Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phospho-ERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.
Monitoring of single cell signal transduction in leukemic cellular subsets has been proposed to provide deeper understanding of disease biology and prognosis, but has so far not been tested in a clinical trial of targeted therapy. We developed a complete mass cytometry analysis pipeline for characterization of intracellular signal transduction patterns in the major leukocyte subsets of chronic phase chronic myeloid leukemia. Changes in phosphorylated Bcr-Abl1 and the signaling pathways involved were readily identifiable in peripheral blood single cells already within three hours of the patient receiving oral nilotinib. The signal transduction profiles of healthy donors were clearly distinct from those of the patients at diagnosis. Furthermore, using principal component analysis, we could show that phosphorylated transcription factors STAT3 (Y705) and CREB (S133) within seven days reflected BCR-ABL1IS at three and six months. Analyses of peripheral blood cells longitudinally collected from patients in the ENEST1st clinical trial showed that single cell mass cytometry appears to be highly suitable for future investigations addressing tyrosine kinase inhibitor dosing and effect. (clinicaltrials.gov identifier: 01061177)
Treatment with ultrasound and microbubbles (sonoporation) to enhance therapeutic efficacy in cancer therapy is rapidly expanding, but there is still very little consensus as to why it works. Despite the original assumption that pore formation in the cell membrane is responsible for increased uptake of drugs, the molecular mechanisms behind this phenomenon are largely unknown. We treated cancer cells (MOLM-13) and healthy peripheral blood mononuclear cells (PBMCs) with ultrasound at three acoustic intensities (74, 501, 2079 mW/cm2) ± microbubbles. We subsequently monitored the intracellular response of a number of key signaling pathways using flow cytometry or western blotting 5 min, 30 min and 2 h post-treatment. This was complemented by studies on uptake of a cell impermeable dye (calcein) and investigations of cell viability (cell count, Hoechst staining and colony forming assay). Ultrasound + microbubbles resulted in both early changes (p38 (Arcsinh ratio at high ultrasound + microbubbles: +0.5), ERK1/2 (+0.7), CREB (+1.3), STAT3 (+0.7) and AKT (+0.5)) and late changes (ribosomal protein S6 (Arcsinh ratio at low ultrasound: +0.6) and eIF2α in protein phosphorylation). Observed changes in protein phosphorylation corresponded to changes in sonoporation efficiency and in viability, predominantly in cancer cells. Sonoporation induced protein phosphorylation in healthy cells was pronounced (p38 (+0.03), ERK1/2 (−0.03), CREB (+0.0), STAT3 (−0.1) and AKT (+0.04) and S6 (+0.2)). This supports the hypothesis that sonoporation may enhance therapeutic efficacy of cancer treatment, without causing damage to healthy cells.
Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.
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