SUMMARY In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both, ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.

Brain invasion by glioblastoma determines prognosis, recurrence, and lethality in patients, but no master factor coordinating the invasive properties of glioblastoma has been identified. Here we report evidence favoring such a role for the noncanonical WNT family member Wnt5a. We found the most invasive gliomas to be characterized by Wnt5a overexpression, which correlated with poor prognosis and also discriminated infiltrating mesenchymal glioblastoma from poorly motile proneural and classical glioblastoma. Indeed, Wnt5a overexpression associated with tumor-promoting stem-like characteristics (TPC) in defining the character of highly infiltrating mesenchymal glioblastoma cells (Wnt5a High ). Inhibiting Wnt5a in mesenchymal glioblastoma TPC suppressed their infiltrating capability. Conversely, enforcing high levels of Wnt5a activated an infiltrative, mesenchymal-like program in classical glioblastoma TPC and Wnt5aLow mesenchymal TPC. In intracranial mouse xenograft models of glioblastoma, inhibiting Wnt5a activity blocked brain invasion and increased host survival. Overall, our results highlight Wnt5a as a master regulator of brain invasion, specifically TPC, and they provide a therapeutic rationale to target it in patients with glioblastoma. Cancer Res; 77(4); 996-1007. Ó2016 AACR.

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders.

Background Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. Methods and findings We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo , in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). Interpretation Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. Fund This work was financially supported by grants from “Ministero della Salute Italiano”(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from “Associazione Italiana Cancro” (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.

Background Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14–15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. Methods By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a “mitogen-independent” phenotype (I-GSCs) from patient’s tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs’ critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher’s exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs’ key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett’s multiple comparison test with the distribution of survival compared by Kaplan–Meier method. Results Here, we assessed that a subset of GSCs from high-grade gliomas is self-sufficient in the activation of regulatory growth signaling. Furthermore, while constitutively present within the same GBM tissue, these GF-independent GSCs cells were endowed with a distinctive functional and molecular repertoire, defined by highly aggressive Wnt5aHigh/EphA2Low profile, as opposed to Wnt5aLow/EphA2High expression in sibling D-GSCs. Regardless of their GBM subtype of origin, I-GSCs, are endowed with a raised in vivo tumorigenic potential than matched D-GSCs, which were fast-growing ex-vivo but less lethal and invasive in vivo. Also, the malignant I-GSCs’ transcriptomic fingerprint faithfully mirrored the original tumor, bringing into evidence key regulators of invasiveness, angiogenesis and immuno-modulators, which became candidates for glioma diagnostic/prognostic markers and therapeutic targets. Particularly, simultaneously counteracting the activity of the tissue invasive mediator Wnt5a and EphA2 tyrosine kinase receptor addictively hindered GSCs’ tumorigenic and invasive ability, thus increasing survival. Conclusion We show how the preservation of a mitogen-independent phenotype in GSCs plays a central role in determining the exacerbated tumorigenic and high mobility features distinctive of GBM. The exploitation of the I-GSCs' peculiar features shown here offers new ways to identify novel, GSCs-specific effectors, whose modulation can be used in order to identify novel, potential molecular therapeutic targets. Furthermore, we show how the combined use of PepA, the anti-Wnt5a drug, and of ephrinA1-Fc to can hinder GSCs’ lethality in a clinically relevant xenogeneic in vivo model thus being conducive to perspective, novel combinatorial clinical application.

Background Colorectal cancer (CRC) harboring BRAFV600E mutation exhibits low response to conventional therapy and poorest prognosis. Due to the emerging correlation between gut microbiota and CRC carcinogenesis, we investigated in serrated BRAFV600E cases the existence of a peculiar fecal microbial fingerprint and specific bacterial markers, which might represent a tool for the development of more effective clinical strategies. Methods By injecting human CRC stem-like cells isolated from BRAFV600E patients in immunocompromised mice, we described a new xenogeneic model of this subtype of CRC. By performing bacterial 16S rRNA sequencing, the fecal microbiota profile was then investigated either in CRC-carrying mice or in a cohort of human CRC subjects. The microbial communities’ functional profile was also predicted. Data were compared with Mann-Whitney U, Welch’s t-test for unequal variances and Kruskal-Wallis test with Benjamini–Hochberg false discovery rate (FDR) correction, extracted as potential BRAF class biomarkers and selected as model features. The obtained mean test prediction scores were subjected to Receiver Operating characteristic (ROC) analysis. To discriminate the BRAF status, a Random Forest classifier (RF) was employed. Results A specific microbial signature distinctive for BRAF status emerged, being the BRAF-mutated cases closer to healthy controls than BRAF wild-type counterpart. In agreement, a considerable score of correlation was also pointed out between bacteria abundance from BRAF-mutated cases and the level of markers distinctive of BRAFV600E pathway, including those involved in inflammation, innate immune response and epithelial-mesenchymal transition. We provide evidence that two candidate bacterial markers, Prevotella enoeca and Ruthenibacterium lactatiformans, more abundant in BRAFV600E and BRAF wild-type subjects respectively, emerged as single factors with the best performance in distinguishing BRAF status (AUROC = 0.72 and 0.74, respectively, 95% confidence interval). Furthermore, the combination of the 10 differentially represented microorganisms between the two groups improved performance in discriminating serrated CRC driven by BRAF mutation from BRAF wild-type CRC cases (AUROC = 0.85, 95% confidence interval, 0.69–1.01). Conclusion Overall, our results suggest that BRAFV600E mutation itself drives a distinctive gut microbiota signature and provide predictive CRC-associated bacterial biomarkers able to discriminate BRAF status in CRC patients and, thus, useful to devise non-invasive patient-selective diagnostic strategies and patient-tailored optimized therapies.

<p>Table S1: Molecular Classification of Low-Grade and Anaplastic Gliomas; Table S2: WNT5A and FRIZZLED5 gene copy number in glioma tissues and hGBM TPCs; Table S3: Ingenuity analysis of the LVWnt5a classical TPCs genes demonstrating the associated cancer genes and molecular and cellular functions (5% FDR); Table S4: Ingenuity analysis of the LVWnt5a U87 and U87 vector genes demonstrating the associated cancer genes and molecular and cellular functions related to Cellular Movement (5% FDR); Table S5: Ingenuity Analysis of the Wnt5a^high Genes Demonstrating The Associated Cancer Genes Molecular and Cellular Function (5% FDR).</p>

<p>Suppl Fig S1: Levels of Wnt5a expression in brain tumors correlate to aggressiveness or outcome; Suppl Fig S2: Wnt5a overexpression sustains TPCs invasive capability; Suppl Fig S3: Elevated Wnt5a expression promotes migratory capability in otherwise poorly motile U87MG, both in vitro and in vivo; Suppl Fig S4: Pathways enrichment of invasive TPCs progeny based on Wnt5a levels; Suppl Fig S5: Mesenchymal hGBM TPCs are endowed with enhanced dispersal ability to distribute widely in the brain as compared to classical TPCs; Suppl Fig S6: Hypothetical mechanism of Wnt5a regulation that confers the infiltratory phenotype in hGBM TPCs;</p>