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.