Stem cells can stay quiescent for a long period of time or proliferate and differentiate into multiple lineages. The activity of stage-specific metabolic programs allows stem cells to best adapt their functions in different microenvironments. Specific cellular phenotypes can be, therefore, defined by precise metabolic signatures. Notably, not only cellular metabolism describes a defined cellular phenotype, but experimental evidence now clearly indicate that also rewiring cells towards a particular cellular metabolism can drive their cellular phenotype and function accordingly. Cellular metabolism can be studied by both targeted and untargeted approaches. Targeted analyses focus on a subset of identified metabolites and on their metabolic fluxes. In addition, the overall assessment of the oxygen consumption rate (OCR) gives a measure of the overall cellular oxidative metabolism and mitochondrial function. Untargeted approach provides a large-scale identification and quantification of the whole metabolome with the aim to describe a metabolic fingerprinting. In this review article, we overview the methodologies currently available for the study of in vitro stem cell metabolism, including metabolic fluxes, fingerprint analyses, and single-cell metabolomics. Moreover, we summarize available approaches for the study of in vivo stem cell metabolism. For all of the described methods, we highlight their specificities and limitations. In addition, we discuss practical concerns about the most threatening steps, including metabolic quenching, sample preparation and extraction. A better knowledge of the precise metabolic signature defining specific cell population is instrumental to the design of novel therapeutic strategies able to drive undifferentiated stem cells towards a selective and valuable cellular phenotype.

Vitiligo is a chronic autoimmune skin disorder whose diagnosis is often psychologically upsetting. The efficacy of the available therapies, including topical corticosteroids and topical calcineurin inhibitors, has historically been limited and the management of vitiligo is still challenging. As vitiligo is a chronic disease limited to the skin, topical rather than systemic therapies may be preferable (especially among patients with localised lesions) to avoid the long‐term side‐effects of the latter. A topical formulation of ruxolitinib, a selective JAK1/2 inhibitor, has recently been approved in the United States for the treatment of non‐segmental vitiligo in patients aged >12 years based on data from the phase III TRuE‐V1 and TRuE‐V2 clinical trials. The aim of this review is to describe the current evidence concerning the efficacy and safety of topical ruxolitinib in the treatment of vitiligo, and discuss issues regarding its use in younger children and pregnant or breastfeeding women, as well as the duration and durability of treatment. The promising results obtained so far suggest that 1.5% ruxolitinib cream is an effective means of treating vitiligo.

Recent evidence from our laboratory provided proof-of-concept for therapeutic potential for glioblastoma (GBM) of a combination strategy based on radiation and adjuvant doxorubicin-loaded liposomes (LIPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate Blood Brain Barrier (BBB)-crossing. Significant glioma stem cell (GSC) apoptosis, tumor growth inhibition and increased overall survival were observed in vivo upon combined treatment offering attractive and innovative therapeutic possibilities for GBM. To strengthen therapeutic efficacy and lower off-target effects, we implemented mApoE-LIPs with a matrix metalloproteinases (MMP)-activable element that allows controlled payload release only in the MMPs rich tumor microenvironment, thus concurring to reduce unspecific interaction in healthy tissue where MMPs are low or absent Given the MMP2 overexpression in GBM, a MMP2-activable block (M2AB) was included in the phospholipid bilayer of mApoE-LIPs. The M2AB efficacy was evaluated on patient-derived GSCs displaying different MMP2 enzymatic activities by means of calcein-loaded M2AB/mApoE-LIPs. Human endothelial cells (hCMEC/d3), not expressing MMP2, were included to validate the targeted strategy as well as to assay the cytotoxicity on non-tumoral cells. Intracellular calcein quantification showed that: 1) calcein uptake correlates with MMP2 activity level; 2) M2AB/mApoE functionalization augmented calcein internalization into GSCs compared to mApoE alone. The MEK/ERK pathway, known to supports GBM cell survival, migration, and radio-resistance was considered as target strategy against GBM. A survey of the MEK/ERK inhibitors Trametinib (TRAM) and Pimasertib (PIMA) in several GSC lines indicated significant induction of GSC apoptosis associated to reduction of ERK phosphorylation. TRAM and PIMA were then encapsulated into mApoE-LIPs and their anti-GSC activity was investigated. Indeed, a dose dependent inhibition of GSC survival and induction of apoptosis combined to a significant lower level of phospho-ERK was observed upon 72h treatment. In conclusion, M2AB/mApoE-LIPs demonstrate: 1) a MMP2-dependent payload release; 2) stability when MMP2 lacks in the cellular milieu; 3) a synergic effect of the double M2AB/mApoE functionalization. TRAM and PIMA encapsulation into LIPs does not alter their anti-GSC activity. Funding by FRRB grant NEVERMIND (CP2_16/2018) Citation Format: Milena Mattioli, Marco Pizzocri, Elisabetta Stanzani, Valentino Ribecco, Simone Olei, Maria Pia Tropeano, Sabrina Giofrè, Antonio Renda, Sara Pellegrino, Pierfausto Seneci, Francesca Re, Federico Pessina, Michela Matteoli, Lorena Passoni. Improving glioblastoma treatment specificity and efficacy of mApoE-targeted liposome by MMP2-controlled drug releasee [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 368.

