Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.

Extracellular vesicles produced by irradiated endothelial or Glioblastoma stem cells promote tumor growth and vascularization modulating tumor microenvironment.

BACKGROUND: Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. METHODS: In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. RESULTS: We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. CONCLUSION: In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

L1077P CFTR pathogenic variant function rescue by Elexacaftor-Tezacaftor-Ivacaftor in cystic fibrosis patient-derived air-liquid interface (ALI) cultures and organoids: in vitro guided personalized therapy of non-F508del patients.

Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

Agile workflow for interactive analysis of mass cytometry data.

MOTIVATION: Single-cell proteomics technologies, such as mass cytometry, have enabled characterization of cell-to-cell variation and cell populations at a single-cell resolution. These large amounts of data, require dedicated, interactive tools for translating the data into knowledge. RESULTS: We present a comprehensive, interactive method called Cyto to streamline analysis of large-scale cytometry data. Cyto is a workflow-based open-source solution that automates the use of state-of-the-art single-cell analysis methods with interactive visualization. We show the utility of Cyto by applying it to mass cytometry data from peripheral blood and high-grade serous ovarian cancer (HGSOC) samples. Our results show that Cyto is able to reliably capture the immune cell sub-populations from peripheral blood and cellular compositions of unique immune- and cancer cell subpopulations in HGSOC tumor and ascites samples. AVAILABILITYAND IMPLEMENTATION: The method is available as a Docker container at https://hub.docker.com/r/anduril/cyto and the user guide and source code are available at https://bitbucket.org/anduril-dev/cyto. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells.

BACKGROUND: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. METHODS: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. RESULTS: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD's catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. CONCLUSIONS: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.

Theratyping cystic fibrosis in vitro in ALI culture and organoid models generated from patient-derived nasal epithelial conditionally reprogrammed stem cells.

QUESTION: Cystic fibrosis (CF) is due to pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent improvements have enabled pharmacological therapy aiming at restoring mutated CFTR expression and function. CFTR "modulators" have revolutionised the CF therapeutic landscape, particularly the last approved, Trikafta. This drug combination is indicated by the United States Food and Drug Administration and very recently by the European Medicines Agency for genotypes carrying at least one copy of CFTR with the F508del pathogenic variant. However, several genotypes are not yet eligible for Trikafta treatment. MATERIALS/PATIENTS AND METHODS: We exploited an innovative cellular approach allowing highly efficient in vitro expansion of airway epithelial stem cells (AESCs) through conditional reprogramming from nasal brushing of CF patients. This approach, coupled to the development of AESC-derived personalised disease models, as organoids and air-liquid interface (ALI) cultures, revealed highly suitable for CFTR pharmacological testing. RESULTS AND ANSWER TO THE QUESTION: We fully validated the experimental models and implemented the CFTR functional assays and biochemical CFTR protein characterisation, which allowed the evaluation of the efficacy of clinically available modulators in restoring CFTR maturation and function of each patient-derived "avatar" (theratyping). F508del homozygous genotypes, used as controls, confirmed the higher clinical activity of Trikafta in comparison with older modulators. In addition, Trikafta showed its efficacy on three rare genotypes previously not eligible for treatment with modulators, opening the way to clinical translation. Finally, encouraging results for innovative drug combinations were obtained.

qSNE: quadratic rate t-SNE optimizer with automatic parameter tuning for large datasets.

MOTIVATION: Non-parametric dimensionality reduction techniques, such as t-distributed stochastic neighbor embedding (t-SNE), are the most frequently used methods in the exploratory analysis of single-cell datasets. Current implementations scale poorly to massive datasets and often require downsampling or interpolative approximations, which can leave less-frequent populations undiscovered and much information unexploited. RESULTS: We implemented a fast t-SNE package, qSNE, which uses a quasi-Newton optimizer, allowing quadratic convergence rate and automatic perplexity (level of detail) optimizer. Our results show that these improvements make qSNE significantly faster than regular t-SNE packages and enables full analysis of large datasets, such as mass cytometry data, without downsampling. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are openly available at https://bitbucket.org/anthakki/qsne/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Variability of treatment modalities and intensity in patients with severe haemophilia A on prophylaxis: Results from the Italian national registry.

