Frank K. H. van Landeghem

Extended Data relating to Main Figure 2/Assessment of ALDH1A1+ cells from primary, treatment-naive glioblastoma. A, Bar plot shows ALDH-bright (ALDHbr) vital cells from two additional paired cases of naive vs. experimental (TMZ→eR) and clinical (cR) relapse. Data as mean ± SD. p values by one-way analysis of variance (ANOVA) with Tukey´s post-hoc test. B, Dotplot shows ALDH1A1 mean fluorescence intensity (MFI) in paired BN46 treatment-naive vs. experimental relapse conditions (in vitro exposure to TMZ (TMZ→eR) or irradiation (RT→eR)). Data are normalized to isotype control, mean ± SD. p values calculated by Kruskal-Wallis test with Dunn's post-hoc test. C, Relative ALDH1A1 expression in -knockdown (shALDH1A1) and -overexpression (Ovx) BN46 cells used for indicated experiment. Data shown as mean ± SD, normalized to their respective controls (ALDH1A1 Ovx to GFP Ctrl | shALDH1A1 to shNT Ctrl). D, Left: Brightfield image of BN46 cells in 96-well plates during monitoring by software-based cell recognition in the limiting dilution assay (NyOne®). An exemplary single cell/well is shown at one day post seeding; representative monoclonal colonies of ALDH1A1-knockdown (sh) and -overexpressing (Ovx) cells at day 16 after seeding. Right: Doubling time estimated at day 16 after seeding (see Methods). Data as mean ± SD, p values calculated by Kruskal-Wallis test with Dunn's post-hoc test. E, Left: Cartoon describes the neurosphere experiments shown in Fig. 2H. Right: Phase contrast microscopic appearance of plated 2° neurosphere and respective immunofluorescence visualization of antibody labeling on neurosphere-derived cells. Neuronal phenotype, β3-tubulin (β3-tub); glial phenotypes, glial fibrillary acidic protein (GFAP). Nuclei exposed with DAPI. Scale bars: left: 100 µM, right: 50 µM. F, Table showing quantification data of 1° and 2° neurosphere generations from (E) in the respective treatment-naive BN46 cells. G, All plated 2° neurospheres from assay (E,F) generated neuronal and glial cell phenotypes.

Extended Data relating to Main Figure 3/AKT-driven progression of ALDH1A1+ cells in glioblastoma. A, Flow cytometry histograms showing ALDH1A1 expression in the paired treatment-naive vs. clinical relapse patient samples quantified in main Fig. 3A. Isotype controls in gray. B, Flow cytometry histograms of ALDH1A1 derived from treatment-naive and paired experimental relapse (TMZ→eR) BN46 cells. Data quantification in Supplementary Fig. S2B. C, Cartoon illustrating course of neurosphere experiments, quantified in main Fig. 3B and in Supplementary Fig. S3D, applying treatment-naive and paired experimental and clinical relapse patient cells. Right: Exemplary qRT-PCR data presenting knockdown efficacy of the siRNA approach in respective cells, normalized to siNT control. Mean of duplicates ± SD. D, Source data for Fig. 3B, and for Supplementary Fig. S3E. Dotplots show percent neurosphere-forming cells estimated from neurospheres at 12 days after seeding. Paired patient cell analysis, evaluating 1° and 2° neurospheres of indicated knockdown (siALDH1A1) and respective control (siNT) cell samples. Data shown as mean ± SD. E, Neurosphere assay, similar to main Fig. 3B on paired treatment-naive vs. experimental relapse (TMZ→eR) BN46 cells. Source data in Supplementary Fig. S3D. Data as mean ± SD. F, Protein patterns, similar to Fig. 3D. on paired cells from 2 additional discovery cohort samples. G, Flow cytometry histograms depicting pAKT(Ser473) expression in the paired treatment-naive vs. clinical relapse patient samples quantified in main Fig. 3E. Isotype controls in gray. H, Flow cytometry histograms of pAKT(Ser473) and, right, quantification of data derived from treatment-naive and paired experimental relapse (TMZ→eR) BN46 cells. Data represent mean fluorescence intensities (MFI), ± SD, normalized to isotype control (gray). I, Representative source data for Fig. 3F. Flow cytometry profiles of ALDH1A1/pAKT(Ser473)-labeled, paired treatment-naive vs. clinical relapse cell samples. J, Flow cytometry profiles of of ALDH1A1/pAKT(Ser473)-labeled, paired treatment-naive vs. experimental relapse conditions of BN46 cells (in vitro exposure to TMZ (TMZ→eR) or irradiation (RT→eR)).

Extended Data relating to Main Figure 4/Targeting of AKT-driven subclonal progression of ALDH1A1+ cells. A, Collected source data for main Figs. 4B,C,F-H. Left, diagrams: in vitro cell confluence dynamics of paired treatment naive vs. TMZ-related experimental (TMZ→eR; BN46) or clinical (RT/TMZ; BN118, BN123, BN132) relapse patient cells. Monitoring of cell confluence by software-based cell recognition. Assay specified in Fig. 4A. Right, graphs: Readout results from the indicated cases in line, representing source data for Figs. 4B (Cell Confluence), 4C (Cell Viability), and 4H (Apoptosis). Respective assays specified in the main Figure Legends. Data as mean ± SD. p values calculated by pairwise Wilcoxon rank-sum test followed by multiple testing correction using Benjamini-Hochberg method. B, IF of ALDH1A1 and Ki67 in the BN46-donor cell tumor PDX model. Shown is an example from a TMZ+MK2206-treated mouse (OS = 148 days; low levels of cellular co-expression). Scale bar: 20 µm. C, Comparison of overall survival under the investigated treatment conditions (refer to Fig. 4J). Hazard ratio 95% confidence interval estimates and significance levels derived from Cox regression are shown.

