RESUMO
Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Terapia de Alvo Molecular , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica/métodos , Análise de Célula Única/métodos , Adaptação Fisiológica , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Butadienos/administração & dosagem , Dasatinibe/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Perfilação da Expressão Gênica , Genes erbB-1 , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Modelos Biológicos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/fisiologia , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Nitrilas/administração & dosagem , Pirazinas/administração & dosagem , Seleção Genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady-state model that describes energy potential in single cells and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low-dose (1 µM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glutamina/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Carbocianinas/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/farmacologia , Transferência Ressonante de Energia de Fluorescência , Glioblastoma/tratamento farmacológico , Humanos , Sondas MolecularesRESUMO
The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose (18F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro18F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling. Further, we find a weak positive correlation between cell size and rate of glycolysis.
