Elisabeth Roider

While the UV-MITF pathway is well-established as a key player of pigmentation, implication of other MITF independent pathways in this biological | POSTER

Two-photon fluorescence lifetime imaging microscopy (FLIM) is a technique that not only probes the intensity of fluorophores, but also provides the temporal decay trace of said fluorophores on a pixel-by-pixel basis. These traces can then be transformed into the frequency domain for subsequent analysis, resulting in a scatterplot of phasor coordinates where each phasor corresponds to a single image pixel. With this in mind, it follows that individual fluorophores result in distinct clusters in the phasor plot, and a mixture of two fluorophores results in phasors that fall somewhere along a line linking the two clusters depending on the relative fluorophore concentrations. Until now, distinction of fluorescent species has relied mainly on computing the Euclidean distance between a given phasor and the mean coordinates of reference phasor clusters. However, this approach becomes inadequate in cases where one fluorophore has a much wider lifetime distribution than the other. As such, we propose the use of the Mahalanobis distance as an alternative to the Euclidean distance, as this metric additionally factors in the relative spread of each reference phasor cluster. This method has been applied to studying the oxidative response of ex vivo human skin via endogenous NADH fluorescence as it is exposed to chemical sun filters, the active ingredients in sunscreens. Given that both NADH and sun filters are fluorescent under the same excitation and emission conditions, the proposed Mahalanobis distance approach was used to distinguish the source of fluorescence in images of human skin. This allowed for the assessment of oxidative response as well as the tracking and monitoring of the sun filter formulation as it permeated throughout the skin.

Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients’ skin.

Therapy-resistant cancer cell states identified across diverse contexts are selectively vulnerable to ferroptotic cell death induced by inhibition of lipid peroxidase pathways converging on GPX4. Cancer cells can assume different biological states, which can affect their resistance to therapies. A mesenchymal phenotype has been associated with drug resistance but the mechanism behind this state is not well understood. Stuart Schreiber and colleagues now show that tumour cells with a mesenchymal phenotype are selectively sensitive to inhibition of GPX4, an enzyme that alters lipid metabolism. GPX4 dissipates lipid peroxides and therefore prevents the iron-mediated reactions which induce ferroptotic cell death. These findings offer new perspectives on targeting cancers that have undergone a transition to a mesenchymal state to evade other therapeutic agents. Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness1,2,3,4. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood1,2,3,4,5,6. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides7,8. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes8,9. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death8. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial–mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Germline mutations in the Folliculin (FLCN) tumour suppressor gene result in fibrofolliculomas, lung cysts and renal cancers, but the precise mechanisms of tumour suppression by FLCN remain elusive. Here we identify Rab7A, a small GTPase important for endocytic trafficking, as a novel FLCN interacting protein and demonstrate that FLCN acts as a Rab7A GTPase-activating protein. FLCN^−/− cells display slower trafficking of epidermal growth factor receptors (EGFR) from early to late endosomes and enhanced activation of EGFR signalling upon ligand stimulation. Reintroduction of wild-type FLCN, but not tumour-associated FLCN mutants, suppresses EGFR signalling in a Rab7A-dependent manner. EGFR signalling is elevated in FLCN^−/− tumours and the EGFR inhibitor afatinib suppresses the growth of human FLCN^−/− cells as tumour xenografts. The functional interaction between FLCN and Rab7A appears conserved across species. Our work highlights a mechanism explaining, at least in part, the tumour suppressor function of FLCN.

Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1r^e/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1r^e/e; Tyr^c/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas.