Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging

Abstract

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 μm spatial resolution. Protein markers of proliferation ( K i -67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.

Comparison of Osteosarcoma Aggregated Tumour Models with Human Tissue by Multimodal Mass Spectrometry Imaging

“…From the segmentation analysis, differences of the phenotypical regions in each model were observed ( Figure 1 ). As reported previously [ 14 ], the segmented image of an HCC827 aggregoid model comprised three regions: a distinctive inner core, an annular zone and an outer region, which were also observable in the histological image. The segmentation of the MG63 model showed a similar pattern, where a core, two annular regions and a periphery were observable in the segmented MSI data.…”

“…Furthermore, we have previously employed multimodal MSI techniques to demonstrate how obtaining a large amount of molecular information with a complementary nature can enhance the understanding of the biological processes within a tissue. The detection of metabolites, proteins and metals determined the interplay between molecules and established key pathways that defined a novel HCC827 lung adenocarcinoma ‘aggregoid’ model [ 14 ]. A similar approach could therefore give a valuable insight into 3D tumour aggregoid models of OS, providing comprehensive information of biochemical pathways that influence the cancer pathogenesis.…”

Characterization of the Tumor Immune Microenvironment in Lung Squamous Cell Carcinoma Using Imaging Mass Cytometry

“…Imaging mass cytometry (IMC) can provide additional information regarding cellular localization within tissues and can help clarify the spatial interactions between different cell types ( 17 ). In addition, IMC has been used to define the metabolite distribution in a lung adenocarcinoma model ( 18 ) and to determine that cancer-associated fibroblasts interact with monocytic myeloid cells to induce immune suppression in the TIME of patients with LUSC ( 19 ). Therefore, we used IMC to characterize the TIME’s immune atlas in specimens from patients with primary LUSC, which may help clarify the TIME landscape and explore the cancer–immune interactions.…”

Role of MALDI-MSI in combination with 3D tissue models for early stage efficacy and safety testing of drugs and toxicants

“…Although, it is an understandable requirement to use more advanced models that can be grown large enough to study the spatial distribution of molecules. For example, a recent study by Flint et al [43] reported the use of multimodal MSI techniques: DESI, imaging mass cytometry (IMC) and laser ablation inductively coupled plasma (LA-ICP)-MS to characterize a novel aggregated 3D culture model of lung adenocarcinoma. The in vitro model, termed 'aggregoid' is formed via the aggregation of clonal tumor spheroids to create a more heterogeneous tissue of approximately 1 mm diameter.…”