BACKGROUND: A shift from a standard to a personalized prophylaxis has been increasingly adopted in patients with severe haemophilia A (SHA). This approach has raised the likelihood of a significant variability in the prophylactic approaches and the relative Factor VIII (FVIII) consumptions. The aim of our study was to assess the treatment variability of SHA patients without inhibitors and on prophylaxis regimen in Italy. MATERIAL AND METHODS: Data reported in the National Registry of Congenital Coagulopathies (NRCC) were analysed to assess treatment distribution within SHA patients without inhibitors, focussing on FVIII consumption in 2017, associated with prophylaxis regimen. The analysis was stratified based on age groups and Italian regions to describe the variability of FVIII consumption in Italy. RESULTS: In 2017, the Registry reported the therapeutic plans of 1068 SHA patients without inhibitors on prophylaxis. The mean (95% CI) individual consumption ranges from 123 127 IU (99 736-146 518) in the age group 0-6 years to 345 000 IU (336 000-354 000) in the age group >20 years. A significant FVIII consumption variability was identified within the adult population. Regions with less than 50 patients reported the higher variability in mean FVIII consumption per patient-year within the different age groups. Similar difference in FVIII consumption variability was reported also in the age groups comparing "low," "middle" and "high" patient volume regions. DISCUSSION: A reliable estimation of FVIII consumption for patients' treatment is necessary to manage and plan the appropriate budget and keep treatment's costs affordable. However, without the implementation of a methodology aiming to assess the overall value produced by these FVIII consumptions, the scenario will keep driven by FVIII consumptions, its costs and the budget available. An effort by haemophilic community, haemophilia treatment centres and institutions is required to develop and share this cultural shift in improving haemophilia management and assessment.

Molecular tests and target therapies in oncology: recommendations from the Italian workshop.

Next-generation sequencing (NGS) and liquid biopsy are new technologies that can allow overall tumor profiling in a single analysis and play an important role in the implementation of precision oncology. However, the lack of guidelines in this setting has limited the development of precision oncology in Italy. This article summarizes recommendations for the appropriate use of NGS in tumor gene profiling, as well as access to tests and target drugs, that were prepared by a group of key opinion leaders and relevant stakeholders. In particular, the need to create laboratory networks capable of carrying out NGS tests in Italy is highlighted. It also appears necessary to establish an adequate reimbursement system for NGS tests. However, the expert panel recommends that the use of NGS tests in clinical practice should be limited to specific tumor types, based on the number and complexity of biomarkers and the availability of treatments.

Lay abstract The increasingly precise and extensive characterization of tumors through gene profiling allows a greater understanding of the molecular mechanisms underlying tumor growth, thus permitting better, more personalized therapeutic options. In the past two decades, tests to individually profile genes (molecular alterations) of different tumors ­ including lung, stomach, colorectal, breast, ovarian cancer and melanoma ­ into clinical practice have been introduced, allowing patients who carry specific genomic alterations greater access to more effective therapies. The first phase of the era of genomic profiling was limited to the identification of molecular alterations, each detectable with a specific test, aiming to define the sensitivity/resistance to a single drug and for a specific cancer site. The second phase of precision medicine determined several molecular alterations tested for single cancer types, often with different techniques. We have now reached a third phase, characterized by important technological developments and, in particular, by the introduction of next-generation sequencing (NGS) and liquid biopsy (using patients' blood). These techniques allow a comprehensive genomic profile of the tumor in a single analysis using the same biological sample. These new techniques have led to the selection of increasingly precise patient candidates for target therapy and then to the monitoring of their treatment, together with identification of resistant tumor clones. However, the lack of guidelines in this setting has limited the development of precision medicine in Italy. This article reports a summary of recommendations for appropriate indications in tumor gene profiling, as well as for access to tests and target drugs, that were prepared by a group of key opinion leaders and relevant stakeholders.

Two-Step Coimmunoprecipitation (TIP) Enables Efficient and Highly Selective Isolation of Native Protein Complexes.

Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.

Different Mechanisms Underlie the Metabolic Response of GBM Stem-Like Cells to Ionizing Radiation: Biological and MRS Studies on Effects of Photons and Carbon Ions.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with very poor prognosis, high recurrence rate, and failure of chemo-radiotherapy, mainly due to a small fraction of cells with stem-like properties (GSCs). To study the mechanisms of GSCs resistance to radiation, two GSC lines, named line #1 and line #83, with different metabolic patterns and clinical outcome, were irradiated with photon beams and carbon ions and assessed by 1H Magnetic Resonance Spectroscopy (MRS). Both irradiation modalities induced early cytotoxic effects in line #1 with small effects on cell cycle, whereas a proliferative G2/M cytostatic block was observed in line #83. MR spectroscopy signals from mobile lipids (ML) increased in spectra of line #1 after photon and C-ion irradiation with effects on lipid unsaturation level, whereas no effects were detected in line #83 spectra. Gamma-Aminobutyric Acid (GABA), glutamic acid (glu) and Phosphocreatine (pCr) signals showed a significant variation only for line #1 after carbon ion irradiation. Glucose (glc) level and lactate (Lac) extrusion behaved differently in the two lines. Our findings suggest that the differences in irradiation response of GSCs #1 and #83 lines are likely attributable to their different metabolic fingerprint rather than to the different radiation types.

Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1.

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

Two-Year Follow-Up of Macaques Developing Intermittent Control of the Human Immunodeficiency Virus Homolog Simian Immunodeficiency Virus SIVmac251 in the Chronic Phase of Infection.

UNLABELLED: Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy and two experimental drugs, i.e., auranofin and buthionine sulfoximine, was able to reduce the viral reservoir, elicit efficient cell-mediated antiviral responses, and induce intermittent posttherapy viral load control in chronically SIVmac251-infected macaques. We here show that the macaques that had received this drug combination and then stopped antiretroviral therapy were also able to maintain low numbers of activated CD4+ T cells at viral rebound. Moreover, these macaques consistently displayed low-level simian immunodeficiency virus (SIV) diversity, which was in line with the strong and broadly reactive cell-mediated immune responses against conserved Gag antigens. Extended follow-up showed that the two macaques that had received the complete drug combination remained healthy and did not develop AIDS in 2 years of follow-up after therapy suspension. This disease-free survival is longer than twice the average time of progression to AIDS in SIVmac251-infected rhesus macaques. These results suggest that limited numbers of activated T cells at viral rebound and subsequent development of broadly reactive cell-mediated responses may be interrelated in reducing the viral reservoir. IMPORTANCE: The HIV reservoir in CD4+ T cells represents one main obstacle to HIV eradication. Recent studies, however, show that a drastic reduction of this reservoir is insufficient for inducing a functional cure of AIDS. In the present work, we thoroughly studied and subjected to long-term follow-up two macaques showing intermittent control of the virus following suspension of antiretroviral therapy plus an experimental antireservoir treatment, i.e., the gold salt auranofin and the investigational chemotherapeutic agent buthionione sulfoximine (BSO). We found that these drugs were able to decrease the number of activated CD4+ T cells, which are preferential targets for HIV infection. Then, efficient immune responses against the virus were developed in the macaques, which remained healthy during 2 years of follow-up. This result may furnish another building block for future attempts to cure HIV/AIDS.

Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells.

Glioblastoma is a highly angiogenetic malignancy, the neoformed vessels of which are thought to arise by sprouting of pre-existing brain capillaries. The recent demonstration that a population of glioblastoma stem-like cells (GSCs) maintains glioblastomas indicates that the progeny of these cells may not be confined to the neural lineage. Normal neural stem cells are able to differentiate into functional endothelial cells. The connection between neural stem cells and the endothelial compartment seems to be critical in glioblastoma, where cancer stem cells closely interact with the vascular niche and promote angiogenesis through the release of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (refs 5-9). Here we show that a variable number (range 20-90%, mean 60.7%) of endothelial cells in glioblastoma carry the same genomic alteration as tumour cells, indicating that a significant portion of the vascular endothelium has a neoplastic origin. The vascular endothelium contained a subset of tumorigenic cells that produced highly vascularized anaplastic tumours with areas of vasculogenic mimicry in immunocompromised mice. In vitro culture of GSCs in endothelial conditions generated progeny with phenotypic and functional features of endothelial cells. Likewise, orthotopic or subcutaneous injection of GSCs in immunocompromised mice produced tumour xenografts, the vessels of which were primarily composed of human endothelial cells. Selective targeting of endothelial cells generated by GSCs in mouse xenografts resulted in tumour reduction and degeneration, indicating the functional relevance of the GSC-derived endothelial vessels. These findings describe a new mechanism for tumour vasculogenesis and may explain the presence of cancer-derived endothelial-like cells in several malignancies.

