# Sparing effects of FLASH irradiation in patient-derived lung tissue

Maxime Dubail, Chloé Lafouasse, Sophie Heinrich, Vincent Favaudon, Arturo Londoño-Vallejo, Marie Dutreix, Delphine Colin, Jean-François Côté, Jérôme Didier, Christelle Pouliquen, et al.

► To cite this version:

Maxime Dubail, Chloé Lafouasse, Sophie Heinrich, Vincent Favaudon, Arturo Londoño-Vallejo, et al. Sparing effects of FLASH irradiation in patient-derived lung tissue. Radiotherapy & Oncology, 2025, 212, pp.111126. 10.1016/j.radonc.2025.111126. hal-05286985

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Submitted on 27 Sep 2025

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have extended to large animal models and certain veterinary cases [10–12], but its mechanisms remain under investigation, and its clinical application in human tissues is still exploratory [13–15].

Recent clinical trials, such as those conducted at the CHUV in Lausanne [16,17] and the University of Cincinnati (FAST-01 and FAST-02), have demonstrated the feasibility of FLASH radiotherapy, reporting no unexpected toxicities or loss of anti-tumoral efficacy compared to conventional radiotherapy [15,18,19]. However, the protective effect of ultra-high dose rate in human tissues has yet to be unequivocally demonstrated. Moreover, mechanistic studies are needed to refine the clinical implementation of FLASH and optimize its benefits.

To address this gap, we used precision-cut lung slice (PCLS) systems as ex vivo surrogate to assess the acute effects of ultra-high dose rate on healthy human lung tissue. Although this model does not recapitulate chronic tissue remodeling, it enables analysis of early cellular responses such as proliferation, which have been associated with regenerative capacity and reduced radiation toxicity in preclinical models [20,21], including our previous murine PCLS study demonstrating a FLASH sparing effect within 24-hours post-irradiation [22]. Therefore, we investigate whether this beneficial effect is present in healthy lung tissue from patients. To this end, precision-cut lung slices (Hu-PCLS) were prepared from lung samples collected during lobectomies and irradiated at a dose of 9 Gy delivered either in conventional or FLASH modes. Analysis of cell division revealed a sparing effect of FLASH irradiation in nearly all patients (95 %). RNAseq analysis, performed on a subset of patients, confirmed an attenuated activation of DNA damage response pathways, pro-apoptotic genes, and oxidative stress signaling in FLASH-treated samples. Altogether, these results obtained from patient-derived lung samples indicate that FLASH irradiation limit effects of radiation at the transcriptional and cellular levels in human lung tissue, fostering the development of FLASH radiotherapy for the treatment of thoracic malignancies.

## Materials and methods

### Ethical consent

All samples were collected from the Institut Mutualiste Montsouris under a dedicated protocol for lung cancer specimens approved by the French Ethics and Informatics Commission (EUdract 2017 - A03081-52). All patients in this study provided written informed consent for sample collection for research purposes.

### Human Precision-Cut Lung Slices (Hu-PCLS)

Tumor-free lung tissues were consistently obtained from the same distal area (i.e. tip of the lobe), away from the resection margin, to avoid tumoral contamination, and were used to prepare precision-cut lung slices (Hu-PCLS) following a protocol adapted from previous publications [23,24]. Briefly, lung tissues were cleaned twice in Phosphate Buffered Saline (PBS) to remove blood. Then, on ice, 2.5 % low-melting agarose (A9414-50G) diluted in Hu-PCLS medium (DMEM F12 from Invitrogen (#31331-028) supplemented with 1 % SVF (CVFSVF00-0U), 1 % penicillin/streptomycin (CABPES01-0U), 1 % non-essential amino acids (11140035), and 1 % L-glutamine (25030-024)) was injected slowly into small bronchi to inflate the lung resection. 8 mm punches were embedded in 5 % agarose, and 500 µm slices were made using a vibratome (Leica VT1000S). Hu-PCLS were placed into a 24-well plate containing 500 µL of Hu-PCLS medium and cultured at 37 °C in 5 % CO2 up to 24 h. Between 15 and 25 Hu-PCLS were obtained from each patient sample and they were randomly assigned to each treatment group.

### Irradiation

Hu-PCLS were irradiated in a 24-well plate using a vertical 7-MeV electron beam produced by the linear accelerator ElectronFLASH (SIT

S.p.A., R&D Dept., Italy) at Institut Curie, capable of delivering both conventional (CONV) and ultra-high dose rate irradiation. A total dose of 9 Gray (Gy) in a single fraction was delivered in both CONV and FLASH modalities. This dose was selected based on prior murine PCLS studies showing optimal detection of FLASH effects on proliferating lung cells. Dose measurements were performed using Gafchromic™ EBT-XD films (Ashland, NJ, USA) and a flashDiamond Detector T60025 (PTW Freiburg, Germany), placed inside the wells at the position of the lung samples for planning and at the top of the plate for online monitoring. Irradiation duration was measured using a Bergoz in-flange Current Transformer mounted in the beam line and connected to a Teledyne Lecroy Wavesurfer 4054HD oscilloscope. The parameters used are summarized in the Supplementary Material. For each patient, a minimum of 5 Hu-PCLS slices were analyzed per condition.

### Cell division analysis

To estimate the proportion of cells that replicate after irradiation in the Hu-PCLS, a Click-IT chemistry protocol was used to monitor cell proliferation via 5-ethynyl-2'-deoxyuridine (EdU) incorporation (BaseClick, #BCK-EdUPro-FC647). Hu-PCLS were incubated 1 h after irradiation in 500 µL of Hu-PCLS medium containing 10 µM EdU. 24 h post-irradiation, Hu-PCLS were fixed in 4 % PFA, and EdU incorporation was revealed according to the manufacturer's instructions. Hu-PCLS were then washed in PBS and incubated with DAPI. The samples were transferred into a glass support adapted for microscopy (μ-Slide 4 Well Glass Bottom, Ibidi) and imaged on an inverted Nikon Spinning Disk TIRF-FRAP using a 10X objective. Four to five fields of view (FOV) were acquired per Hu-PCLS. Image post-processing and EdU quantification were performed using a semi-automatic method combining 3D reconstruction and segmentation of the nuclei with IMARIS software (Bit-plane). For the analysis and for each patient, to compare the effects of FLASH versus CONV irradiation, we normalized the proportion of cycling cells relative to the average proportion of EdU+ cells per FOV in untreated conditions. This normalization allowed us to estimate a relative proportion of cells still cycling under irradiated conditions.

### RNA extraction

For transcriptional analysis induced by conventional and FLASH irradiation, lung resections were cut into small pieces of 2–3 mm, irradiated either in CONV or FLASH modes, and cultured at 37 °C, 5 % CO2 for 24 h. After a PBS wash, lung tissues were incubated in RNAprotect solution (Qiagen, Hilden, Germany) overnight at 4 °C. Disruption and lysis of the lung tissue were performed in 600 µL of RLT lysis buffer (Qiagen, Hilden, Germany) using a Tissue Ruptor II (Qiagen, Hilden, Germany). Lung homogenate was then processed according to manufacturer's instructions using an RNeasy Mini Kit. RNA integrity and concentration were estimated using the Agilent Bioanalyzer following the manufacturer's instructions (RNA 6000 Nano Kit #5067-1512).

### RNAseq analysis

All samples presented a RIN > 7. Sequencing libraries were prepared from 500 ng of total RNA using the Illumina TrueSeq Stranded mRNA Library preparation kit, following the manufacturer's recommendations. 100 bp paired-end sequencing was performed with the Illumina NovaSeq 6000 instrument. Reads were mapped to the reference human genome (hg19/GRCh37) using STAR 2.7.6a (https://github.com/alexdobin/STAR). In the raw count matrix, background genes (expression < 1) in most patient replicates were filtered out. For Gene Set Enrichment Analysis (GSEA) and heatmap generation, data were normalized using the DESeq2 package (https://github.com/thelovelab/DESeq2). To explore the signaling pathways differentially impacted after FLASH versus conventional dose rate, GSEA was performed comparing CONV vs. Non-Irradiated (NI), FLASH vs. NI, or CONV vs. FLASH. Significantly
enriched pathways were selected based on p-value < 0.05 and FDR q-value < 0.1 with permutations (n = 1000, permutation type = gene set).

## Statistical analysis

Statistical analyses were performed using the ggpubr package (https://rpkgs.datanovia.com/ggpubr/) in R. For EdU+ nuclei count data, the Wilcoxon Mann-Whitney test was used to compare the means of these counts between conventional and FLASH conditions.

## Results

### Analysis of patient-derived lung samples

To analyze the acute effect of ultra-high dose rate in human healthy lung tissue, we established a cohort of 19 lung cancer patients, from whom healthy lung tissue was resected after surgery at the Institut Mutualiste Montsouris (Table 1). The cohort included 10 men and 9 women, with ages ranging from 48 to 86 years. Most patients were diagnosed with lung adenocarcinoma (13/19), with a smaller number presenting with squamous cell carcinoma (5/19) and large cell carcinoma (1/19). Notably, two patients had a history of breast cancer and had previously undergone radiotherapy and chemotherapy. Three patients received neoadjuvant therapy, including the anti-PD1 antibody (Nivolumab), prior to surgery. To assess the heterogeneity of the cohort, we first quantified the basal level of cell division in the lung. Hu-PCLS were prepared from fresh lung samples from each patient, and the proportion of dividing cells was measured 24 h post-culture (Fig. 1). The basal level of cell division in untreated Hu-PCLS varies across patients, ranging from an average of 61 EdU+ cells per FOV in patient 12 to 8 EdU+ cells per FOV in the patient with the lowest rate of cycling cells. The heterogeneity between patients is well-known, as many intrinsic factors can affect the basal division rate in the patient's lung. Interestingly, Hu-PCLS from the three patients treated with neoadjuvant therapies exhibited a higher number of dividing EdU+ cells. While a positive pearson correlation was observed between neoadjuvant therapy and increased cell division, statistical analysis did not show significant

correlations between cell division and other clinical data such as sex or age (Supplementary Fig. 1). Overall, this cohort represents an heterogeneous subset of lung cancer patients who may be subjected to ultra-high dose rate treatment in future studies.

### FLASH irradiation limits the decrease of cycling cells in patient-derived lung sample

Our previous study, using analysis of cycling cells in mouse lung ex vivo, reported that compared to conventional radiotherapy (CONV), FLASH irradiation limits the decrease in the capacity of lung cells to divide 24-hours after whole thorax irradiation [22]. To determine the impact of ultra-high dose rate in human lung samples, we applied similar irradiation parameters as well as the endpoints used for the analysis of mouse lung samples. Briefly, Hu-PCLS were prepared from the 19 patients of the described cohort, irradiated with a dose of 9 Gy delivered in either CONV or FLASH mode, and the proportion of dividing cells was quantified 24-hours after irradiation (Fig. 2). Conventional radiotherapy triggered a sharp decrease in the proportion of dividing cells, with EdU+ cells ranging from 9% to 33% in women and 18% to 42% in men. In contrast, ultra-high dose rate induced an attenuated response, with EdU+ cells ranging from 17% to 56% in women and 28% to 57% in men. In 18 out of 19 patients (≈95%), FLASH limits the decrease in the proportion of dividing cells after irradiation. In one case, Hu-PCLS from a single patient did not exhibit a significant difference in cell division between FLASH and CONV irradiation ex vivo. Of note, this patient had the highest rate of cell division at the basal level and received immunotherapy a few weeks prior to the treatment but further investigation is needed. Altogether, these results indicate that FLASH, compared to conventional radiotherapy, is associated with a higher preservation of cycling cells in the lung from the vast majority of patients tested.

### RNAseq analysis confirms a differential impact of FLASH irradiation in patient-derived lung

To provide exploratory insights into the underlying mechanisms of

<caption>**Table 1**</caption>

Patient information included in this study. M: Male; F: Female; PY: Number of Packs/Year; *Patients' samples selected for RNAseq analysis.

<table><thead><tr><th>Sample ID</th><th>Age (years)</th><th>Sex</th><th>Smoking status</th><th>Cancer</th><th>Neoadjuvant therapy</th><th>Previous thoracic radiotherapy</th><th>Lung comorbidities</th></tr></thead><tbody><tr><td>Patient 1</td><td>72</td><td>M</td><td>35 PY</td><td>Squamous cell carcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 2</td><td>61</td><td>F</td><td>35 PY</td><td>Large cell carcinoma</td><td>—</td><td>—</td><td>Lung infection in childhood</td></tr><tr><td>Patient 3</td><td>72</td><td>M</td><td>60 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>Stented ischemic heart disease</td></tr><tr><td>Patient 4</td><td>70</td><td>F</td><td>10 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 5</td><td>76</td><td>M</td><td>56 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>Sars-COV infection</td></tr><tr><td>Patient 6</td><td>86</td><td>M</td><td>0 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 7</td><td>57</td><td>M</td><td>60 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>Chronic bronchitis</td></tr><tr><td>Patient 8</td><td>56</td><td>F</td><td>40 PY</td><td>Adenocarcinoma</td><td>—</td><td>Breast cancer (2019)</td><td>—</td></tr><tr><td>Patient 9</td><td>67</td><td>M</td><td>40 PY</td><td>Squamous cell carcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 10</td><td>72</td><td>M</td><td>45 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 11</td><td>66</td><td>F</td><td>20 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>Sars-COV infection</td></tr><tr><td>Patient 12</td><td>60</td><td>F</td><td>60 PY</td><td>Adenocarcinoma</td><td>Cisplatin/Pémétrexd/ Nivolumab</td><td>—</td><td>—</td></tr><tr><td>Patient 13</td><td>76</td><td>M</td><td>50 PY</td><td>Squamous cell carcinoma</td><td>Carboplatin/Taxol/Nivolumab</td><td>—</td><td>—</td></tr><tr><td>Patient 14</td><td>48</td><td>M</td><td>30 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 15*</td><td>67</td><td>F</td><td>45 PY</td><td>Squamous cell carcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 16*</td><td>64</td><td>F</td><td>20 PY</td><td>Squamous cell carcinoma</td><td>Carboplatin/Pémétrexd/ Nivolumab</td><td>Breast cancers (1999, 2003)</td><td>—</td></tr><tr><td>Patient 17</td><td>72</td><td>M</td><td>55 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 18</td><td>62</td><td>F</td><td>0 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Patient 19*</td><td>57</td><td>F</td><td>15 PY</td><td>Adenocarcinoma</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Analysis of EdU incorporation in Hu-PCLS reveals heterogeneous cell division rates across patients. (A) Schematic illustrating the workflow to obtain, process, and analyze Hu-PCLS. (B) Representative images showing the variation in the proportion of EdU+ cells across patients. Images were acquired on a confocal microscope with a 10× objective. The scale bar represents 200 µm. (C) Quantification of the number of EdU+ cells within Hu-PCLS 24 h after slicing showed an average of 18 EdU+ cells per field of view (FOV). Notably, patients 12, 13, and 16, who received neoadjuvant immunotherapy, exhibited higher numbers of EdU+ cells, ranging from 27 to 61. (n = 4 to 5 FOV per slice for a total of 5–8 slices per patient). Each dot represents the quantification for one field of view.</figcaption></figure>

this protective effect observed after FLASH irradiation, RNAseq analysis was conducted from patient-derived lung samples collected 24 hours following exposure to either conventional or ultra-high dose rate at a dose of 9 Gy (Fig. 3A). To identify molecular responses specific of FLASH, we selected genes that are differentially expressed between FLASH and CONV irradiation in at least 2 out of 3 patients analyzed. Differentially expressed genes analysis revealed that conventional radiotherapy triggered significant upregulation of more than 2000 genes compared to Non-Irradiated (NI) condition. In contrast, FLASH induced an increase in the expression of only 55 genes (Fig. 3B). Gene Set Enrichment Analysis (GSEA) analysis revealed that conventional radiotherapy downregulated cell cycle-related genes such as TOP2A, MKI67, and CDK1, whereas FLASH attenuated this decrease, consistent with the results obtained from the cell division analysis in patient Hu-PCLS (Fig. 3C). Simultaneously, while conventional radiotherapy strongly upregulated p53 and DNA damage-related pathways, FLASH limits the activation of these genes, showing a reduced level of stress and DNA damage. To determine whether this sparing effect of FLASH irradiation is specific to healthy tissue, we analyzed previously published RNAseq data from xenografted human brain tumors resected 24 h after exposure to conventional or FLASH irradiation [1]. In tumor, similar GSEA showed that FLASH induced a downregulation of genes associated to cell cycle progression as well as an upregulation of DNA damage and

p53 pathways similarly to conventional radiotherapy (Supplementary Fig. 2). Altogether, RNAseq analysis from patient-derived lung tissue confirms, at the transcriptional level, that FLASH limits the impact of radiation on cell cycle arrest, consistently with the incorporation of EdU in Hu-PCLS.

### FLASH irradiation limits activation of oxidative stress-related genes

While radiation is known to trigger a burst of reactive oxygen species, accumulating evidence indicates that FLASH irradiation generates a reduced oxidative burden compared to conventional dose rates [25,26]. To further explore this mechanism in human lung tissue, we analyzed differentially expressed genes associated with oxidative stress responses in lung samples exposed to conventional or FLASH irradiation. (Fig. 4). This analysis revealed that NRF2 signaling, a pathway playing a critical role in Reactive Oxygen Species (ROS) detoxification as well as genes in controlling stem cell self-renewal, proliferation, and differentiation [27], were selectively upregulated after conventional radiotherapy (Fig. 4A–C). In particular, KEAP1, one of the key players of the NRF2 pathway [28], was found significantly consistently upregulated in lung samples from three distinct patients exposed to conventional radiotherapy (Fig. 4B). In the FLASH-treated samples, no activation of NRF2 pathway were found at the transcriptional level. Similarly, GSEA
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** FLASH radiotherapy preserves a higher proportion of cycling cells in almost all patients at 24 h post-treatment. (A) Quantification of the proportion of EdU+ cells in Hu-PCLS prepared from female patients and analyzed 24 h after conventional (CONV), FLASH and NI conditions. Analysis of cycling cells after irradiation shows that 8 out of 9 patients exhibit a higher proportion of EdU+ cells after FLASH compared to CONV. (B) A similar analysis performed on Hu-PCLS obtained from 10 male patients confirmed that FLASH irradiation attenuates the reduction in the proportion of cycling cells compared to conventional radiotherapy. ns = not significant; \* p-value < 0.05; \*\* p-value < 0.01; \*\*\* p-value < 0.001; \*\*\*\* p-value < 0.0001.</figcaption></figure>

analysis of genes linked to ROS detoxification lead to the observation of a strong upregulation of genes such as PRDX1 and SOD1, known NRF2 target genes as well as canonical thioredoxin reductase genes, TXNRD2 and TXNRD3, after conventional irradiation. Again, no activation of these ROS-induced genes were found in lung samples exposed to FLASH. These results indicate that, unlike conventional treated samples, FLASH does not induce, at the transcriptional level, the expression of genes classically induced by oxidative stress, confirming that FLASH reduces radiation-induced ROS burden in patient-derived lung samples.

Discussion

This study indicates that FLASH irradiation is associated with preserved cell division in patient-derived lung samples, which can serve as an early marker of normal tissue sparing response. By analyzing a cohort of 19 lung cancer patients, we showed that compared to conventional radiotherapy, FLASH irradiation limits the decrease in the proportion of cycling cells in almost all patients-derived PCLS (18/19). This beneficial effect was confirmed by RNAseq analysis 24-hours after treatment,

showing the preservation of the expression cell cycle-related genes (e.g., MKI67, TOP2A) after FLASH, compared to conventional radiotherapy. Additionally, the molecular analysis highlighted an attenuated upregulation of p53 pro-apoptotic transcriptional targets (e.g., BAX, GADD45A), as well as genes associated with DNA damage (e.g., ATR, PARP1, CHEK2). Furthermore, FLASH irradiation triggered limited activation of oxidative stress-related pathways, suggesting a reduction in reactive species compared to conventional treatment. In the last decade, FLASH has shown great potential in widening the therapeutic window of radiation therapy. Preclinical studies have demonstrated that FLASH reduces toxicities in organs such as the lung, gut, brain, skin, and esophagus [5,8,29,30]. Not only in mouse models, but also in larger mammals, including mini-pigs as well as dogs and cats cancer patients [10,11,31]. Several studies investigating the molecular and cellular mechanisms underlying the sparing of healthy tissue induced by FLASH have reported the preservation of the cycling capabilities of stem/progenitor cells. In mouse brain models, FLASH irradiation limits the radiation impact on progenitor cells, which correlated with attenuated astrogliosis and maintained neurocognitive function [30,32,33].
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** RNAseq analysis confirms a differential effect of FLASH radiotherapy 24 h post-radiation in healthy lung tissue. (A) Workflow for RNAseq processing and analysis from 3 selected patients. Each patient's sample was split across 3 conditions (NI, FLASH, CONV) with 3 replicates per condition. (B) Differentially expressed genes (DEG) analysis revealed that conventional radiotherapy (CONV) upregulates 40 times more genes than FLASH. (C) Heatmap showing downregulation of cell cycle gene expression after conventional (CONV) radiotherapy compared to FLASH and non-irradiated (NI) conditions, as well as attenuated activation of genes related to cell cycle checkpoints, p53 pro-apoptotic pathway, and DNA damage response. Rows and columns are ordered based on predefined clustering of pathways and conditions.</figcaption></figure>

Similarly, in mouse gut models, FLASH reduced intestinal damage, limited ferroptosis, and increased the proportion of intestinal stem cells capable of regenerating the crypts after radiation injury [29,34,35]. Previous preclinical studies in mouse lung models showed that FLASH irradiation spares progenitor cells, reduces persistent DNA damage, reduces senescence, and limits radio-induced pulmonary fibrosis [36]. In this work, we observed similar preservation of cycling cells in lung tissue resected from patients, suggesting that the mechanism underlying the beneficial effect of FLASH irradiation is conserved across organs and species. Regarding mechanistic hypothesis, RNAseq analysis of human lung tissue indicates that ultra-high dose rate irradiation limits the transcriptional activation of genes involved in reactive oxygen species (ROS) detoxification, confirming reduced ROS burden after FLASH

compared to conventional radiotherapy. Similar results were obtained in mouse brain models after FLASH irradiation [33]. Recent studies have reported lower radiolytic yields of H₂O₂ under FLASH conditions as well as oxygen depletion effects due to radical recombination at ultra-high dose rates, both of which support a biochemical basis for reduced oxidative stress [25,26]. Results obtained in this study therefore align with and strengthen the mechanistic hypothesis that lower ROS production under FLASH irradiation contributes to the tissue-sparing effect observed in multiple preclinical settings. At the biochemical level, a recent study reported that FLASH irradiation induces less lipid peroxidation [37]. In line with these previous studies and the mechanistic model of the FLASH effect [38], our study provides further evidence that FLASH irradiation produces fewer oxidative species, reducing the
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** FLASH radiotherapy reduces induction of anti-oxidant program in human lung at the transcriptional level. (A) Gene Set Enrichment Analysis (GSEA) comparing differentially expressed genes after conventional (CONV) vs. FLASH radiotherapy revealed an enrichment of the NRF2 pathway specifically after CONV. (B) Heatmap representing the expression of NRF2-related genes, which were strongly upregulated after conventional radiotherapy but not after FLASH radiotherapy. Rows and columns are ordered based on predefined clustering of signature and conditions. (C) Concomitantly, pathways involved in the management of Reactive Oxygen Species (ROS) are selectively enriched after conventional radiotherapy. (D) Heatmap illustrating the upregulation of genes responsible for ROS detoxification specifically after conventional radiotherapy. Rows and columns are ordered based on predefined clustering of signatures and conditions.</figcaption></figure>

oxidative pressure.

In terms of transcriptional responses in the previously published U87 tumor model [1], no notable differences were observed in the activation of cell cycle arrest or p53 signaling pathways 24-hours after exposure to 20 Gy, whether delivered at conventional or ultra-high dose rate. These findings suggest that, unlike normal lung tissue response, FLASH irradiation does not attenuate stress responses in tumor cells. To fully validate the selective sparing effect of FLASH in human tissues, future studies analyzing healthy and tumor tissues side by side within the same patient context are needed. In a clinical perspective, few patients have been treated with FLASH for skin lesions or bone metastases [15–17], but clinical studies demonstrating reduced toxicities after FLASH have not been reported yet. To our knowledge, results obtained from patient-derived lung samples are the first to provide information suggesting that preserved cell proliferation can serve as an early indicator of a differential response to FLASH irradiation in human tissue. The ex vivoHPCLS model presents inherent limitations in assessing chronic outcomes such as fibrosis or late inflammatory remodeling. Nevertheless, prior studies have shown that cell division capacity analyzed shortly after irradiation can serve as a relevant surrogate for tissue regenerative

potential. In intestinal and neural tissues, early preservation of cycling cells following exposure to FLASH irradiation has been correlated with improved long-term function. Given the practical and ethical challenges of studying FLASH effects longitudinally in human for the moment, acute analysis of patients-derived lung samples provides a valuable alternative. These results are promising for future clinical trials and will be, hopefully, validated in patients.

Although preclinical in vivo studies in rodents often use single doses between 14 and 30 Gy to assess late toxicities such as fibrosis, such outcomes cannot be evaluated in an ex vivo system limited to short-term culture. The choice of the 9 Gy dose in this study was based on both biological relevance and the technical limitations of the ex vivo model. In previous experiments using murine PCLS, this dose provided optimal sensitivity to detect FLASH-specific effects on proliferating cells. In contrast, higher doses such as 12 Gy or above caused a marked suppression of cell division under both irradiation conditions, making it difficult to distinguish treatment-specific effects. Importantly, the 9 Gy dose used here corresponds to single-fraction doses commonly applied in clinical hypofractionated radiotherapy protocols, such as stereotactic body radiotherapy for lung cancer, supporting the clinical relevance of
this approach.

## Conclusions

This study presents initial evidence of the beneficial effects of FLASH irradiation in human tissue. Although clinical data are required to demonstrate the potential of FLASH, the results obtained from patient-derived lung holds great promise for the clinical translation of FLASH irradiation.

## Funding statement

This work was funded by INSERM (contract N°19CR057-00) and received support from La Ligue contre le Cancer. It has received fundings from ANR in the frame of France 2030 under the reference ANR-24-FRAT-0001 as well as from INCa under the INCa-AMED ASPIRE funding scheme (2025-014).

## CRediT authorship contribution statement

**Maxime Dubail:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. **Chloé Lafouasse:** Resources, Methodology, Formal analysis, Data curation. **Sophie Heinrich:** Writing – review & editing, Resources, Methodology, Formal analysis, Data curation. **Vincent Favaudon:** Validation, Methodology, Data curation. **Arturo Londoño-Vallejo:** Visualization, Investigation, Formal analysis. **Marie Dutreix:** Funding acquisition, Conceptualization. **Delphine Colin:** Resources. **Jean-François Côté:** Resources. **Jérôme Didier:** Resources. **Christelle Pouliquen:** Resources. **Abdelali Benali:** Resources. **Pierre Verrelle:** Validation, Resources, Methodology, Data curation. **Marine Lefevre:** Resources. **Nicolas Girard:** Resources, Methodology, Data curation. **Agathe Seguin-Givelet:** Resources, Methodology, Data curation. **Gilles Créhange:** Supervision, Resources, Investigation. **Charles Fouillade:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The contribution of the bioinformatics core facility at U900 Inserm-Institut Curie is gratefully acknowledged. High-throughput sequencing was performed by the ICGex NGS platform of the Institut Curie, supported by grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the Agence Nationale de la Recherche (“Investissements d’Avenir” program), by the ITMO-Cancer Aviesan (Plan Cancer III), and by the SiRIC-Curie program (SiRIC Grant INCA-DGOS-465 and INCa-DGOS-Inserm_12554). Data management, quality control, and primary analysis were performed by the Bioinformatics platform of the Institut Curie. The authors are grateful to Marie-Noëlle Soler and Laetitia Besse from the Multimodal Imaging Center-Light Microscopy Facility of the Institut Curie (CNRS UMS2016/InermUS43/Institut Curie/Université Paris-Saclay), as well as the Cell and Tissue Imaging Platform – PICT-IBiSA (member of France-Bioimaging – ANR-10-INBS-04) of the U934/UMR3215 of Institut Curie for their guidance with light microscopy. The authors wish to thank Sarah Lagha and Kheira Bouaziz from the Institut du Thorax Curie-Montsouris, as well as Simon Lefranc from the Centre de Ressources Biologiques of Institut Mutualiste Montsouris, for their help in providing patient samples. Thanks are also due to the members of the

Londoño-Vallejo team for scientific exchanges and discussions.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.radonc.2025.111126.

## Data availability

RNAseq data from tumor-free human lung treated with either conventional or at ultra-high dose rate treatment have been deposited in NCBI's Gene Expression Omnibus under the accession number GSE263484. All data and code are available upon request. The RNAseq dataset previously published [1] from xenografted human U-87 MG glioblastoma tumors exposed to conventional or FLASH irradiation is accessible through the accession number GSE223607.

## References

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