Single-cell metabolomics by mass spectrometry: ready for primetime?

Single-cell metabolomics (SCMs) is a powerful tool for studying cellular heterogeneity by providing insight into the differences between individual cells. With the development of a set of promising SCMs pipelines, this maturing technology is expected to be widely used in biomedical research. However, before SCMs is ready for primetime, there are some challenges to overcome. In this review, we summarize the trends and challenges in the development of SCMs. We also highlight the latest methodologies, applications, and sketch the perspective for integration with other omics and imaging approaches.Toxicolog

Crosstalk between hypoxia and extracellular matrix in the tumor microenvironment in breast cancer

Even though breast cancer is the most diagnosed cancer among women, treatments are not always successful in preventing its progression. Recent studies suggest that hypoxia and the extracellular matrix (ECM) are important in altering cell metabolism and tumor metastasis. Therefore, the aim of this review is to study the crosstalk between hypoxia and the ECM and to assess their impact on breast cancer progression. The findings indicate that hypoxic signaling engages multiple mechanisms that directly contribute to ECM remodeling, ultimately increasing breast cancer aggressiveness. Second, hypoxia and the ECM cooperate to alter different aspects of cell metabolism. They mutually enhance aerobic glycolysis through upregulation of glucose transport, glycolytic enzymes, and by regulating intracellular pH. Both alter lipid and amino acid metabolism by stimulating lipid and amino acid uptake and synthesis, thereby providing the tumor with additional energy for growth and metastasis. Third, YAP/TAZ signaling is not merely regulated by the tumor microenvironment and cell metabolism, but it also regulates it primarily through its target c-Myc. Taken together, this review provides a better understanding of the crosstalk between hypoxia and the ECM in breast cancer. Additionally, it points to a role for the YAP/TAZ mechanotransduction pathway as an important link between hypoxia and the ECM in the tumor microenvironment, driving breast cancer progression.Toxicolog

Acute vs. chronic vs. intermittent hypoxia in breast cancer: a review on its application in in vitro research

Hypoxia has been linked to elevated instances of therapeutic resistance in breast cancer. The exposure of proliferating cancer cells to hypoxia has been shown to induce an aggressive phenotype conducive to invasion and metastasis. Regions of the primary tumors in the breast may be exposed to different types of hypoxia including acute, chronic or intermittent. Intermittent hypoxia (IH), also called cyclic hypoxia, is caused by exposure to cycles of hypoxia and reoxygenation (H-R cycles). Importantly, there is currently no consensus amongst the scientific community on the total duration of hypoxia, the oxygen level, and the possible presence of H-R cycles. In this review, we discuss current methods of hypoxia research, to explore how exposure regimes used in experiments are connected to signaling by different hypoxia inducible factors (HIFs) and to distinct cellular responses in the context of the hallmarks of cancer. We highlight discrepancies in the existing literature on hypoxia research within the field of breast cancer in particular and propose a clear definition of acute, chronic, and intermittent hypoxia based on HIF activation and cellular responses: (i) acute hypoxia is when the cells are exposed for no more than 24 h to an environment with 1% O-2 or less; (ii) chronic hypoxia is when the cells are exposed for more than 48 h to an environment with 1% O-2 or less and (iii) intermittent hypoxia is when the cells are exposed to at least two rounds of hypoxia (1% O-2 or less) separated by at least one period of reoxygenation by exposure to normoxia (8.5% O-2 or higher). Our review provides for the first time a guideline for definition of hypoxia related terms and a clear foundation for hypoxia related in vitro (breast) cancer research.Toxicolog

Targeting the heterogeneous genomic landscape in triple-negative breast cancer through inhibitors of the transcriptional machinery

