Lipid Droplet Biosynthesis Impairment through DGAT2 Inhibition Sensitizes MCF7 Breast Cancer Cells to Radiation.

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.

Invasiveness modulation of glioma cells by copper complex-loaded nanoarchitectures.

Among the tumors with the highest lethality, gliomas are primary brain tumors associated with common recurrence inclined to metastasize along the neuraxis and occasionally out of the central nervous system. Even though metastasis is the main responsible for death in oncological patients, few dedicated treatments are approved. Therefore, the establishment of effective anti-metastasis agents is the final frontier in cancer research. Interestingly, some copper complexes have demonstrated promising efficacy as antimetastatic agents, but they may cause off-site effects such as the alteration of copper homeostasis in healthy tissues. Thus, the incorporation of copper-based antimetastatic agents in rationally designed nano-architectures can increase the treatment localization reducing the side effects. Here, copper complex loaded hybrid nano-architectures (CuLNAs) are presented and employed to assess the impact of an intracellular copper source on glioma cell invasiveness. The novel CuLNAs are fully characterized and exploited for cell migration modulation in a glioma cell line. The results demonstrate that CuLNAs significantly reduce cell migration without impairing cell proliferation compared to standard gold and copper NAs. A concomitant antimigratory-like regulation of the epithelial-to-mesenchymal transition genes confirmed these results, as the gene encoding for the epithelial protein E-cadherin was upregulated and the other explored mesenchymal genes were downregulated. These findings, together with the intrinsic behaviors of NAs, demonstrate that the inclusion of metal complexes in the nano-architectures is a promising approach for the composition of a family of agents with antimetastatic activity.

Linear Energy Transfer Measurements and Estimation of Relative Biological Effectiveness in Proton and Helium Ion Beams Using Fluorescent Nuclear Track Detectors.

PURPOSE: Our objective was to develop a methodology for assessing the linear energy transfer (LET) and relative biological effectiveness (RBE) in clinical proton and helium ion beams using fluorescent nuclear track detectors (FNTDs). METHODS AND MATERIALS: FNTDs were exposed behind solid water to proton and helium (4He) ion spread-out Bragg peaks. Detectors were imaged with a confocal microscope, and the LET spectra were derived from the fluorescence intensity. The track- and dose-averaged LET (LETF and LETD, respectively) were calculated from the LET spectra. LET measurements were used as input on RBE models to estimate the RBE. Human alveolar adenocarcinoma cells (A549) were exposed at the same positions as the FNTDs. The RBE was calculated from the resulting survival curves. All measurements were compared with Monte Carlo simulations. RESULTS: For protons, average relative differences between measurements and simulations were 6% and 19% for LETF and LETD, respectively. For helium ions, the same differences were 11% for both quantities. The position of the experimental LET spectra primary peaks agreed with the simulations within 9% and 14% for protons and helium ions, respectively. For the RBE models using LETD as input, FNTD-based RBE values ranged from 1.02 ± 0.01 to 1.25 ± 0.04 and from 1.08 ± 0.09 to 2.68 ± 1.26 for protons and helium ions, respectively. The average relative differences between these values and simulations were 2% and 4%. For A549 cells, the RBE ranged from 1.05 ± 0.07 to 1.47 ± 0.09 and from 0.89 ± 0.06 to 3.28 ± 0.20 for protons and helium ions, respectively. Regarding the RBE-weighted dose (2.0 Gy at the spread-out Bragg peak), the differences between simulations and measurements were below 0.10 Gy. CONCLUSIONS: This study demonstrates for the first time that FNTDs can be used to perform direct LET measurements and to estimate the RBE in clinical proton and helium ion beams.

Lipid droplets and small extracellular vesicles: More than two independent entities.

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.

Human Cancer Cell Radiation Response Investigated through Topological Analysis of 2D Cell Networks.

Clonogenic assays are routinely used to evaluate the response of cancer cells to external radiation fields, assess their radioresistance and radiosensitivity, estimate the performance of radiotherapy. However, classic clonogenic tests focus on the number of colonies forming on a substrate upon exposure to ionizing radiation, and disregard other important characteristics of cells such their ability to generate structures with a certain shape. The radioresistance and radiosensitivity of cancer cells may depend less on the number of cells in a colony and more on the way cells interact to form complex networks. In this study, we have examined whether the topology of 2D cancer-cell graphs is influenced by ionizing radiation. We subjected different cancer cell lines, i.e. H4 epithelial neuroglioma cells, H460 lung cancer cells, PC3 bone metastasis of grade IV of prostate cancer and T24 urinary bladder cancer cells, cultured on planar surfaces, to increasing photon radiation levels up to 6 Gy. Fluorescence images of samples were then processed to determine the topological parameters of the cell-graphs developing over time. We found that the larger the dose, the less uniform the distribution of cells on the substrate-evidenced by high values of small-world coefficient (cc), high values of clustering coefficient (cc), and small values of characteristic path length (cpl). For all considered cell lines, [Formula: see text] for doses higher or equal to 4 Gy, while the sensitivity to the dose varied for different cell lines: T24 cells seem more distinctly affected by the radiation, followed by the H4, H460 and PC3 cells. Results of the work reinforce the view that the characteristics of cancer cells and their response to radiotherapy can be determined by examining their collective behavior-encoded in a few topological parameters-as an alternative to classical clonogenic assays.

A theoretical study of H2 O2 as the surrogate of dose in minibeam radiotherapy, with a diffusion model considering radical removal process.

