Radiation treatment curtails pro-tumorigenic functions from cancer-associated fibroblasts in preclinical models

Radiation treatment curtails pro-tumorigenic functions from cancer-associated fibroblasts in preclinical models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Radiation treatment curtails pro-tumorigenic functions from cancer-associated fibroblasts in preclinical models Rodrigo Berzaghi, Kristin Lode, Vera Maia, Indusmita Routray, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8680198/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and active drivers of tumor progression, metastasis, and resistance to therapy. However, the effects of radiotherapy (RT) on CAFs and the consequent impact on tumor cell behavior remain controversial. Methods: Primary cultures of human CAFs isolated from non-small cell lung cancer (NSCLC) tumor samples were exposed to ionizing radiation by a single-high dose or a hypofractionated regimen. Different lung tumor cell lines were exposed to control or irradiated CAFs in direct co-cultures or to CAFs conditioned medium (CM), and changes in tumor cell proliferation, migration, epithelial-mesenchymal transition (EMT) and clonogenic survival were measured. The impact of RT on intratumoral CAF levels in different subcutaneous tumor models was measured by α-SMA expression in excised tumor specimens. In addition, pirfenidone, a CAF-reprogramming agent, was investigated for its capacity to influence tumor responses to radiation. Finally, correlations of CAF markers with treatment outcomes was studied in a cohort of RT-treated non-small cell lung cancer (NSCLC) patients. Results: Ionizing radiation induced premature senescence in CAFs; however, the expression of established CAF activation markers remained unchanged. Exposure of lung tumor cell lines to CM from irradiated CAFs did not alter their proliferation, migratory capacity, or clonogenic survival. Conversely, CAF-driven epithelial–mesenchymal transition (EMT) was attenuated in all tumor cell lines after incubations with CM from irradiated CAFs. Radiation delivered to subcutaneously transplanted tumors (1x12 Gy) did not change intra-tumoral CAF abundance in the LLC and CT26 murine models, whereas CAF numbers were significantly reduced in the stroma-rich 4662PDA pancreatic model. Pirfenidone-treated animals exhibited enhanced tumor radio-resistance, along with decreased collagen levels within tumors, and unchanged numbers of aSMA+CAFs. In clinical specimens, baseline tissue expression levels of the CAF markers FAP and aSMA were not associated with disease-specific survival. Conclusions: Our findings suggest that ionizing radiation may lessen certain pro-tumorigenic CAF functions—such as EMT induction—, whereas pharmacologic CAF reprogramming with pirfenidone paradoxically confers increased tumor radio-resistance, highlighting potential negative implications from CAF-targeted therapies. Radiotherapy cancer-associate fibroblasts epithelial-mesenchymal transition in vivo models Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Radiotherapy (RT) is a long-established and widely applied treatment modality in oncology, used in more than half of all cancer patients and responsible for roughly 40% of curative outcomes [ 1 ]. External beam radiotherapy (EBRT), the most common form of RT, has benefited from major technological advances in recent years, resulting in improved tumor control and enhanced patient quality of life [ 2 ]. Contemporary EBRT techniques—such as three-dimensional conformal radiotherapy (3DCRT), intensity-modulated radiotherapy (IMRT), stereotactic body radiotherapy (SBRT), image-guided radiotherapy (IGRT), and particle therapy—rely on sophisticated imaging modalities (CT, MRI, PET) to accurately define tumor volumes and optimize dose delivery while minimizing exposure to surrounding healthy tissues [ 3 ]. Although the primary objective of RT is to induce lethal damage in malignant cells and inhibit their proliferation, it also triggers complex biological responses in the non-malignant components of the tumor microenvironment (TME). Increasing evidence indicates that RT-induced modifications within the TME and the resulting cellular interactions are key determinants of therapeutic success [ 4 , 5 ]. Cancer-associated fibroblasts (CAFs) represent a major stromal population within the TME and are known to influence tumor progression and resistance to therapy [ 6 ]. Beyond establishing a supportive niche for cancer cell growth, CAFs contribute to immunosuppression and can directly promote angiogenesis and metastasis through the secretion of cytokines, growth factors, extracellular matrix (ECM) components, and matrix-modifying enzymes [ 7 ]. In clinical settings, elevated expression of CAF-specific markers or CAF-related gene signatures has been associated with responses to (chemo)radiotherapy and is frequently linked to poor prognosis across multiple cancer types [ 8 , 9 ]. Despite these correlations, the mechanisms underlying CAF-mediated radio-resistance remain insufficiently understood. Compared with other TME constituents, the effects of radiotherapy on CAFs have been less extensively investigated. Existing studies suggest that radiation can induce phenotypic and functional changes in CAFs that may enhance tumor radio-resistance. Irradiated CAFs have been shown to release factors that support cancer cell survival and proliferation [ 10 – 12 ]. Moreover, radiation-induced senescence in CAFs can lead to the secretion of pro-inflammatory cytokines and growth factors, further promoting tumor growth and potentially contributing to treatment resistance [ 13 , 14 ]. Conversely, some preclinical animal studies report that irradiation may diminish the tumor-promoting properties of CAFs [ 15 , 16 ]. In this study, we explore how irradiation influences the tumor-supportive functions of CAFs using 2D in vitro culture systems. We further examine radiation-induced changes in CAF infiltration within tumors in in vivo models. Additionally, we evaluate the impact of CAF modulation on radiotherapy outcomes by pre-treating animals with pirfenidone, a CAF-reprogramming agent. Collectively, our findings provide new insights into how RT affects CAF biology and clarify the potential role of CAFs in shaping radiotherapy responses Material & methods Human material, CAF isolation, and cell cultures Human cancer-associated fibroblasts (CAFs) were derived from surgically resected non-small cell lung cancer (NSCLC) tissues obtained from patients at the University Hospital of Northern Norway (UNN), as previously described [ 17 ] (clinical and patient characteristics can be found in supplementary material, Table S1 ). Briefly, CAFs were isolated by enzymatic digestion of tissues and the outgrowth method, followed by phenotypic characterization. Patient consent was obtained in writing, and the study was conducted in compliance with ethical guidelines approved by the Regional Ethical Committee of Northern Norway (REK Nord 2016/2307). CAFs were isolated using enzymatic digestion and the outgrowth method, followed by phenotypic characterization. Tumor samples from ten patients were processed (Table S1 ). CAFs were cultured in DMEM high glucose medium (Sigma-Aldrich, Cat. # D5796) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Experiments were conducted with low-passage cells (passages 3–6). Donor-derived tumor cells (Donor-TC) were separated from mixed fibroblast cultures through differential detachment and characterized by cytokeratin expression, epithelial morphology, and colony-forming ability. Human lung adenocarcinoma cell lines A549 and HCC827 were obtained from LGC Standards AB and cultured in DMEM or RPMI-1640 medium, respectively, supplemented with 10% FBS and antibiotics. Irradiation of cell cultures Adherent cells were seeded in T-75 flasks or 6-well plates at 80% confluency one day prior to irradiation. High-energy photons were delivered using a clinical Varian linear accelerator, employing either a single dose of 18 Gy or a fractionated regimen of 3 × 6 Gy at 24-hour intervals. Standard dose delivery parameters included a depth of 30 mm, beam quality of 15 MV, dose rate of 6 Gy/min, and a field size of 20 × 20 cm. Western blots Whole-cell lysates were prepared using RIPA buffer (Cell Signaling) supplemented with protease and phosphatase inhibitors (ThermoFisher, Cat. # 78440). Proteins were separated via 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 1% BSA in TBS-T for 2 hours at room temperature and incubated overnight at 4°C with primary antibodies (Table S2 ) diluted 1:1000. After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:2000; Cell Signaling, #7074) for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence and quantified with ImageJ software. β-galactosidase and apoptosis assay CAFs were seeded in 6-well plates (10,000 cells/well) and irradiated the following day. Seven days post-irradiation, cells were fixed with 4% formaldehyde and stained for β-galactosidase activity using the Cellular Senescence Assay Kit (Sigma Aldrich, Cat. # KA0002). Senescent cells were identified by blue staining under a light microscope. Apoptosis was assessed in parallel using the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher, Cat. # C10740) and analyzed by flow cytometry. Tumor cell treatments with CAFs and preparation of CAFs conditioned medium In some experimental settings, tumor cells were directly treated with CAF conditioned medium (CAF-CM), and in other settings tumor cells were incubated with CAFs in co-cultures for some phases of the experiment. Co-culture experiments were conducted by combining tumor cells (A549, HCC827, or donor-TC) with irradiated or non-irradiated CAFs at a 1:4 ratio. After three days, conditioned medium (CM) was collected, centrifuged, filtered (0.22 µm), and stored at -80°C. CAFs were selectively detached using enzyme-free dissociation solution, and tumor cells were analyzed for purity based on morphology and CD90 negativity by flow cytometry. The procedure resulted in purities > 98%. Proliferation of tumor cells Proliferation of tumor cells exposed to CAFs and CAF-CM was assessed using the xCELLigence Real-Time Cell Analysis system (Agilent). Tumor cells were seeded in E-plates (A549 and HCC827: 2,000 cells/well; donor-TC: 1,000 cells/well) and cultured in a 50:50 mixture of CM and fresh medium. Cell index (CI) was recorded every 30 minutes for seven days. Cell migration assay Migratory potential of tumor cells following co-cultures with irradiated and sham-irradiated CAFs was assessed by a scratch assay. Tumor cells were pre-treated with irradiated or untreated CAFs for three days and then replated in 6-well plates at 20.000, 30.000, and 30.000 cells/well for A549, HCC827, and donor-TCs, respectively, to ensure confluency 24 h after seeding. Each of the cultures were seeded with 3 technical replicates, and cells were maintained in the CM from the co-cultures. Twenty four hours post-seeding, a vertical scratch was made in each well using a 100 µL pipette-tip. Each well was photographed (100x magnification) using an Idea SPOT digital camera (Spot Imaging, Michigan, USA) immediately after the scratch-making, to designate the start of migration, and next 24 h post-scratch. Open areas from the scratch at the different time points were quantified using ImageJ Software and used to determine relative migration rates for each tumor cell type from the different CAF co-culture conditions. Clonogenic survival of tumor cells Tumor cells were plated at low densities as monocultures, and CAFs were added 24h later, in a proportion according to Table 1 . Tumor cells were plated 48 h before IR, whereas CAFs were plated 24 h before IR, to ensure that all cells were properly attached before IR exposure. Ten days post-IR, cultures were fixed (formaldehyde 4%, 10 min, 20°C) Cultures were then stained with crystal violet (Sigma-Aldrich, St Louis, MO, USA; Cat. #V5265) for 2h and washed with PBS. Colonies of tumor cells in the different cultures were counted and normalized according to the number of cells seeded in the well. Table 1 Number of cells seeded per well in 6well/plates used for the clonogenic assay. Cell type Cells/well 0 Gy Cells/well 3 Gy Cells/well 9 Gy Cells/well 12 Gy A549 500 500 1.000 1.000 CAFs 10.000 10.000 20.000 20.000 Donor TCs 400 400 2.000 4.000 CAFs 10.000 10.000 20.000 20.000 In vivo models Female C57BL/6J and BALB/cJ mice (age 6–8 weeks), were purchased from Charles River (Sulzfeld, Germany), and acclimatized in the local animal facility for a minimum of five days before experimentation. All procedures and experiments involving animals were conducted according to regulations by the Federation of European Laboratory Animal Science Association (FELASA) and approved by the National Animal Research Authority (permission ID 6373, 6942, and 7873). Maximum permitted size of tumors was 1000mm3, and animals were sacrificed if this humane endpoint was reached by cervical dislocation while being sedated. Animal sedation was achieved by short exposure to 2% isoflurane gas. The CT26 mouse colon carcinoma cells and the LL/2-Luc2 mouse luciferase-expressing Lewis lung carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM high glucose basal medium (Sigma-Aldrich, St Louis, MO, USA, Cat. # D5796) supplemented with 10% FBS, 100 U/mL penicillin/100 µg/mL streptomycin; plus blasticidin (10 ng/mL) for LL/2-Luc2 cells. All cells used for implantation were tested for pathogens by Idexx Bioanalytics (Mice Comprehensive test). Before transplantation, LL/2-Luc2 and CT26 cells were prepared in RPMI culture medium plus Matrigel (1:1, GelTrex, Thermo Fisher Scientific, Cat. #A1413202) and injected (5x10 5 cells/100 µL/mouse) subcutaneously into the right flank of animals. Tumors were measured three times per week using a digital caliper, and tumor volumes calculated by the modified ellipsoidal formula (V= ½ (Length x width 2 ). Animals were sacrificed on the day of irradiation (baseline) or 7 days post-irradiation as indicated above. Pirfenidone treatment Immediately after tumor cell inoculation, animals received intraperitoneal injections of pirfenidone (Sigma-Aldrich, Cat# P2116) 200 mg/kg in PBS, 100 µL intraperitoneally) daily for 2 weeks (as indicated in scheme in Fig. 5 A). In vivo RT treatment Tumor-bearing mice received tumor-specific radiation exposure by the image-guided irradiator X-RAD SmART system (Precision X-Ray irradiation, Madison, USA), as described previously [ 18 ]. Radiation treatments were performed when tumors reached 5–6 mm in diameter (~ 100 mm3, approx. 8–10 days cell post-implantation). Structural imaging measurements (CT), as well as digital caliper measurements, were used for monitoring tumor growth. A single dose of 12 Gy was delivered to tumors using two opposing photon beams (maximum energy 225 kV, 13 mA), at dose-rate of 3 Gy/min. The treatment-planning system SmART-plan (version 1.3.9 Precision X-ray, North Branford, CT) was used for dose calculations of the plan with selected beam-angles and to deliver the treatment. Before delivering radiation to tumors, animals were sedated by continuous isoflurane gas anesthesia via induction in an anesthesia chamber (0.5 L/min oxygen, 4.0% isoflurane). During the imaging/radiation procedure, the animals were maintained sedated by continuous isoflurane anesthesia gas via a nose cone (0.4 L/min oxygen, 2% isoflurane). Flow cytometry CAF-specific markers were analyzed by flow cytometry on BD FACSAria III using the FlowJo software, Ver.7.2.4 (Tree Star, Ashland, OR, USA). Briefly, tumors were excised and minced with scalpels in 2.5 mL PBS, and the resulting cells and fragments digested in Accutase® solution (Sigma-Aldrich, Cat. #A6964) for 1 h at 37°C on a platform rocker. Digested tumors were further dissociated by mechanical disruption and sieving through 70-µm cell strainers (BD Biosciences, Bedford, MA, USA). Single-cell suspensions were labeled with panels of specific antibodies(Miltenyi Biotec, Table S2 ). CAF-specific markers consisted of FAP-1 and α-SMA. Isotype controls consisted of REA control and IgG2a (Cat. no. 130-113-450 and 130-104-612, respectively). Data were obtained by flow cytometry using the following gating strategy: cells gated according to their scatter properties (FSC-A vs SSC-A), doublets exclusion (FSC-A vs FSC-H), and analyzed by the percentage of viable cells expressing FAP-1 and α-SMA, and also by median fluorescence intensity (Median FI). Immunohistochemistry For immunohistochemistry evaluations, tumors were fixed in paraformaldehyde (4% in PBS) immediately after resection, incubated for at least 24h before embedding in paraffin blocks. Tissue sections (4µm thickness) were made and sections deparaffinized and rehydrated before staining. The Ventana Discovery-Ultra Research instrument (Roche 05987750001) was used for automated preparation and immunohistochemical staining of tumor tissue samples. Anti-mouse α-SMA antibody (D4K9N, cell signaling) was manually applied at a dilution of 1:100 followed by incubation at 36°C for 30 min. The antibody was validated for IHC-P (formalin-fixed and paraffin-embedded tissue) by the supplier. Stained slides were digitalized by an Olympus VS120 scanner. Quantification of positive areas/cells per square mm2 of intact tumor tissue was determined electronically using the QuPath Software for Bioimage Analysis (version v.0.5.1, tool: positive cell detection). Clinical cohort Seventy-six patients who underwent adjuvant radiotherapy after curative intent surgery were identified from a larger retrospective cohort of lung-tumor specimens from NSCLC patients collected at the University Hospital of North-Norway and Nordland central hospital between 1990–2010. The cohort is previously described by Hald et al. and approved by The Regional Committee for Medical and Health Research Ethics (REK-Nord Project-ID: 2016/2307/REK-Nord) [ 