American Association for Cancer Research
ccr-22-0611_supplementary_figure_s3_supps3.png (1.92 MB)

Supplementary Figure S3 from A Sequential Targeting Strategy Interrupts AKT-Driven Subclone-Mediated Progression in Glioblastoma

Download (1.92 MB)
posted on 2023-04-01, 00:02 authored by Sied Kebir, Vivien Ullrich, Pia Berger, Celia Dobersalske, Sarah Langer, Laurèl Rauschenbach, Daniel Trageser, Andreas Till, Franziska K. Lorbeer, Anja Wieland, Timo Wilhelm-Buchstab, Ashar Ahmad, Holger Fröhlich, Igor Cima, Shruthi Prasad, Johann Matschke, Verena Jendrossek, Marc Remke, Barbara M. Grüner, Alexander Roesch, Jens T. Siveke, Christel Herold-Mende, Tobias Blau, Kathy Keyvani, Frank K.H. van Landeghem, Torsten Pietsch, Jörg Felsberg, Guido Reifenberger, Michael Weller, Ulrich Sure, Oliver Brüstle, Matthias Simon, Martin Glas, Björn Scheffler

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)).


Deutschen Konsortium für Translationale Krebsforschung (DKTK)

Deutsche Forschungsgemeinschaft (DFG)

Bundesministerium für Bildung und Forschung (BMBF)

Volkswagen Foundation (VolkswagenStiftung)

Deutsche Krebshilfe (German Cancer Aid)

Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)



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. 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. 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. 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.

Usage metrics

    Clinical Cancer Research





    Ref. manager