B is accumulated without damaging the surrounding healthy tissues, after being irradiated with low energy thermal neutrons. Since Na[o-COSAN] displays a strong and characteristic ν(B-H) frequency in the infrared range 2.600–2.500 cm⁻¹, we studied the uptake of Na[o-COSAN] followed by its interaction with biomolecules and its cellular biodistribution in two different glioma initiating cells (GICs), mesenchymal and proneural respectively, by using Synchrotron Radiation-Fourier Transform Infrared (FTIR) micro-spectroscopy (SR-FTIRM) facilities at the MIRAS Beamline of ALBA synchrotron light source. The spectroscopic data analysis from the bands in the regions of DNA, proteins, and lipids permitted to suggest that after its cellular uptake, Na[o-COSAN] strongly interacts with DNA strings, modifies proteins secondary structure and also leads to lipid saturation. The mapping suggests the nuclear localization of [o-COSAN]⁻, which according to reported Monte Carlo simulations may result in a more efficient cell-killing effect compared to that in a uniform distribution within the entire cell. In conclusion, we show pieces of evidence that at low doses, [o-COSAN]⁻ translocates GIC cells’ membranes and it alters the physiology of the cells, suggesting that Na[o-COSAN] is a promising agent to BNCT for glioblastoma cells.

BACKGROUND: In the last decade, the cancer stem cell hypothesis has gained progressively agreement towards researchers. Cancer stem cells (CSCs) or tumour initiating cells (TICs) are considered to be responsible for tumor initiation, propagation and therapeutic resistance. In glioblastoma multiforme (GBM), the most frequent and malignant primary brain tumour in adults, TICs are related with chemo- and radioresistance. Integrins are cell surface receptors involved in the crosstalk of tumoral cells with their extracellular matrix (ECM). In particular integrin α6 (ITGA6) is known to be extremely important in embryonic, hematopoietic and neural stem cells. In brain it is involved in maintaining adhesion to the ventricular zone and ensures proper cell division. Besides, recently it has been proposed as a functional marker of TICs. Given the raising evidence of the ECM and perivascular niche role in the maintenance and guidance of TICs, we focused on ITGA6 to determine whether ITGA6 expression is associated with radiation-resistance of TICs in glioblastoma patients. METHODS: Four different GBM cultures obtained from post-surgical specimens were growth in two different growth conditions: a differentiating FBS-containing media and an FBS-free media supplemented with bFGF and EGF which is considered to be selective for TICs. Self-renewal capability and proliferative potential has been evaluated. Tumour initiating capability has been assessed through injection of low number of cells in immunocompromised mice. ITGA6 expression has been analyzed at RNA and protein levels. TICs have been sorted in order to get a population enriched for ITGA6high cells and dose-response to ionizing radiation (IR) has been evaluated and compared to ITGA6low cells by clonogenic assays. RESULTS: TICs have shown noticeable self-renewal capability, extensive proliferative potential, and a remarkable radioresistance compared to the same primary cultures maintained under differentiated conditions. In TICs, ITGA6 RNA and protein levels were statistically significant higher than in differentiated glioblastoma cells. In addition, this expression is decreasing consistently when TICs were cultured under differentiating conditions. Response to radiotherapy assessed by the clonogenic surviving fraction showed that ITGA6high cells are remarkably more radioresistant than ITGA6low cells (p < 0.001). In addition, ITGA6high cells showed a higher α/β ratio than ITGA6low cells (30 Gy vs. 6 Gy, respectively). In conclusion, these results suggest that ITGA6 is involved in the radioresistant phenotype of glioblastoma stem cells.