Among the main aberrations occurring in GBM, those in MEK/ERK and PI3K/akt/mTOR pathways predominate and confer GBM Stem-like Cells (GSCs) sustained proliferation and resistance to therapy. A panel of eight patient-derived primary GSCs lines have been screened for their sensitivity to a small kinase MEK inhibitor (MEKi) with AnnexinV/PI staining. Among these, five display a sensitive phenotype with at least 50% reduction on cell viability after 72 hours of treatment. Then, four cell lines, two MEKi-sensitive (ICH001 and ICH003) and two MEKi-resistant (ICH013 and ICH027) were selected for a deeper molecular characterization based on MGMT methylation status, mesenchymal index and main hotspot mutations associated with GBM pathology MEKi incubation on GSC caused a prompt phospho-ERK reduction already after 3 hours. Of note, we report a concomitant activation of AKT and downstream molecules pointing to an ERK-mTOR redundant activity. To this end, we combined MEKi to PI3K/akt/mTOR inhibitor and we observed an increased cell death even in GSCs displaying moderate sensitivity to MEKi as single-agent (MEKi: 90% vs MEKi-PI3K/akt/mTOR inhibitor: 30% cell viability). Then, MEKi ability to cross the Blood Brain Barrier (BBB) and target GBM cells was investigated using a transwell BBB in vitro model. The PI3K/akt/mTOR pathway inhibitor, known from the literature to readily cross the BBB, was included as positive control. Obtained results showed MEKi inability to efficiently cross the BBB, thus limiting its utility as GBM therapy. These results suggest the need for a specific drug delivery strategy in the brain that might be therapeutically effective. Recently our laboratory has provided proof-of-concept of a combination strategy based on radiation and adjuvant drug-loaded liposomes (LPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate BBB crossing. To strengthen therapeutic efficacy and to lower off-target effects, we implemented mApoE-LPs with a matrix metalloproteinases 2 and 9 sensitive lipopeptide (M2-9SLP) that allows controlled payload release only in the tumor microenvironment rich in MMPs. To this end, MEKi was encapsulated into the M2-9SLP/mApoE-LPs, and its capacity to promote cell death was evaluated. M2-9SLP/mApoE-MEKi-LPs caused in all the sensitive cell lines GSCs proliferation inhibition and induction of apoptosis upon 72h in vitro treatment indicating that the encapsulation process did not alter drug efficacy. In conclusion, our in vitro results support MEKi encapsulation into M2P/mApoE-LP as nanotherapeutic strategy that could guarantee specific delivery of MEKi in a MMP2-enriched tumor microenvironment without altering its capacity to inhibit GSC proliferation and survival. Funding by FRRB grant NEVERMIND (CP2_16/2018) Citation Format: Milena Mattioli, Elisabetta Stanzani, Valentino Ribecco, Marco Pizzocri, Eliana Lauranzano, Margherita Maria Ravanelli, Simone Olei, Maria Pia Tropeano, Pierfausto Seneci, Francesca Re, Federico Pessina, Michela Matteoli, Lorena Passoni. Smart encapsulation of a MEK inhibitor into M2-9SLP/mApoE-liposomes for specific GBM targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2711.