Bogdan Munteanu

Label-Free in Situ Monitoring of Histone Deacetylase Drug Target Engagement by Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Biotyping and Imaging

Measurements of target activation in cells or tissues are key indicators of efficacy during drug development. In contrast to established methods that require reagents and multiple preprocessing steps, reagent-free in situ analysis of engaged drug targets or target-proximal pharmacodynamic signatures in solid tumors remains challenging. Here, we demonstrate that label-free quantification of histone acetylation-specific mass shifts by matrix-assisted laser desorption ionization (MALDI) mass spectrometry biotyping can be used for measurement of cellular potency of histone deacetylase inhibitors in intact cells. Furthermore, we employ MALDI mass spectrometry imaging of these mass shifts to visualize the spatiotemporal distribution of acetylated histones and thus the tumor-selective pharmacodynamic responses in a mouse model of gastrointestinal cancer. Taken together, our study suggests that the monitoring of drug-induced mass shifts in protein ion intensity fingerprints or images may be a powerful analytical tool in pharmacology and drug discovery.

Emergence of whole-cell MALDI - MS biotyping for high-throughput bioanalysis of mammalian cells?

Since their inception in the 1970s, methods for classification of microorganisms based on mass spectral fingerprints obtained by MALDI-TOF MS have become a mainstay in environmental as well as in clinical microbiology. Recently, related whole-cell MALDI-TOF fingerprinting workflows have been adopted for the classification of mammalian cells. In this report we summarize this work and discuss the challenges of adapting whole-cell MS fingerprinting methods for the successful classification of mammalian cells. We highlight current limitations as well as opportunities and emerging applications of this technology in industrial and clinical settings, such as cell-line authentication, clinical diagnostics, and quality and productivity control in bioprocesses.

Monitoring CHO cell cultures: cell stress and early apoptosis assessment by mass spectrometry.

Mammalian cells, especially CHO (Chinese Hamster Ovary), are an important host for the production of biopharmaceuticals. Early detection of cellular stress and the onset of apoptosis, ultimately leading to a reduced viability of the culture, are important with respect to process development and monitoring. In this work, intact cell MALDI mass spectrometry (ICM MS) biotyping was used to rapidly and sensitively detect cell stress and the onset of apoptosis at line in CHO cell cultures. We describe the identification of specific and highly reproducible stress and apoptosis related changes in m/z signal intensities that allowed prediction of upcoming cell viability changes approximately 24h earlier than standard culture monitoring. Furthermore, early identification of apoptosis onset was comparable to that using a sensitive, albeit offline, detection method. By comparison with ICM MS analysis of apoptosis induced cultures, many of the m/z values were identified as apoptosis-specific. A classification model for discrimination of unknown samples regarding their cellular viability/apoptosis status was developed based on a condensed set of 51 m/z values. The fast, robust and automated acquisition of cell state specific MS signatures could become a promising tool for CHO culture monitoring.

The stress kinase GCN2 does not mediate suppression of antitumor T cell responses by tryptophan catabolism in experimental melanomas

ABSTRACT Tryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific Gcn2 knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of Tdo did not differentially affect GCN2-proficient vs. GCN2-deficient T cells in vivo. Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer

Gastric cancer (GC) remains a malignant disease with high mortality. Patients are frequently diagnosed in advanced stages where survival prognosis is poor. Thus, there is high medical need to find novel drug targets and treatment strategies. Recently, the comprehensive molecular characterization of GC subtypes revealed mutations in the small GTPase RHOA as a hallmark of diffuse-type GC. RHOA activates RHO-associated protein kinases (ROCK1/2) which regulate cell contractility, migration and growth and thus may play a role in cancer. However, therapeutic benefit of RHO-pathway inhibition in GC has not been shown so far. The ROCK1/2 inhibitor 1-(5-isoquinoline sulfonyl)-homopiperazine (HA-1077, fasudil) is approved for cerebrovascular bleeding in patients. We therefore investigated whether fasudil (i.p., 10 mg/kg per day, 4 times per week, 4 weeks) inhibits tumor growth in a preclinical model of GC. Fasudil evoked cell death in human GC cells and reduced the tumor size in the stomach of CEA424-SV40 TAg transgenic mice. Small animal PET/CT confirmed preclinical efficacy. Mass spectrometry imaging identified a translatable biomarker for mouse GC and suggested rapid but incomplete in situ distribution of the drug to gastric tumor tissue. RHOA expression was increased in the neoplastic murine stomach compared with normal non-malignant gastric tissue, and fasudil reduced (auto) phosphorylation of ROCK2 at THR249 in vivo and in human GC cells in vitro. In sum, our data suggest that RHO-pathway inhibition may constitute a novel strategy for treatment of GC and that enhanced distribution of future ROCK inhibitors into tumor tissue may further improve efficacy.

