Although much progress has been made in cancer treatment, the molecular mechanisms underlying cancer radioresistance (RR) as well as the biological signatures of radioresistant cancer cells still need to be clarified. In this regard, we discovered that breast, bladder, lung, neuroglioma, and prostate 6 Gy X-ray resistant cancer cells were characterized by an increase of lipid droplet (LD) number and that the cells containing highest LDs showed the highest clonogenic potential after irradiation. Moreover, we observed that LD content was tightly connected with the iron metabolism and in particular with the presence of the ferritin heavy chain (FTH1). In fact, breast and lung cancer cells silenced for the FTH1 gene showed a reduction in the LD numbers and, by consequence, became radiosensitive. FTH1 overexpression as well as iron-chelating treatment by Deferoxamine were able to restore the LD amount and RR. Overall, these results provide evidence of a novel mechanism behind RR in which LDs and FTH1 are tightly connected to each other, a synergistic effect that might be worth deeply investigating in order to make cancer cells more radiosensitive and improve the efficacy of radiation treatments.

Breast cancer is the most frequent cancer in women worldwide and late diagnosis often adversely affects the prognosis of the disease. Radiotherapy is commonly used to treat breast cancer, reducing the risk of recurrence after surgery. However, the eradication of radioresistant cancer cells, including cancer stem cells, remains the main challenge of radiotherapy. Recently, lipid droplets (LDs) have been proposed as functional markers of cancer stem cells, also being involved in increased cell tumorigenicity. LD biogenesis is a multistep process requiring various enzymes, including Diacylglycerol acyltransferase 2 (DGAT2). In this context, we evaluated the effect of PF-06424439, a selective DGAT2 inhibitor, on MCF7 breast cancer cells exposed to X-rays. Our results demonstrated that 72 h of PF-06424439 treatment reduced LD content and inhibited cell migration, without affecting cell proliferation. Interestingly, PF-06424439 pre-treatment followed by radiation was able to enhance radiosensitivity of MCF7 cells. In addition, the combined treatment negatively interfered with lipid metabolism-related genes, as well as with EMT gene expression, and modulated the expression of typical markers associated with the CSC-like phenotype. These findings suggest that PF-06424439 pre-treatment coupled to X-ray exposure might potentiate breast cancer cell radiosensitivity and potentially improve the radiotherapy effectiveness.

Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols.

Despite increasing knowledge about small extracellular vesicle (sEV) composition and functions in cell–cell communication, the mechanism behind their biogenesis remains unclear. Here, we reveal for the first time that sEV biogenesis and release into the microenvironment are tightly connected with another important organelle, Lipid Droplets (LDs). The correlation was observed in several human cancer cell lines as well as patient‐derived colorectal cancer stem cells (CR‐CSCs). Our results demonstrated that external stimuli such as radiation, pH, hypoxia or lipid‐interfering drugs, known to affect the number of LDs/cell, similarly influenced sEV secretion. Importantly, through multiple omics data, at both mRNA and protein levels, we revealed RAB5C as a potential important molecular player behind this organelle connection. Altogether, the potential to fine‐tune sEV biogenesis by targeting LDs could significantly impact the amount, cargos and properties of these sEVs, opening new clinical perspectives.

Despite an increasing gain of knowledge regarding small extracellular vesicle (sEV) composition and functions in cell-cell communication, the mechanism behind their biogenesis remains unclear. Here, we revealed for the first time that the sEV biogenesis and release into the microenvironment are tightly connected with another important organelle: Lipid Droplets (LD). We have observed this correlation using different human cancer cell lines as well as patient-derived colorectal cancer stem cells (CR-CSCs). Our results showed that the use of external stimuli such as radiation, pH, hypoxia, or lipid interfering drugs, known to affect the LD content, had a similar effect in terms of sEV secretion. Additional validations were brought using multiple omics data, at the mRNA and protein levels. Altogether, the possibility to fine-tune sEV biogenesis by targeting LDs, could have a massive impact on the amount, the cargos and the properties of those sEVs, paving the way for new clinical perspectives. Significance Statement: