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Updated: Sep 10, 2025
Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells
Published on: July 12, 2022
High content-imaging drug synergy screening identifies specific senescence-related vulnerabilities of mesenchymal
Sonja Herter1,2,3,4, Marta Emperador1,2,3,4, Kyriaki Smyrilli1,2,3,4
1Hopp Children's Cancer Center Heidelberg (KiTZ), 69120, Heidelberg, Germany.
Abstract:
Neuroblastomas encompass malignant cells with varying degrees of differentiation, ranging from adrenergic (adr) cells resembling the sympathoadrenal lineage to undifferentiated, stem-cell-like mesenchymal (mes) cancer cells. Relapsed neuroblastomas, which often have mesenchymal features, have a poor prognosis and respond less to anticancer therapies, necessitating the development of novel treatment strategies. To identify novel treatment options, we analyzed the sensitivity of 91 pediatric cell models, including patient-derived tumoroid cultures, to a drug library of 76 anti-cancer drugs at clinically relevant concentrations. This included 24 three-dimensionally cultured neuroblastoma cell lines representing the range of mesenchymal to adrenergic subtypes. High-throughput ATP-based luminescence measurements were compared to high-content confocal imaging. With machine learning-supported imaging analysis, we focused on changes in the lysosomal compartment as a marker for therapy-induced senescence and assessed the basal lysosomal levels in a subset of untreated mesenchymal versus adrenergic cells. We correlated these findings with pathway activity signatures based on bulk RNA and scRNAseq. Comprehensive image-based synergy screens with spheroid cultures validated the combined effects of selected drugs on proliferation and cytotoxicity. Mesenchymal models presented high basal lysosomal levels correlating with senescence-associated secretory phenotype (SASP) and sphingolipid metabolism pathways. Chemotherapy treatment further increased lysosome numbers, indicative of therapy-induced senescence. Furthermore, the mesenchymal subtypes correlated with MAPK activity and sensitivity to MAPK pathway inhibitors. Lysosomal and SASP signaling is druggable by inhibitors of lysosomal acid sphingomyelinase (SLMi) or senolytics, including BCL2-family inhibitors. Especially the sequential combination of MEK inhibitors (MEKi) with BCL2-family inhibitors was the most effective on relapsed neuroblastoma cell lines. Gene expression analysis of 223 patient samples, drug sensitivity profiling of five patient-derived fresh tissue cultures, and in vivo zebrafish embryo neuroblastoma xenograft models confirmed these findings. Inhibition of MAPK signaling in combination with BCL2-family inhibitors is a novel treatment option for patients suffering from relapsed neuroblastomas.
Insights
Novel treatments for relapsed neuroblastoma, a cancer with poor prognosis, were identified. Combining MEK inhibitors with BCL2-family inhibitors effectively targeted mesenchymal neuroblastoma cells by modulating lysosomal and MAPK pathways.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Neuroblastomas exhibit diverse differentiation, from adrenergic to mesenchymal subtypes.
- Relapsed neuroblastomas, often mesenchymal, show poor prognosis and resistance to therapy.
- Novel therapeutic strategies are crucial for improving outcomes in relapsed neuroblastoma.
Purpose of the Study:
- To identify novel therapeutic targets and drug combinations for relapsed neuroblastoma.
- To analyze drug sensitivity across a spectrum of neuroblastoma subtypes.
- To investigate the role of lysosomal and MAPK pathways in neuroblastoma treatment response.
Main Methods:
- Screened 76 anti-cancer drugs against 91 pediatric neuroblastoma cell models, including 3D cultures.
- Utilized high-content imaging and machine learning to analyze lysosomal compartment changes.
- Correlated cellular phenotypes with pathway activity signatures from RNA sequencing data.
- Validated drug synergy using spheroid cultures and in vivo zebrafish xenograft models.
Main Results:
- Mesenchymal neuroblastoma models showed high basal lysosomal levels, correlating with SASP and sphingolipid metabolism.
- Chemotherapy increased lysosome numbers, indicating therapy-induced senescence.
- Mesenchymal subtypes were sensitive to MAPK pathway inhibitors.
- Sequential combination of MEK inhibitors and BCL2-family inhibitors demonstrated significant efficacy in relapsed neuroblastoma models.
Conclusions:
- Lysosomal and SASP signaling are druggable targets in neuroblastoma.
- Inhibition of MAPK signaling combined with BCL2-family inhibitors represents a promising novel treatment for relapsed neuroblastomas.
- Findings were validated across patient samples, fresh tissue cultures, and in vivo models.
