Abstract The intensity of the lipid signal in tumors is spatially heterogeneous, partially due to the physiologic tumor microenvironment. Cancer cells can significantly contribute to the abnormal tumor microenvironment through increased glycolysis, upregulation of inflammatory pathways, and secretion of proteolytic enzymes. Here, we have investigated the relationship between hypoxia and lipid metabolites in a human breast cancer model by combining in vivo magnetic resonance imaging (MRI), MR spectroscopic imaging (MRSI), ex vivo mass spectrometric imaging (MSI) and optical imaging of hypoxia and necrosis. Human MDA-MB-231-HRE-tdTomato breast cancer cells were orthotopically grown in nude mice. We performed 3D MRSI to detect metabolites. Each tumor was sectioned to obtain fiducially marked 2-mm thick slices, which were imaged by microscopy to visualize hypoxic tumor regions. Each 2-mm thick slice was cryosectioned into fiducially marked 10-um thick slices to perform MSI, and histologically hematoxylin/eosin stained to visualize the necrotic area in the tumor. For MSI, cryosections were mounted on indium tin oxide-coated glass slides, and covered with matrix. Matrix-assisted laser desorption ionization (MALDI) MSI was performed to detect small molecules as well as intact proteins up to 20,000 m/z. We built a coregistration platform based on fiducial markers and shape characteristics that allowed us to fuse MRI, MRSI, MSI and optical images. Lactate/lipid CH2 and lipid CH3 were observed in MRSI. Several triacylglyceride (TG) species were observed in MSI. Lactate/lipid CH2 and lipid CH3 obtained by MRSI were mostly located at the rim of the tumors, while some TG signals from MSI co-localized with hypoxic regions, and some other MSI-detected TG signals co-localized with necrotic regions. MRSI in vivo detects CH2 and CH3 groups in fatty acids, which are located mostly in TG and other lipids that form mobile lipid droplets. Our ultimate goal in this study is to determine if MRSI-detected lipid CH2 and CH3 groups are predominantly localized in hypoxic, necrotic, or normoxic tumor regions, and, with the help of co-registered MSI, what molecular TG species contribute to these MRSI-detected lipid signals. Our data revealed that at least three TG species, namely TG(18:1/16:2/20:0), TG(20:4/18:2/20:4), and TG(18:2/20:0/20:0) partially localized to hypoxic regions, and two others (TG(18:0/16:1/18:0), TG(18:2/16:2/20:2)) to necrotic regions. In this particular tumor slice, MRSI-detected lipid signals localized to viable tumor regions, and overlapped with TG(18:1/16:2/20:0) and TG(18:2/20:0/20:0). Our fiducially marked co-registration platform allowed us to fuse MRI, MRSI, MSI and optical images in 2D. We are currently extending this platform for application in 3D. This work was supported by NIH R01 CA134695. We thank Tiffany Blackwell for technical laboratory support. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5272. doi:10.1158/1538-7445.AM2011-5272