MALDI-Mass Spectrometric Imaging Revealing Hypoxia-Driven Lipids and Proteins in a Breast Tumor Model

Abstract

Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of lipids and proteins by using principal component analysis-linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study, we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis, and telomere stress induced senescence. In parallel, we also identified colocalization of hypoxic regions and various lipid species such as PC(16:0/18:0), PC(16:0/18:1), PC(16:0/18:2), PC(16:1/18:4), PC(18:0/18:1), and PC(18:1/18:1), among others. Our findings shed light on the biomolecular composition of hypoxic tumor regions, which may be responsible for a given tumor's resistance to radiation or chemotherapy.

Figure 1

Overview of the experimental and data analysis workflow for 3D MALDI-MSI in MDA-MB-231-HRE-tdTomato breast tumor xenografts. The workflow includes sample preparation of breast tumor tissue, 3D MALDI-MSI, and data processing.

Figure 2

Display of PC1, PC2, DA1, and DA2 score images of a representative MDA-MB-231-HRE-tdTomato breast tumor xenograft. The top row shows examples from the lipid range of m/z 100 to m/z 1000, and the bottom row shows examples from the tryptic peptide range of m/z 1000 to m/z 3000, as well as the corresponding tdTomato tryptic peptide image at m/z 2225.0.

Figure 3

Functional protein–protein interaction network of the hypoxia-up-regulated proteins that were identified by our 3D MALDI-MSI analysis of the MDA-MB-231-HRE-tdTomato breast tumor xenograft model. The hypoxia-up-regulated proteins are also listed in Table S1, Supporting Information. The Reactome Database was used to generate this network display (http://www.reactome.org/) and clusters the discovered proteins into distinct biological pathways that were up-regulated in hypoxic regions in these breast tumors.

Figure 4

Display of representative biomolecular MALDI-MS images in (A) 2D and (B) 3D. PS(14:0/22:6) at m/z 818.4 and a tryptic peptide of hypoxia up-regulated protein (HYOU1) at m/z 1047.4, as well as LDHA at m/z 1071.5, colocalized with hypoxic regions, in which high levels of a tdTomato tryptic peptide at m/z 2225.0 were detected in MDA-MB-231-HRE-tdTomato breast tumor xenografts. This was evident in the 2D as well as the 3D display. The entire list of m/z values of hypoxia-up-regulated lipids and proteins, including all detected tryptic peptides per protein, is given in Tables S1 and S2, Supporting Information, respectively.