Supplementary Table S1. Discovery cohort of vital cells; Supplementary Table S2. Validation cohort/FFPE tissue samples; Supplementary Table S3. Differentially regulated genes; Supplementary Table S4. Selected gene variants compared between the paired treatment-naive vs. relapse cell samples.

Validation cohort, patient flow diagram. Patient enrollment and final inclusion of FFPE tissue samples in this study is shown. n = 3 patients were excluded from enrollment for reasons listed. The validation cohort comprised paired tissue samples from a total of n = 38 patients. Detailed characteristics in Supplementary Table S4. Note, the separate discovery cohort of paired vital cells is detailed in Supplementary Table S1. The GLASS cohort was used as an additional, external reference comprising data on n=37 patients extracted from (10).

Purpose:Therapy resistance and fatal disease progression in glioblastoma are thought to result from the dynamics of intra-tumor heterogeneity. This study aimed at identifying and molecularly targeting tumor cells that can survive, adapt, and subclonally expand under primary therapy.Experimental Design:To identify candidate markers and to experimentally access dynamics of subclonal progression in glioblastoma, we established a discovery cohort of paired vital cell samples obtained before and after primary therapy. We further used two independent validation cohorts of paired clinical tissues to test our findings. Follow-up preclinical treatment strategies were evaluated in patient-derived xenografts.Results:We describe, in clinical samples, an archetype of rare ALDH1A1+ tumor cells that enrich and acquire AKT-mediated drug resistance in response to standard-of-care temozolomide (TMZ). Importantly, we observe that drug resistance of ALDH1A1+ cells is not intrinsic, but rather an adaptive mechanism emerging exclusively after TMZ treatment. In patient cells and xenograft models of disease, we recapitulate the enrichment of ALDH1A1+ cells under the influence of TMZ. We demonstrate that their subclonal progression is AKT-driven and can be interfered with by well-timed sequential rather than simultaneous antitumor combination strategy.Conclusions:Drug-resistant ALDH1A1+/pAKT+ subclones accumulate in patient tissues upon adaptation to TMZ therapy. These subclones may therefore represent a dynamic target in glioblastoma. Our study proposes the combination of TMZ and AKT inhibitors in a sequential treatment schedule as a rationale for future clinical investigation.

Glioblastoma is the most common and aggressive malignant brain tumor in adults, with an increasing incidence and a poor prognosis. Current challenges in glioblastoma management include rapid tumor growth, limited treatment effectiveness, high recurrence rates, and a significant impact on patients’ quality of life. Given the complexity of glioblastoma care and recent advancements in diagnostic and treatment modalities, updated guidelines are needed in Canada. This Delphi study aimed to develop Canadian consensus recommendations for the diagnosis, classification, and management of newly diagnosed and recurrent glioblastoma. A multidisciplinary panel of 14 Canadian experts in glioblastoma care was convened, and a comprehensive literature review was conducted to synthesize evidence and formulate initial recommendations. Consensus was achieved through three Delphi rounds, in which panelists rated their agreement with recommendation statements on a five-point Likert scale. Statements with ≥75% agreement were accepted, and others were revised for re-voting. Final recommendations were formulated based on the consensus level, strength of evidence, clinical expertise, and consideration of the Canadian healthcare context. These recommendations aim to standardize glioblastoma diagnosis and classification across Canada, provide evidence-based guidance for optimal treatment selection, integrate novel therapies, and enhance the overall quality of care for glioblastoma patients.

Extended Data relating to Main Figure 2/Assessment of ALDH1A1+ cells from primary, treatment-naive glioblastoma. A, Bar plot shows ALDH-bright (ALDHbr) vital cells from two additional paired cases of naive vs. experimental (TMZ→eR) and clinical (cR) relapse. Data as mean ± SD. p values by one-way analysis of variance (ANOVA) with Tukey´s post-hoc test. B, Dotplot shows ALDH1A1 mean fluorescence intensity (MFI) in paired BN46 treatment-naive vs. experimental relapse conditions (in vitro exposure to TMZ (TMZ→eR) or irradiation (RT→eR)). Data are normalized to isotype control, mean ± SD. p values calculated by Kruskal-Wallis test with Dunn's post-hoc test. C, Relative ALDH1A1 expression in -knockdown (shALDH1A1) and -overexpression (Ovx) BN46 cells used for indicated experiment. Data shown as mean ± SD, normalized to their respective controls (ALDH1A1 Ovx to GFP Ctrl | shALDH1A1 to shNT Ctrl). D, Left: Brightfield image of BN46 cells in 96-well plates during monitoring by software-based cell recognition in the limiting dilution assay (NyOne®). An exemplary single cell/well is shown at one day post seeding; representative monoclonal colonies of ALDH1A1-knockdown (sh) and -overexpressing (Ovx) cells at day 16 after seeding. Right: Doubling time estimated at day 16 after seeding (see Methods). Data as mean ± SD, p values calculated by Kruskal-Wallis test with Dunn's post-hoc test. E, Left: Cartoon describes the neurosphere experiments shown in Fig. 2H. Right: Phase contrast microscopic appearance of plated 2° neurosphere and respective immunofluorescence visualization of antibody labeling on neurosphere-derived cells. Neuronal phenotype, β3-tubulin (β3-tub); glial phenotypes, glial fibrillary acidic protein (GFAP). Nuclei exposed with DAPI. Scale bars: left: 100 µM, right: 50 µM. F, Table showing quantification data of 1° and 2° neurosphere generations from (E) in the respective treatment-naive BN46 cells. G, All plated 2° neurospheres from assay (E,F) generated neuronal and glial cell phenotypes.