Multimodal mass spectrometric imaging of small molecules reveals distinct spatio-molecular signatures in differentially metastatic breast tumor models

Abstract

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.

Figure 1

(A) Representative in vivo ¹H MR spectrum demonstrating that the total choline signal cannot be spectrally resolved in vivo. (B) Representative ¹H MRSI data set showing the spatial distribution of tCho displayed in red within an MDA-MB-231 tumor as described in the experimental section. (C) High-resolution MR spectrum obtained from a representative water-soluble MDA-MB-231 cell extract resolving GPC, PC, and Cho spectrally.

Figure 2

Optical images of H&E stained sections of (A) a representative MDA-MB-231 tumor and (B) a representative MCF-7 tumor. The MS spectra are provided for two selected areas, containing two expanded mass regions (m/z 15 to 65) with peak assignment of Na⁺ and K⁺, and the total area spectrum (m/z 1 to 1000) including two expanded spectral regions (m/z 300 to 640, m/z 640 to 1000) with peak assignments for Cho, PC, cholesterol, 1,2-Diacylglycerol and PtdCho.

Figure 3

(A) MALDI-MS microscope mode data set of a representative MDA-MB-231 tumor showing the H&E image, the corresponding MS imaging of Cho and PC, and the PCA image of the function +2. (B) SIMS microprobe data set of a representative MCF-7 tumor showing the H&E image, the corresponding MS imaging of Cho and PC, and the PCA images of the functions +2 and −2 providing inverse images, in which tumor regions are defined by masses other than Cho and PC. Scale bar: 1 mm

Figure 4

(A) SIMS microprobe data set of a representative MDA-MB-231 tumor showing the H&E image, the corresponding MS imaging of Na⁺ and K⁺, and the PCA image of the function −2. (B) SIMS microprobe data set of a representative MCF-7 tumor showing the H&E image, the corresponding MS imaging of Na⁺ and K⁺, and the inverse PCA images of the functions +2 and −2. Scale bar: 1 mm.

Figure 5

(A) Optical image of a small part of a representative MDA-MB-231 tumor, stained with H&E after concluding a MetA-SIMS experiment with (1) necrotic region and (2) viable tumor areas selected. (B) Na⁺ shown in blue and (C) K⁺ distribution shown in gray from MetA-SIMS data and overlay with microscopic image in regions (1) and (2) are shown in the upper panel; the individual ion distributions in areas (1) and (2) are shown in the bottom panel. Due to previous gold coating of the tissue, the hematoxylin staining was less efficient, and therefore the visibility of cell nuclei is less pronounced. Scale bar: (A) 100 μm; (B) and (C) 25 μm.