figure
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 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.
Funding
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)
History
ARTICLE ABSTRACT
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.