BACKGROUND: Minibeam radiation therapy (MBRT) is an innovative dose delivery method with the potential to spare normal tissue while achieving similar tumor control as conventional radiotherapy. However, it is difficult to use a single dose parameter, such as mean dose, to compare different patterns of MBRT due to the spatially fractionated radiation. Also, the mechanism leading to the biological effects is still unknown. PURPOSE: This study aims to demonstrate that the hydrogen peroxide (H2 O2 ) distribution could serve as a surrogate of dose distribution when comparing different patterns of MBRT. METHODS: A free diffusion model (FDM) for H2 O2 developed with Fick's second law was compared with a previously published model based on Monte Carlo & convolution method. Since cells form separate compartments that can eliminate H2 O2 radicals diffusing inside the cell, a term describing the elimination was introduced into the equation. The FDM and the diffusion model considering removal (DMCR) were compared by simulating various dose rate irradiation schemes and uniform irradiation. Finally, the DMCR was compared with previous microbeam and minibeam animal experiments. RESULTS: Compared with a previous Monte Carlo & Convolution method, this analytical method provides more accurate results. Furthermore, the new model shows H2 O2 concentration distribution instead of the time to achieve a certain H2 O2 uniformity. The comparison between FDM and DMCR showed that H2 O2 distribution from FDM varied with dose rate irradiation, while DMCR had consistent results. For uniform irradiation, FDM resulted in a Gaussian distribution, while the H2 O2 distribution from DMCR was close to the dose distribution. The animal studies' evaluation showed a correlation between the H2 O2 concentration in the valley region and treatment outcomes. CONCLUSION: DMCR is a more realistic model for H2 O2 simulation than the FDM. In addition, the H2 O2 distribution can be a good surrogate of dose distribution when the minibeam effect could be observed.

Radio-resistance of hypoxic tumors: exploring the effects of oxygen and x-ray radiation on non-small lung cancer cell lines.

BACKGROUND: Solid tumors are often riddled with hypoxic areas, which develops as a result of high proliferation. Cancer cells willingly adapt and thrive in hypoxia by activating complex changes which contributes to survival and enhanced resistance to treatments, such as photon radiation. Photon radiation primarily relies on oxygen for the production of reactive oxygen species to induce DNA damage. The present in-vitro study aimed at investigating the biochemical responses of hypoxic non-small cell lung cancer (NSCLC) cells, particularly the effects on the DNA damage repair systems contributing to more radioresistant phenotypes and their pro- and anti-oxidant potential, within the first 24 h post-IR. METHODS: NSCLC cell lines (H460, A549, Calu-1) were irradiated using varying X-ray doses under normoxia (21% O2) and hypoxia (0.1% O2). The overall cell survival was assessed by clonogenic assays. The extent of irradiation (IR)-induced DNA damage was evaluated by analyzing γ-H2AX foci induction and the altered expression of repair genes involved in non-homologous end joining and homologous recombination pathways. Moreover, cell-altered responses were investigated, including the nuclear and cytosolic hydrogen peroxide (H2O2) production, as well as the associated anti-oxidant potential, in particular some components related to the glutathione system. RESULTS: Analysis of clonogenic survival revealed an enhanced radioresistance of the hypoxic NSCLC cells associated with reduced DNA damage and a downregulation of DNA repair genes. Moreover, nuclear H2O2 levels were IR-induced in a dose-dependent manner only under normoxia, and directly correlated with the DNA double-strand breaks. However, the observed nuclear H2O2 reduction in hypoxia appeared to be unaffected by IR, thus highlighting a possible reason for the enhanced radioresistance of the hypoxic NSCLC cells. The cellular antioxidant capacity was upregulated by IR in both oxygen conditions most likely helping to counteract the radiation effect on the cytosolic H2O2. CONCLUSIONS: In conclusion, our data provide insight into the adaptive behavior of radiation-resistant hypoxic NSCLC cells, in particular their DNA repair and oxidative stress responses, which could contribute to lower DNA damage and higher cell survival rates following X-ray exposure. These findings may therefore help to identify potential targets for improving cancer treatment outcomes.

Human lung-cancer-cell radioresistance investigated through 2D network topology.

Radiation therapy (RT) is now considered to be a main component of cancer therapy, alongside surgery, chemotherapy and monoclonal antibody-based immunotherapy. In RT, cancer tissues are exposed to ionizing radiation causing the death of malignant cells and favoring cancer regression. However, the efficiency of RT may be hampered by cell-radioresistance (RR)-that is a feature of tumor cells of withstanding RT. To improve the RT performance, it is decisive developing methods that can help to quantify cell sensitivity to radiation. In acknowledgment of the fact that none of the existing methods to assess RR are based on cell graphs topology, in this work we have examined how 2D cell networks, within a single colony, from different human lung cancer lines (H460, A549 and Calu-1) behave in response to doses of ionizing radiation ranging from 0 to 8 Gy. We measured the structure of resulting cell-graphs using well-assessed networks-analysis metrics, such as the clustering coefficient (cc), the characteristic path length (cpl), and the small world coefficient (SW). Findings of the work illustrate that the clustering characteristics of cell-networks show a marked sensitivity to the dose and cell line. Higher-than-one values of SW coefficient, clue of a discontinuous and inhomogeneous cell spatial layout, are associated to elevated levels of radiation and to a lower radio-resistance of the treated cell line. Results of the work suggest that topology could be used as a quantitative parameter to assess the cell radio-resistance and measure the performance of cancer radiotherapy.

Changes in Radical Levels as a Cause for the FLASH effect: Impact of beam structure parameters at ultra-high dose rates on oxygen depletion in water.

The influence of different average and bunch dose rates in electron beams on the FLASH effect was investigated. The present study measures O2 content in water at different beam pulse patterns and finds strong correlation with biological data, strengthening the hypothesis of radical-related mechanisms as a reason for the FLASH effect.

Lipid droplets and ferritin heavy chain: a devilish liaison in human cancer cell radioresistance.

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.