Lung cancers are highly resistant to radiotherapy, necessitating the use of high doses, which leads to radiation toxicities such as radiation pneumonitis and fibrosis. Caffeic Acid Phenethyl Ester (CAPE) has been suggested to have anti-proliferative and pro-apoptotic effects in tumour cells, while radioprotective anti-inflammatory and anti-oxidant effects in the normal tissue. We investigated the radiosensitizing and radioprotective effects of CAPE in lung cancer cell lines and normal tissue in vitro and ex vivo, respectively. The cytotoxic and radiosensitizing effects of CAPE in lung cancer were investigated using viability and clonogenic survival assays. The radioprotective effects of CAPE were assessed in vitro and ex vivo using precision cut lung slices (PCLS). Potential underlying molecular mechanisms of CAPE focusing on cell cycle, cell metabolism, mitochondrial function and pro-inflammatory markers were investigated. Treatment with CAPE decreased cell viability in a dose-dependent manner (IC₅₀ 57.6±16.6μM). Clonogenic survival assays showed significant radiosensitization by CAPE in lung adenocarcinoma lines (p<0.05), while no differences were found in non-adenocarcinoma lines (p≥0.13). Cell cycle analysis showed an increased S-phase (p<0.05) after incubation with CAPE in the majority of cell lines. Metabolic profiling showed that CAPE shifted cellular respiration towards glycolysis (p<0.01), together with mitochondrial membrane depolarization (p<0.01). CAPE induced a decrease in NF-κB activity in adenocarcinomas and decreased pro-inflammatory gene expression in PCLS. The combination of CAPE and radiotherapy may be a potentially effective approach to increase the therapeutic window in lung cancer patients.

To rapidly assess healthy tissue toxicities induced by new anti-cancer therapies (i.e., radiation alone or in combination with drugs), there is a critical need for relevant and easy-to-use models. Consistent with the ethical desire to reduce the use of animals in medical research, we propose to monitor lung toxicity using an ex vivo model. Briefly, freshly prepared organotypic lung slices from mice were irradiated, with or without being previously exposed to chemotherapy, and treatment toxicity was evaluated by analysis of cell division and viability of the slices. When exposed to different doses of radiation, this ex vivo model showed a dose-dependent decrease in cell division and viability. Interestingly, monitoring cell division was sensitive enough to detect a sparing effect induced by FLASH radiotherapy as well as the effect of combined treatment. Altogether, the organotypic lung slices can be used as a screening platform to rapidly determine in a quantitative manner the level of lung toxicity induced by different treatments alone or in combination with chemotherapy while drastically reducing the number of animals. Translated to human lung samples, this ex vivo assay could serve as an innovative method to investigate patients’ sensitivity to radiation and drugs.

Radiation toxicities, such as pneumonitis and fibrosis, are major limitations affecting patients' quality of life. Developed a decade ago, FLASH radiotherapy is an innovative method that, by delivering radiation at ultrafast dose rate, reduces radiation toxicities on healthy tissue while preserving the anti-tumoral effect of radiotherapy. This so-called FLASH effect has been described in different preclinical models but has not been observed in human tissue. This study aims to determine if FLASH irradiation can induce a sparing effect on human healthy lung tissue. To address this question, precision-cut lung slices (Hu-PCLS) were prepared from healthy lung samples collected from 19 lung cancer patients undergoing lobectomy. These Hu-PCLS were irradiated ex vivo at a dose of 9 Gy using the ElectronFLASH (SIT) device operated either in conventional or FLASH mode. We monitored cell division for each patient and performed RNAseq analysis to uncover some mechanistic insights. Analysis of cell division 24 h after treatment with conventional or ultra-high dose rate showed a higher proportion of dividing cells in Hu-PCLS after FLASH irradiation. Consistently, RNAseq analysis from irradiated lung samples confirmed an attenuated cell cycle checkpoint inhibition, p53 pro-apoptotic genes, DNA damage, and antioxidant pathways after ultra-high dose rate compared to conventional treatment. Altogether, this study shows that, using freshly isolated patient-derived lung samples, cell proliferation can serve as an early marker of the normal lung response to FLASH irradiation. These findings hold great promises for future applications of FLASH radiotherapy in the clinic.

