Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.

Drug tolerant persister (DTP) cells enter into a reversible slow-cycling state after drug treatment. We performed proteomic characterization of the breast cancer (BC) DTP cell secretome after eribulin treatment. We showed that the growth differentiation factor 15 (GDF15) is a protein significantly over-secreted upon eribulin treatment. The biomarker potential of GDF15 was confirmed in 3D-cell culture models using BC cells lines and PDXs, as well as in a TNBC in vivo model. We also found that GDF15 is required for survival of DTP cells. Direct participation of GDF15 and its receptor GFRAL in eribulin-induction of DTPs was established by the enhanced cell killing of DTPs by eribulin seen under GDF15 and GFRAL loss of function assays. Finally, we showed that combination therapy of eribulin plus an anti-GDF15 antibody kills BC-DTP cells. Our results suggest that targeting GDF15 may help eradicate DTP cells and block the onset of acquired resistance.

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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.