METABOLOMIC ANALYSIS OF LIVE SINGLE CELLS AND SPHEROID SECRETIONS USING MASS SPECTROMETRY: TOWARDS UNDERSTANDING OF CELLULAR METABOLISM
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This dissertation describes the single cell metabolomics analysis and microscale extracellular metabolites analysis using the Single-probe mass spectrometry (MS) techniques. Chapter one summarizes the current technique for single cell analysis, including the exploration of single-cell genome, transcriptome, proteome, and metabolome. Techniques used for extracellular metabolites analysis were also briefly described. Chapter two introduces the metabolomic analysis of single cancer stem cells (CSCs) using the Single-probe MS technique. CSCs are rare types of cells responsible for tumor development, relapse, and metastasis. However, current research in CSC biology is largely limited by the difficulty of obtaining sufficient CSCs. Single-cell analysis techniques are promising tools for CSC-related studies. The Single-probe MS technique was used to investigate the metabolic features of live colorectal CSCs at the single-cell level. Experimental data were analyzed using statistical analysis methods, including the t-test and partial least squares discriminant analysis. The overall metabolic profiles of CSCs are distinct from non-stem cancer cells (NSCCs). Specifically, tricarboxylic acid (TCA) cycle metabolites are more abundant in CSCs compared to NSCCs, indicating their major energy production pathways are different. Moreover, CSCs have relatively higher levels of unsaturated lipids. Inhibiting the activities of stearoyl-CoA desaturase-1 (SCD1), nuclear factor κB (NF-κB), and aldehyde dehydrogenases (ALDH1A1) in CSCs significantly reduced the abundances of unsaturated lipids and hindered the formation of spheroids, resulting in reduced stemness of CSCs. Our techniques and experimental protocols can be potentially used for metabolomic studies of other CSCs and rare types of cells and provide a new approach to discovering functional biomarkers as therapeutic targets. Chapter three demonstrates the investigation of metabolic features of single algae cells under different environmental stress. Traditional approaches for the assessment of physiological responses of microbes in the environment rely on bulk filtration techniques that obscure differences among populations as well as among individual cells. The Single-probe MS technique was used to directly extract metabolites from living, individual phytoplankton cells for analysis. Marine dinoflagellate Scrippsiella trochoidea cells were grown under different illumination levels and under nitrogen (N) limiting conditions. In both experiments, significant differences in the cellular metabolome of individual cells could readily be identified, though the vast majority of detected metabolites could not be assigned to KEGG pathways. Using the same approach, significant changes in cellular lipid complements were observed, with individual lipids being both up- and down-regulated under light vs. dark conditions. Conversely, lipid content increased across the board under N limitation, consistent with an adjustment of Redfield stoichiometry to reflect higher C:N and C:P ratios. Overall, these data suggest that the Single-probe MS technique has the potential to allow for near in situ metabolomic analysis of individual phytoplankton cells, opening the door to targeted analyses that minimize cell manipulation and sampling artifacts, while preserving metabolic variability at the cellular level. Chapter four describes comprehensive studies of early-stage drug resistance cells using single cell MS metabolomics combined with other techniques. Efficacy of chemotherapy is often limited by de novo or acquired drug resistance in clinic treatment. Studying the underlying mechanisms of drug resistance is necessary for better development of novel therapeutic strategies. Studies in this field are generally conducted through bulk analysis of samples obtained from long-term chemotherapy. Investigating the metabolic changes occurred in the early stage of drug resistance at the single-cell level provides insights into drug-resistant mechanisms, and the developed techniques can be potentially used for early monitoring of drug resistance in the clinic. Comprehensive studies were carried out using multiple techniques, including single cell MS metabolomics, western blotting, flow cytometry, and reverse transcription polymerase chain reaction (RT-PCR), to explore mechanisms of irinotecan resistance at the initial stage at the single cell level. Results illustrate that SCD1 is upregulated in the irinotecan-resistant cells, which show increased levels of stemness and decreased degrees of ROS. Chapter 5 demonstrates the critical roles of extracellular compounds in intercellular communication, tumor proliferation, and cancer cell metastasis. However, the lack of appropriate techniques leads to limited studies of extracellular metabolite. Here, we introduced a microscale collection device, the Micro-funnel, fabricated from biocompatible fused silica capillary. With a small probe size (∼25 μm), the Micro-funnel can be implanted into live multicellular tumor spheroids to accumulate the extracellular metabolites produced by cancer cells. Metabolites collected in the Micro-funnel device were then extracted by a microscale sampling and ionization device, the Single-probe, for real-time mass spectrometry (MS) analysis. We successfully detected the abundance change of anticancer drug irinotecan and its metabolites inside spheroids treated under a series of conditions. Moreover, we found that irinotecan treatment dramatically altered the composition of extracellular compounds. Specifically, we observed the increased abundances of a large number of lipids, which are potentially related to the drug resistance of cancer cells. This study provides a novel way to detect the extracellular compounds inside live spheroids, and the successful development of our technique can benefit the research in multiple areas, including the microenvironment inside live tissues, cell−cell communication, biomarker discovery, and drug development.
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