Abstract To survive in the nutrient- and oxygen-deprived niche, cancer cells undergo a drastic metabolic reprogramming where they upregulate their endogenous lipid synthesis and exogenous lipid uptake. Population-wide mass spectrometry lipid analyses reveal that amounts of lipid stores, degree of lipid saturation, and acyl-chain length provide survival advantages by making cells metabolically independent of external sources of energy, protecting against oxidative stress, and stimulating immune response suppression. Hence, cancer cell lipid profiling emerges as a powerful diagnostic tool and fundamental research goal. However, current technologies lack single-cell resolution, do not provide spatial information, and are incapable of linking the cancer cell phenotype to the lipid composition. Here, we describe a unique platform for simultaneous lipid profiling of multiple cell types in intact tissue samples at sub-cellular level by probing inherent molecular vibrations using spectral coherent anti-Stokes Raman scattering (CARS) microscopy. It promises transformative information on how lipid profiles are shaped in different tumor microenvironments (TMEs) in both cancer cells and cancer- associated cells, and relates this quantitative, spatial information to patterns of protein and gene expression. We combine a custom, automatically tunable laser system with a commercial confocal microscope to enable convenient mapping of a range of molecular vibrations to assess (i) amounts of lipids with sub-femtoliter precision, (ii) degree of lipid unsaturation at single carbon double-bond precision, and (iii) acyl chain length at single methylene group precision, which can be scaled up by programmable multipoint measurements of multiple samples. Further, by implementing simultaneous single-cell RNA imaging, metabolic profiles can be linked to upstream RNA transcripts controlling cell metabolism, including known oncogenes. For this purpose, we will design a series of novel RNA probes, each with an isotope-specific nitrile group, which can be targeted with high specificity through its distinct molecular vibration by stimulated Raman excited fluorescence (SREF). Thanks to a multitude of possible combinations for isotope labeling, this innovative approach allows simultaneous detection of a higher number of RNA transcripts than can be done with current fluorescence-based RNA labeling techniques without disturbing the lipid landscape. Simultaneously, standard fluorophores can be used to probe cell-type markers at the protein level with conventional immunocytochemistry. As a result, multiple RNA transcripts can be mapped simultaneously with lipid profiles and protein profiles for multiple different cell types. The development will be conducted in collaboration with cancer biologists, cancer metabolism experts and clinicians, ensuring the biological and clinical relevance of our approach and data analysis.

Project Narrative To survive in the nutrient- and oxygen-deprived tumor microenvironment (TME), cancer cells undergo a drastic metabolic reprogramming where they upregulate both their endogenous lipid synthesis and exogenous uptake of lipids. Although this altered lipid metabolism has the potential to serve as a powerful diagnostic approach and research tool in cancer biology, current methods to evaluate lipid profiles are unable to provide single-cell analysis and lack spatial precision. Here, we describe a unique platform for lipid profiling of cancer and cancer- associated cells in intact tissue samples at single-cell level by probing inherent molecular vibrations using spectral coherent anti-Stokes Raman scattering (CARS) microscopy, simultaneously with RNA imaging by stimulated Raman excited fluorescence (SREF).