Genomic Exploration of Distinct Molecular Phenotypes Steering Temozolomide Resistance Development in Patient-Derived Glioblastoma Cells

Chemotherapy using temozolomide is the standard treatment for patients with glioblastoma. Despite treatment, prognosis is still poor largely due to the emergence of temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. To induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells’ molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell RNA sequencing on four of the cell cultures to track the evolution of temozolomide resistance. The induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as “adaptive” (ADA) or “non-adaptive” (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. The adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumor’s ability to survive. Depending on the tumor’s adaptability potential, subpopulations with acquired resistance mechanisms may arise.</p

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers

Background: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). Methods: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. Results: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. Conclusion:GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.</p

Author Correction:Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts (Nature Genetics, (2021), 53, 1, (86-99), 10.1038/s41588-020-00750-6)

This paper was originally published without open access. As of the date of this correction, the paper is available online as an open-access paper under a Creative Commons Attribution 4.0 International License. *Lists of authors and their affiliations appear online

A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate

<div><p>Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [¹⁸F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [¹¹C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.</p></div

Physiological effects of sunitinib in two rodent models.

<p>Effects of sunitinib treatment on MAP (A, n = 9/group) and LVEF (B, n = 13/group) in female Balb/CJ mice. D-E) Effect of sunitinib treatment on systolic blood pressure (C), LVEF (D) and heart rate (E) in female Sprague-Dawley rats (n = 6/group, Significant difference between groups (unpaired t-test, *p<0.05 **p<0.01 ***p<0.001), Significant change from pre-treatment values (paired t-test, †p<0.05 ††p<0.01 †††p<0.001). Error bars = SEM)</p

Proteomic analyses of sunitinib effects in myocardial tissue.

<p>The most significantly perturbed canonical pathways from (A) crude membrane and (B) cytosolic fractions from sunitinib treated and control mouse myocardial tissue (n = 4/group) C) Proteins perturbed in the three most significantly enriched canonical pathways. D) Ingenuity Pathway Analysis (IPA) to determine the most significantly enriched network which is centred on Mitochondrial complex 1. Colour intensity indicates the degree of up- (red) or down- (green) regulation. IPA predictions are shown in blue (predicted inhibition) and orange (predicted activation). Black indicates no predicted effect. Continuous lines indicate a direct relationship between two proteins; a discontinuous line indicates indirect association.</p

Effect of sunitinib treatment on myocardial metabolism and perfusion measured by PET.

<p>A) Myocardial metabolic rate of glucose (MMRG) (mice, n = 10/group), B) K_mono (rats, n = 6/group). C-E) The effects of sunitinib treatment on overall perfusion of the myocardium in the rat model: (C) Representative images of the myocardium as seen in Carimas software (version 2.7) which is used to select region of interest (ROI), subsequently used to construct 17 segment heat maps for the 11C-Acetate data (rats) (D) which are used to determine global perfusion values. D) Representative pre- and 5 days post-treatment in one treated and one vehicle rat E) The effects of sunitinib treatment on overall perfusion of the myocardium (rats, n = 6/group, * = significant difference between groups (unpaired t-test, p<0.05), Significant change from pre-treatment (paired t-test, †p<0.05 ††p<0.01), bar* indicates significantly different overall on-treatment values (two-way ANOVA p < 0.05) Error bars = SEM).</p

Confirmation of proteomic findings in mouse myocardial tissue.

<p>A) ELISA to measure Mitochondrial complex 1 levels in myocardial tissue lysates. B) Immunoblot analysis of lysates from the cytosolic fraction of myocardial tissue from vehicle or sunitinib treated animals (n = 4/group) as described. C-G) Densitometry assessment of the presented western blots as described (* p < 0.05, error bars represent SEM).</p