Whole/Intact Cell MALDI MS Biotyping in Mammalian Cell Analysis

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) biotyping of microorganisms has arguably emerged as the premier application of MALDI MS in recent years. It is now widely used in clinical microbiology as an approved diagnostic tool and has recently been applied beyond the clinic, e.g., in environmental microbiology. Not long ago the adaptation of MALDI MS biotyping methods to applications with non-preprocessed, whole mammalian cells has started. Important tasks such as standardization of cell culture, sample preparation, and data acquisition as well as development of suitable databases, classification algorithms, and software are still at an early stage. However, applications of mammalian cell biotyping by MALDI MS in the clinic and beyond have already become visible, in particular in the pharmaceutical and diagnostics industry. They range from general quality assurance of mammalian assay cell lines, to at-line quality control of bioprocesses during development or production, and to the development of MALDI MS-based assays. In this chapter, we describe general methods for whole-cell MALDI MS biotyping of mammalian cells and highlight their application in development of cell-based assays for discovery and profiling of histone deacetylase (HDAC) inhibitors.

Intact cell MALDI mass spectrometry biotyping for "at-line" monitoring of apoptosis progression in CHO cell cultures

Background Mammalian cell cultures, especially Chinese Hamster Ovary (CHO), are the predominant host for the production of biologics. Despite considerable progress in industry and academia alike (also enforced e.g. by the Process Analytical Technology Initiative of the FDA), particularly in the field of process monitoring there is still a need for innovative methods enabling improvement of process monitoring. For optimized process control it would be imperative to know as early as possible “when a cell needs what”, when it is stressed, running into substrate limitations etc., at best in an online or robust at line format. Intact cell MALDI mass spectrometry (ICM-MS) biotyping, a method used successfully in the field of clinical and environmental microbiology, is getting more attention in the context of mammalian cell cultivation. Here we report preliminary results of an assessment of a fast and high throughput at line capable ICM MS method for cell culture monitoring. As a first example, we choose apoptosis monitoring. The identification of specific mass spectrometric signatures related to early stages of apoptosis using ICMMS biotyping as reported here could be a promising tool for CHO culture.

Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.

Automated analysis of lipid drug-response markers by combined fast and high-resolution whole cell MALDI mass spectrometry biotyping

Recent advances in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have enabled whole cell-MALDI mass spectrometry biotyping of drug-treated cultured cells for rapid monitoring of known abundant pharmacodynamic protein markers such as polyacetylated histones. In contrast, generic and automated analytical workflows for discovery of such pharmacodynamic markers, in particular lipid markers, and their use in cellular tests of drug-like compounds are still lacking. Here, we introduce such a workflow and demonstrate its utility for cellular drug-response monitoring of BCR-ABL tyrosine kinase inhibitors in K562 leukemia cells: First, low-molecular mass features indicating drug responses are computationally extracted from groups of MALDI-TOF mass spectra. Then, the lipids/metabolites corresponding to these features are identified by MALDI-Fourier transformation mass spectrometry. To demonstrate utility of the method, we identify the potassium adduct of phosphatidylcholine PC(36:1) as well as heme B, a marker for erythroid differentiation, as markers for a label-free MALDI MS-based test of cellular responses to BCR-ABL inhibitors. Taken together, these results suggest that MALDI-TOF mass spectrometry of lipids and other low molecular mass metabolites could support cell-based drug profiling.

Fourier Transform Infrared Microscopy Enables Guidance of Automated Mass Spectrometry Imaging to Predefined Tissue Morphologies

Multimodal imaging combines complementary platforms for spatially resolved tissue analysis that are poised for application in life science and personalized medicine. Unlike established clinical in vivo multimodality imaging, automated workflows for in-depth multimodal molecular ex vivo tissue analysis that combine the speed and ease of spectroscopic imaging with molecular details provided by mass spectrometry imaging (MSI) are lagging behind. Here, we present an integrated approach that utilizes non-destructive Fourier transform infrared (FTIR) microscopy and matrix assisted laser desorption/ionization (MALDI) MSI for analysing single-slide tissue specimen. We show that FTIR microscopy can automatically guide high-resolution MSI data acquisition and interpretation without requiring prior histopathological tissue annotation, thus circumventing potential human-annotation-bias while achieving >90% reductions of data load and acquisition time. We apply FTIR imaging as an upstream modality to improve accuracy of tissue-morphology detection and to retrieve diagnostic molecular signatures in an automated, unbiased and spatially aware manner. We show the general applicability of multimodal FTIR-guided MALDI-MSI by demonstrating precise tumor localization in mouse brain bearing glioma xenografts and in human primary gastrointestinal stromal tumors. Finally, the presented multimodal tissue analysis method allows for morphology-sensitive lipid signature retrieval from brains of mice suffering from lipidosis caused by Niemann-Pick type C disease.