Biology subjects

Heap, R. E.

Publications and source records attributed to Heap, R. E..

3 recordsLinked to original sources

bioRxiv2021

A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukemia and multiple myeloma.

Inflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukaemia (AML), by tracking several biological features ionizing from only 2,500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later generation BCR-ABL inhibitors inhibit the p38-MK2/3 signalling axis which suppressed the expression of inflammatory cytokines, cell adhesion and innate immunity markers in activated human monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases. Key PointsLabel-free cell-based assay identifies new anti-inflammatory drugs using MALDI-TOF MS. Nilotinib reduces inflammation by inhibition of MAPK14-MK2/3 signalling axis in AML.

cancer biology↗

bioRxiv2020

Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation

Bone marrow-derived macrophages (BMDMs) are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell conditioned media (LCCM) and how it affects BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period, identifying 2,193 proteins. While M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF or LCCM, respectively. Interestingly, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.

immunology↗

bioRxiv2019

Profiling embryonic stem cell differentiation by MALDI-MS: development of a reproducible and robust sample preparation workflow

MALDI-TOF mass spectrometry (MS) is widely used to characterize and biotype bacterial samples, but a complimentary method for profiling of mammalian cells is still underdeveloped. Current approaches vary dramatically in their sample preparation methods and are not suitable for high-throughput studies. In this work, we present a universal workflow for mammalian cell MALDI-TOF MS analysis and apply it to distinguish ground-state naive and differentiating mouse embryonic stem cells (mESCs), which can be used as a model for drug discovery. We employed a systematic approach testing many parameters to evaluate how efficiently and reproducibly each method extracted unique mass features from four different human cell lines. This data enabled us to develop a unique mammalian cell MALDI-TOF workflow involving a freeze-thaw cycle, methanol fixing and CHCA matrix to generate spectra that yield maximum information and are highly reproducible. We applied our optimized workflow to distinguish naive and differentiating populations using multivariate analysis and reproducibly identifying unique features. Consequently, our MALDI-TOF MS profiling method enables identification of unique biomarkers and robust phenotyping of mESC differentiation. This method can in the future be applied to profile other cell types and expanded towards cellular MALDI-TOF MS screening assays.

biochemistry↗