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by lack of the estrogen, progesterone and human epidermal growth factor receptor 2. Although TNBC tumors contain a wide variety of oncogenic mutations and copy number alterations, the direct targeting of these alterations has failed to substantially improve therapeutic efficacy. This efficacy is strongly limited by interpatient and intratumor heterogeneity, and thereby a lack in uniformity of targetable drivers. Most of these genetic abnormalities eventually drive specific transcriptional programs, which may be a general underlying vulnerability. Currently, there are multiple selective inhibitors, which target the transcriptional machinery through transcriptional cyclin-dependent kinases (CDKs) 7, 8, 9, 12 and 13 and bromodomain extra-terminal motif (BET) proteins, including BRD4. In this review, we discuss how inhibitors of the transcriptional machinery can effectively target genetic abnormalities in TNBC, and how these abnormalities can influence sensitivity to these inhibitors. These inhibitors target the genomic landscape in TNBC by specifically suppressing MYC-driven transcription, inducing further DNA damage, improving anti-cancer immunity, and preventing drug resistance against MAPK and PI3K-targeted therapies. Because the transcriptional machinery enables transcription and propagation of multiple cancer drivers, it may be a promising target for (combination) treatment, especially of heterogeneous malignancies, including TNBC.Toxicolog

Density-dependent migration characteristics of cancer cells driven by pseudopod interaction

The ability of cancer cells to invade neighboring tissue from primary tumors is an important determinant of metastatic behavior. Quantification of cell migration characteristics such as migration speed and persistence helps to understand the requirements for such invasiveness. One factor that may influence invasion is how local tumor cell density shapes cell migration characteristics, which we here investigate with a combined experimental and computational modeling approach. First, we generated and analyzed time-lapse imaging data on two aggressive Triple-Negative Breast Cancer (TNBC) cell lines, HCC38 and Hs578T, during 2D migration assays at various cell densities. HCC38 cells exhibited a counter-intuitive increase in speed and persistence with increasing density, whereas Hs578T did not exhibit such an increase. Moreover, HCC38 cells exhibited strong cluster formation with active pseudopod-driven migration, especially at low densities, whereas Hs578T cells maintained a dispersed positioning. In order to obtain a mechanistic understanding of the density-dependent cell migration characteristics and cluster formation, we developed realistic spatial simulations using a Cellular Potts Model (CPM) with an explicit description of pseudopod dynamics. Model analysis demonstrated that pseudopods exerting a pulling force on the cell and interacting via increased adhesion at pseudopod tips could explain the experimentally observed increase in speed and persistence with increasing density in HCC38 cells. Thus, the density-dependent migratory behavior could be an emergent property of single-cell characteristics without the need for additional mechanisms. This implies that pseudopod dynamics and interaction may play a role in the aggressive nature of cancers through mediating dispersal.NWO864.12.013Toxicolog

A Red-Light-Activated Ruthenium-Caged NAMPT Inhibitor Remains Phototoxic in Hypoxic Cancer Cells

Metals in Catalysis, Biomimetics & Inorganic Material

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein (GFP) reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolization and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g., 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.Toxicolog

FAIR high content screening in bioimaging

The Minimum Information for High Content Screening Microscopy Experiments (MIHCSME) is a metadata model and reusable tabular template for sharing and integrating high content imaging data. It has been developed by combining the ISA (Investigations, Studies, Assays) metadata standard with a semantically enriched instantiation of REMBI (Recommended Metadata for Biological Images). The tabular template provides an easy-to-use practical implementation of REMBI, specifically for High Content Screening (HCS) data. In addition, ISA compliance enables broader integration with other types of experimental data, paving the way for visual omics and multi-Omics integration. We show the utility of MIHCSME for HCS data using multiple examples from the Leiden FAIR Cell Observatory, a Euro-Bioimaging flagship node for high content screening and the pilot node for implementing Findable, Accessible, Interoperable and Reusable (FAIR) bioimaging data throughout the Netherlands Bioimaging network.Computer Systems, Imagery and MediaMicrobial Biotechnolog

High-content high-throughput imaging reveals distinct connections between mitochondrial morphology and functionality for oxphos complex I, III, and V inhibitors

Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals.Toxicolog

Circumventing the crabtree effect in cell culture: a systematic review

Metabolic reprogramming and mitochondrial dysfunction are central elements in a broad variety of physiological and pathological processes. While cell culture established itself as a versatile technique for the elaboration of physiology and disease, studying metabolism using standard cell culture protocols is profoundly interfered by the Crabtree effect. This phenomenon refers to the adaptation of cultured cells to a glycolytic phenotype, away from aoxidative phosphorylation in glucose-containing medium, and questions the applicability of cell culture in certain fields of research. In this systematic review we aim to provide a comprehensive overview and critical appraisal of strategies reported to circumvent the Crabtree effect.Toxicolog