Three-Dimensional Mass Spectrometry Imaging Reveals Distributions of Lipids and the Drug Metabolite Associated with the Enhanced Growth of Colon Cancer Cell Spheroids Treated with Triclosan

Abstract

The application of mass spectrometry imaging (MSI) to explore the responses of cancer cell spheroids (CCS) after treatment of exogenous molecules has attracted growing attention. Increasing studies have utilized MSI to image the two-dimensional distributions of exogenous and endogenous molecules in planar CCS sections. However, because CCS are volumetric and heterogenous, maintaining their three-dimensional (3D) information is essential for acquiring a better understanding of the tumor microenvironment and mechanisms of action of exogenous molecules. Here, an established method of 3D MSI was applied to distinguish the distributions of triclosan sulfate and endogenous lipids in three microregions of colon CCS with an enhanced growth induced by the treatment of triclosan, a common antimicrobial agent. The results of 3D MSI showed that triclosan sulfate gradually accumulated from the periphery to the entire structure of CCS and finally localized in the core region. Spatial lipidomics analysis revealed that the upregulated phosphatidylethanolamine (fold change (FD) = 1.26, p = 0.0021), phosphatidylinositol (FD = 1.17, p = 0.0180), and phosphatidylcholine (FD = 1.22, p = 0.0178) species mainly distributed in the outer proliferative region, while the upregulated sphingomyelin (FD = 1.18, p = 0.024) species tended to distribute in the inner necrotic region. Our results suggest that a competitive mechanism between inhibiting and promoting CCS growth might be responsible for the proliferation of CCS treated with triclosan.

Spatial Metabolomics and Lipidomics Reveal the Mechanisms of the Enhanced Growth of Breast Cancer Cell Spheroids Exposed to Triclosan

“…DCTB was used as the MALDI matrix to detect TCS, TCSS, and TCSG in CCS in negative ionization mode according to the previous work. 25 As shown in Figure 2A and Figure S1A, negligible signals of TCSS and TCS were observed in the unexposed CCS, indicating that no endogenous metabolites were interfered with TCSS and TCS detection. Two major ion peaks (Figure 2B and Figure S1B) were found in both [TCSS − H] − and [TCS − H] − owing to the existence of chlorine-37 and chlorine-35.…”

“…We chosen 0–72 h as the exposure time range because the culture medium containing various concentrations of TCS was replaced every 72 h. To investigate the time-dependent penetrations of TCS and its metabolites in CCS, the time range was divided into several time points (0, 1, 3, 6, 12, 24, 48, and 72 h). DCTB was used as the MALDI matrix to detect TCS, TCSS, and TCSG in CCS in negative ionization mode according to the previous work . As shown in Figure A and Figure S1A, negligible signals of TCSS and TCS were observed in the unexposed CCS, indicating that no endogenous metabolites were interfered with TCSS and TCS detection.…”

“…This may be contributing to the concentrated distributions of TCS and TCCS in the inner area of CCS after 6 h. The obtained results were consistent with our previous study. 25 It demonstrated that, when HCT116 colon CCS were exposed to TCS (10 μM), TCSS gradually localized from the periphery area to the full area at 0 to 12 h and accumulated in the inner area after 24 h. With the increasing intensities of TCS and its metabolic wastes (e.g., TCSS) in cell spheroids from 0 to 24 h (Figure 2A,B), the accumulation of TCS and its metabolic wastes may not good for cell spheroids to achieve a fast growth. Hence, the cells in spheroids may tend to eliminate a portion of TCS and metabolic wastes from the spheroids after 24 h. For a better understanding of TCS metabolism in breast CCS, the content of TCSS, TCSG, and TCS in the culture medium and breast CCS were measured by LC−MS/MS.…”

“…A UPLC system coupled with an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific, U.S.A.) was used for the analyses of metabolites and lipids in breast CCS. The detailed analytical protocols were described in our previous published works. , Briefly, an amide and C18 column were used to separate metabolites and lipids, respectively. The sample temperature was set at 4 °C.…”

“…Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique that can simultaneously acquire the information of spatial distribution and abundance for different compounds (e.g., metabolites, lipids, and environmental pollutants) in different biological samples. , However, MALDI suffers from poor ionization efficiencies for many different analysts. Its estimated ionization efficiencies (the ratio of the number of generated ions to the number of desorbed neutrals) are 10 –4 and lower. , Several approaches have been developed to improve the ionization efficiency of MALDI. , The most common approach is to introduce specific functional groups to target analytes by chemical derivatizations.…”