(1) H NMR spectroscopy of glioblastoma stem-like cells identifies alpha-aminoadipate as a marker of tumor aggressiveness.

Patients suffering from glioblastoma multiforme (GBM) face a poor prognosis with median survival of about 14 months. High recurrence rate and failure of conventional treatments are attributed to the presence of GBM cells with stem-like properties (GSCs). Metabolite profiles of 42 GSC lines established from the tumor tissue of adult GBM patients were screened with (1) H NMR spectroscopy and compared with human neural progenitor cells from human adult olfactory bulb (OB-NPCs) and from the developing human brain (HNPCs). A first subset (n=12) of GSCs exhibited a dramatic accumulation of the metabolite α-aminoadipate (αAAD), product of the oxidation of α-aminoadipic semialdehyde catalyzed by the ALDH7A1 aldehyde dehydrogenase (ALDH) family in lysine catabolism. αAAD was low/not detectable in a second GSC subset (n=13) with the same neural metabolic profile as well as in a third GSC subset (n=17) characterized by intense lipid signals. Likewise, αAAD was not detected in the spectra of OB-NPCs or HNPCs. Inhibition of mitochondrial ATP synthase by oligomycin treatment revealed that the lysine degradative pathway leading to αAAD formation proceeds through saccharopine, as usually observed in developing brain. Survival curves indicated that high αAAD levels in GSCs significantly correlated with poor patient survival, similarly to prostate and non-small-cell-lung cancers, where activity of ALDH7A1 correlates with tumor aggressiveness.

Hedgehog controls neural stem cells through p53-independent regulation of Nanog.

Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs.

1H NMR detects different metabolic profiles in glioblastoma stem-like cells.

The metabolic profiles of glioblastoma stem-like cells (GSCs) growing in neurospheres were examined by (1)H NMR spectroscopy. Spectra of two GSC lines, labelled 1 and 83, from tumours close to the subventricular zone of the temporal lobe were studied in detail and compared with those of neural stem/progenitor cells from the adult olfactory bulb (OB-NPCs) and of the T98G glioblastoma cell line. In both GSCs, signals from myoinositol (Myo-I), UDP-hexosamines (UDP-Hex) and glycine indicated an astrocyte/glioma metabolism. For line 1, the presence of signals from N-acetyl aspartate, GABA and creatine pointed to a neuronal fingerprint. These metabolites were almost absent from line 83 spectra, whereas lipid signals, absent from normal neural lineages, were intense in line 83 spectra and remained low in those of line 1, irrespective of apoptotic fate. Spectra of OB-NPC cells displayed strong similarities with those from line 1, with low lipid signals and clearly detectable neuronal signals. In contrast, the spectral profile of line 83 was more similar to that of T98G, displaying high lipids and nearly complete absence of the neuronal markers. A mixed neural-astrocyte metabolic phenotype with a strong neuronal fingerprint was therefore found in line 1, while an astrocytic/glioma-like metabolism prevailed in line 83. We found a signal assigned to the amide proton of N-acetyl galactosamine in GSC lines and in OB-NPC spectra, whereas it was absent from those of T98G cells. This signal may be related to a stem-cell-specific protein glycosylation pattern and is therefore suggested as a marker of cell multipotency. Other GSC lines from patients with different clinical outcomes were then examined. Unsupervised analysis of spectral data from 13 lines yielded two clusters, with six lines resembling spectral features of line 1 and seven resembling those of line 83, suggesting that distinct metabolic phenotypes may be present in GSC lines.