To understand the ultra-early reaction of normal organ lipids during irradiation, we investigated the response of lipids, including polyunsaturated fatty acid (PUFA) chains, which are particularly susceptible to damage by ROS, in mice’s kidneys, lungs, brains, and livers within 5 min of single high-dose irradiation. In this study, we set up three groups of C56BL/6 male mice and conducted whole-body irradiation with 0 Gy, 10 Gy, and 20 Gy single doses. Kidney, lung, brain, and liver tissues were collected within 5 min of irradiation. PUFA-targeted and whole lipidomic analyses were conducted using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The results showed that PUFA chains of kidney phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TG) significantly increased within 5 min of 10 Gy and 20 Gy irradiation. The main components of increased PUFA chains in PC and PE were C18:2, C20:4, and C22:6, and in TG the main component was C18:2. The kidney lipidomes also showed significant changes from the perspective of lipid species, mainly dominated by an increase in PC, PE, TG, and signal lipids, while lipidomes of the lung, brain, and liver were slightly changed. Our results revealed that acute PUFA chains increase and other lipidomic changes in the kidney upon whole-body irradiation within 5 min of irradiation. The significantly increased lipids also showed a consistent preference for possessing PUFA chains. The lipidomic changes varied from organ to organ, which indicates that the response upon irradiation within a short time is tissue-specific.

Purpose: This study systematically investigates the radiobiological effects of Very High Energy Electrons (VHEE) generated by a laser-plasma accelerator (LPA), in comparison with Conventional Intermediate Energy Electrons (CIEE) from a conventional linear accelerator (LINAC). Using in vitro, ex vivo , and in vivo models, we evaluate and compare their potential toxicity on healthy tissues. Methods and Materials: Cell survival, tissue response, and developmental toxicity were assessed across three biological models. In vitro , human fibroblasts (MRC5-hTERT) were used to generate post-irradiation survival curves. Ex vivo , precision-cut lung slices (PCLS) from mice were analyzed for radiation-induced inhibition of cell proliferation. In vivo , zebrafish embryos were used to evaluate developmental toxicity through body length and spinal curvature measurements. VHEE irradiations were performed using a broadband electron beam spanning 50–300 MeV, using the Salle Jaune LPA (Laboratoire d’Optique Appliquée, France), while CIEE exposures were performed with a 7 MeV conventional LINAC (Institut Curie, France). Results: In vitro , MRC5-hTERT cells showed no significant difference in radiosensitivity between VHEE and CIEE, with comparable D ₁₀ values (p-value = 0.7). In the ex vivo model, both beams induced a dose-dependent decrease in cell division with no significant inter-beam differences at any dose level (p-value > 0.99). In vivo , zebrafish embryos exhibited dose-dependent body shortening and increased spinal curvature following both VHEE and CIEE exposure. No significant differences were observed between the two modalities at matched doses for any measured metric (p-value ≥ 0.5). Conclusion: This study presents the first comprehensive radiobiological evaluation of a laser-driven VHEE beam across multiple biological models. Under the investigated conditions, VHEE and CIEE irradiations exhibit similar biological toxicity. These findings support the feasibility and potential of VHEE generated with LPA for future clinical applications.

Organ development relies on complex molecular mechanisms that guide initially homogeneous populations of stem cells to differentiate into specialized cell types within defined spatial patterns. While stable during homeostasis, the proper spatial organization of cell types must be re-established in case of tissue injury for successful regeneration of organ shape and function. How cells commit to a differentiation path is a central question in stem cell research; however, the coordination between tissue geometry and cell fate specification remains enigmatic. To elucidate the molecular mechanisms instructing self-organization and symmetry breaking of epithelial stem cells, we developed a multi-faceted approach combining in vitro organoids, ex vivo embryonic tissue explants, and single-cell quantitative imaging to investigate the dynamic acquisition of cell fate in four bi-layered epithelia, during embryonic development but also in regeneration. Our findings indicate that tissue architecture is the primary determinant of cell fate decisions in these tissues. Upon the initial cell internalization event, the homogeneous population of stem cell break symmetry. Through genetic and pharmacological perturbations, we have demonstrated that a tightly coordinated interplay between Hippo/YAP and Notch signaling is essential for conveying information from tissue architecture to functional cell differentiation and stem cell potency restriction. Globally, this study uncovers the inherent capacity of stem cells to self-organize into multicellular structures, where the precise position of each differentiated cell is critical to instruct their differentiation choices during embryonic development and regeneration.