Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities

Apparent diffusion coefficient for molecular subtyping of non-gadolinium-enhancing WHO grade II/III glioma: volumetric segmentation versus two-dimensional region of interest analysis

OBJECTIVES: To investigate if quantitative apparent diffusion coefficient (ADC) measurements can predict genetic subtypes of non-gadolinium-enhancing gliomas, comparing whole tumour against single slice analysis. METHODS: Volumetric T2-derived masks of 44 gliomas were co-registered to ADC maps with ADC mean (ADCmean) calculated. For the slice analysis, two observers placed regions of interest in the largest tumour cross-section. The ratio (ADCratio) between ADCmeanin the tumour and normal appearing white matter was calculated for both methods. RESULTS: Isocitrate dehydrogenase (IDH) wild-type gliomas showed the lowest ADC values throughout (p < 0.001). ADCmeanin the IDH-mutant 1p19q intact group was significantly higher than in the IDH-mutant 1p19q co-deleted group (p < 0.01). A volumetric ADCmeanthreshold of 1201 × 10-6mm2/s identified IDH wild-type with a sensitivity of 83% and a specificity of 86%; a volumetric ADCratiocut-off value of 1.65 provided a sensitivity of 80% and a specificity of 92% (area under the curve (AUC) 0.9-0.94). A slice ADCratiothreshold for observer 1 (observer 2) of 1.76 (1.83) provided a sensitivity of 80% (86%), specificity of 91% (100%) and AUC of 0.95 (0.96). The intraclass correlation coefficient was excellent (0.98). CONCLUSIONS: ADC measurements can support the distinction of glioma subtypes. Volumetric and two-dimensional measurements yielded similar results in this study. KEY POINTS: • Diffusion-weighted MRI aids the identification of non-gadolinium-enhancing malignant gliomas • ADC measurements may permit non-gadolinium-enhancing glioma molecular subtyping • IDH wild-type gliomas have lower ADC values than IDH-mutant tumours • Single cross-section and volumetric ADC measurements yielded comparable results in this study

Filtration‐histogram based magnetic resonance texture analysis (Mrta) for the distinction of primary central nervous system lymphoma and glioblastoma

Primary central nervous system lymphoma (PCNSL) has variable imaging appearances, which overlap with those of glioblastoma (GBM), thereby necessitating invasive tissue diagnosis. We aimed to investigate whether a rapid filtration histogram analysis of clinical MRI data supports the distinction of PCNSL from GBM. Ninety tumours (PCNSL n = 48, GBM n = 42) were analysed using pre‐treatment MRI sequences (T1‐weighted contrast‐enhanced (T1CE), T2‐weighted (T2), and apparent diffusion coefficient maps (ADC)). The segmentations were completed with proprietary texture analysis software (TexRAD version 3.3). Filtered (five filter sizes SSF = 2–6 mm) and unfil-tered (SSF = 0) histogram parameters were compared using Mann‐Whitney U non‐parametric test-ing, with receiver operating characteristic (ROC) derived area under the curve (AUC) analysis for significant results. Across all (n = 90) tumours, the optimal algorithm performance was achieved using an unfiltered ADC mean and the mean of positive pixels (MPP), with a sensitivity of 83.8%, specificity of 8.9%, and AUC of 0.88. For subgroup analysis with >1/3 necrosis masses, ADC permit-ted the identification of PCNSL with a sensitivity of 96.9% and specificity of 100%. For T1CE‐derived regions, the distinction was less accurate, with a sensitivity of 71.4%, specificity of 77.1%, and AUC of 0.779. A role may exist for cross‐sectional texture analysis without complex machine learning models to differentiate PCNSL from GBM. ADC appears the most suitable sequence, especially for necrotic lesion distinction

Microglia promote glioblastoma via mTOR-mediated immunosuppression of the tumour microenvironment

Tumour-associated microglia/macrophages (TAM) are the most numerous non-neoplastic populations in the tumour microenvironment in glioblastoma multiforme (GBM), the most common malignant brain tumour in adulthood. The mTOR pathway, an important regulator of cell survival/proliferation, is upregulated in GBM, but little is known about the potential role of this pathway in TAM. Here, we show that GBM-initiating cells induce mTOR signalling in the microglia but not bone marrow-derived macrophages in both in vitro and in vivo GBM mouse models. mTOR-dependent regulation of STAT3 and NF-jB activity promotes an immunosuppressive microglial phenotype. This hinders effector T-cell infiltration, proliferation and immune reactivity, thereby contributing to tumour immune evasion and promoting tumour growth in mouse models. The translational value of our results is demonstrated in whole transcriptome datasets of human GBM and in a novel in vitro model, whereby expandedpotential stem cells (EPSC)-derived microglia-like cells are conditioned by syngeneic patient-derived GBM-initiating cells. These results raise the possibility that microglia could be the primary target of mTOR inhibition, rather than the intrinsic tumour cells in GB

Deficient Plakophilin-1 Expression Due to a Mutation in PKP1 Causes Ectodermal Dysplasia-Skin Fragility Syndrome in Chesapeake Bay Retriever Dogs

In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved