daoy cells  (ATCC)

Journal: Acta Neuropathologica Communications

Article Title: Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging

doi: 10.1186/s40478-022-01496-4

Figure Lengend Snippet: The 3D hydrogel models display subgroup-specific metabolic characteristics to those observed in MB patients. Several metabolites are known to be specifically high in MB patients . 3D OrbiSIMS mass spectrometry imaging also confirms relatively high metabolite levels of glucose a . Lactate levels b are elevated in Group 3 nodules (D458, HD-MB03), while glutamate c is specifically high in SHH (DAOY, ONS-76). The Group 3-specific metabolite taurine d is also significantly higher in Group 3 nodules compared to SHH (a–d: mean ± SEM; n = 5; a, b: unpaired t-test, c: Mann Whitney test, d: unpaired t-test with Welch correction; * P < 0.05, ** P < 0.01). Gel supernatants of 3 week-old SHH and Group 3 nodules were collected 6, 24, 48 and 72 h after medium change (containing 5000 µM glucose and 2000 µM glutamine) to measure consumption and secretion of glucose e , lactate f, glutamine g and glutamate h . Note that although both subgroups quickly consume glucose and glutamine, only lactate is secreted by both. Interestingly, glutamate secretion is characteristic for SHH nodules and below detection limit in Group 3 nodules (mean ± SEM; n = 3). i Gene expression for glycolysis (ALDOA, LDHB, GALM, AKR1A1, HKDC1, ENO2, ALDH2, ENO3, PGAM2, PFKM, PDHA1, PCK2, ALDH9A1), TCA cycle (SUCLG1, PDHA1, PCK2, MDH2, PC, MDH1, FH) and ECM genes (COL6A1, COL6A2, COL6A3, COL1A1, TNC) at the single cell level are displayed for the SHH (ONS 76) and Group 3 (HD MB03) model (purple: gene expressing cell; grey: non-expressing cell; percentage indicates relative proportion of total cells expressing the specified gene set). Cluster specific expression of these gene sets is shown in Additional file : Fig. S6

Article Snippet: DAOY cell line was obtained from ATCC (Manassas, USA), ONS-76 from Dr. Annette Künkele (Charité Universitätsmedizin Berlin, Germany), HD-MB03 from Dr Till Milde (DKFZ Heidelberg, Germany) and D458 from Dr. John R. Silber (University of Washington, Seattle, USA).

Techniques: Mass Spectrometry, Imaging, MANN-WHITNEY, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging

doi: 10.1186/s40478-022-01496-4

Figure Lengend Snippet: SHH nodules are uniquely characterized by deposits of sulphur-containing species and a collagen-based outer shell. a 3D OrbiSIMS mass spectrometry imaging identified several sulphur-containing species within the outer shell of ONS 76 nodules that are non-specifically distributed in HD-MB03 nodules. b Similar to the localization of sulphur species, collagens (Col I and Col VI) are specifically located at the outer surface of SHH (DAOY, ONS-76) nodules compared to nonspecific or no expression in Group 3 nodules (D458, HD-MB03). c Expression of COL6A1, COL6A2, COL6A3, COL1A1, COL1A2 and COL3A1 is significantly higher in SHH patients compared to Group 3 patients in the Cavalli data set (Cavalli et al. , SHH: n = 223 and Group 3: n = 144; Kruskal–Wallis test with Dunn’s post hoc test; *** P < 0.001)

Article Snippet: DAOY cell line was obtained from ATCC (Manassas, USA), ONS-76 from Dr. Annette Künkele (Charité Universitätsmedizin Berlin, Germany), HD-MB03 from Dr Till Milde (DKFZ Heidelberg, Germany) and D458 from Dr. John R. Silber (University of Washington, Seattle, USA).

Techniques: Mass Spectrometry, Imaging, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging

doi: 10.1186/s40478-022-01496-4

Figure Lengend Snippet: Fumarate accumulation is predominantly observed in Group 3 models. a 3D OrbiSIMS analysis of TCA metabolites after 16 h reveal low levels of malate (m/z 133.01), pyruvate (m/z 88.02), isocitrate (m/z 174.02) and glutamate (m/z 146.05), intermediate levels of α-ketoglutarate (m/z 145.01), high levels of succinate (m/z 117.02) and very high levels of fumarate (m/z 115.00). b Normalised intensity measurements confirm the accumulation of succinate and fumarate and the concurrent lack of malate after 16 h in ONS-76 and HD-MB03 cells. c Gel supernatants of 3 week-old SHH (DAOY, ONS 76) and Group 3 (D458, HD MB03) nodules were collected 6, 24, 48 and 72 h after medium change to quantify the secretion levels of fumarate and malate. Note the increasingly higher fumarate levels in the group 3 nodules compared to SHH nodules over time. Malate levels were below the assay detection limit at all time points (mean; n = 3)

Article Snippet: DAOY cell line was obtained from ATCC (Manassas, USA), ONS-76 from Dr. Annette Künkele (Charité Universitätsmedizin Berlin, Germany), HD-MB03 from Dr Till Milde (DKFZ Heidelberg, Germany) and D458 from Dr. John R. Silber (University of Washington, Seattle, USA).

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging

doi: 10.1186/s40478-022-01496-4

Figure Lengend Snippet: A combination of chemotherapy and NRF2 inhibition significantly improves long-term treatment of Group 3 models. a A scheme illustrates the course of the long-term drug treatment assay as previously established . After 3 weeks of growth inside the HA hydrogels the SHH cell lines ONS-76 b , DAOY c and the Group 3 cell lines HD-MB-03 d , D458 e were treated four times with either 10 nM vincristine (green), 5 µM ML385 (NRF2 inhibitor; blue) or a combination (red) or vehicle (black) during one week and cell viability was monitored for the following four weeks in the absence of drug/vehicle present anymore. In the p53 wt SHH cell line ONS-76 only the chemotherapeutic reagent vincristine significantly decreases cell viability, while the NRF2 inhibitor (ML385) alone and in combination with vincristine are also effective in the p53mut SHH cell line DAOY. In contrast, in both Group 3 models the combination of vincristine and NRF2 inhibitor significantly reduced cell viability. (mean ± SEM, n = 3; Two-way ANOVA and Dunnett’s post hoc test, * P < 0.05, ** P < 0.01 and *** P < 0.001 all relative to DMSO according to colour code). Analysis of the biggest publicly available MB data base shows that NRF2 (gene name: NFE2L2 ) gene expression does not predict survival of SHH ( f ; logrank test, p = 0.924) patients, but is associated with worse survival in Group 3 patients ( g ; logrank test, p = 0.078). Note the exclusive effect of NRF2 expression in Group 3 patients

Article Snippet: DAOY cell line was obtained from ATCC (Manassas, USA), ONS-76 from Dr. Annette Künkele (Charité Universitätsmedizin Berlin, Germany), HD-MB03 from Dr Till Milde (DKFZ Heidelberg, Germany) and D458 from Dr. John R. Silber (University of Washington, Seattle, USA).

Techniques: Inhibition, Expressing