19 ]. In brief, the cohort comprises a complete set of clinicopathological variables and tissue-microarrays (TMAs) with two to four replicate cores for each patient. The overall cohort was previously evaluated by us for the prognostic impact of several CAF markers including FAP1, αSMA, PDGFRα and PDGFRβ [ 20 , 21 ] Statistical analysis All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Comparison of data between experimental groups was analyzed using the Brown-Forsythe and Welch ANOVA test, and significance values were adjusted by Dunnett’s T3 correction for multiple comparisons. Outcomes of Western blot experiments were analyzed using the 2way ANOVA test, and significance values were adjusted by Dunnett correction for multiple comparisons. Results were presented in graphs, where each donor was plotted as an individual dot in the dataset. Survival data for the clinical cohort was visualized using the Kaplan-Meier method and assessed with the log-rank test. The chosen endpoint was DSS defined as the time from surgical resection to lung cancer specific death. For all analyses, lLevel of significance was set at p < 0.05. Results Radiation effects on CAF viability and phenotype Initial experiments were performed to study the direct effects of radiation exposure on the viability and phenotype of primary NSCLC human CAFs in culture. Analyses performed 5–6 days post-IR exposure show that CAFs are surviving all the given radiation regimens. However, radiation-induced cellular damage was able to trigger cell senescence in a dose-dependent manner (Fig. 1 A). Of note, only marginal levels of apoptosis were detected in CAFs exposed to either 1x18Gy or 3x6Gy (Fig. 1 B). Additionally, expressions of classical CAF activation markers was measured and compared between experimental groups. Results show minor (non-significant) variations in the expression of CAFs markers including FAP-1, FSP-1, α-SMA, PDGFRα and podoplanin on irradiated CAFs (Fig. 1 C-D). Effects of irradiated CAFs on lung cancer cells pro-tumorigenic functions The capacity of CAFs to confer tumor cell radioresistance was investigated in clonogenic assays performed with A549 lung adenocarcinoma and an in-house generated patient-derived tumor cell line (Donor-TC) in the presence or absence of CAFs. Outcomes from clonogenic assays show no differences between experimental groups, indicating that CAFs are not interfering with tumor cell radioresistance (Fig. 2 A-B). In separate experiments, we investigated potential effects of CAFs on tumor cell proliferative properties. Tumors cells and CAFs (irradiated or untreated) were co-cultured for three days followed by measurements of tumor cell proliferation during 7 days in the presence of CAF-CM. CAFs were eliminated from co-cultures by selective detachment as described in the method section. Results show no variations in the proliferation rates in any of the tested lung tumor cell lines (Fig. 2 C). In similar type of experiments, we analyzed tumor cells migration rates following exposure of cancer cells to irradiated and control CAFs in co-cultures in wound healing assays. Once again, migration rates of tumor cells seemed unaffected by CAFs, regardless of whether they were irradiated or not (Fig. 2 D). All experiments presented in Fig. 2 were reproduced three times (3X) with human CAFs from three randomly selected donors. Radiation reduces CAF-mediated induction of EMT on lung cancer cells Epithelial-mesenchymal transition (EMT) is associated with tumor cell aggressiveness and metastatic potential. To explore potential effects of irradiated CAFs on EMT processes, tumor cells were incubated in co-cultures with irradiated or control CAFs, followed by tumor cell isolation and quantification of EMT-related markers in whole cell protein lysates. The epithelial marker E-cadherin was abundantly expressed in untreated A549 and HCC827 cells and to a lesser extent in the in-house made lung tumor cell line (donor-TC) (Fig. 3 A-B). On the contrary, the mesenchymal marker vimentin was non-detectable in untreated A549 and HCC827 cells but detectable in the donor-derived tumor cells, indicating that tumor cells from this patient display an EMT-like phenotype at baseline. Upon incubation with irradiated and control CAFs, A549 cells lost E-cadherin expression (Fig. 3 C, top-left panel). This phenomenon was not observed in HCC827 cells or donor TCs (Fig. 3 C-top middle and right panels). Importantly, incubation of HCC827 and donor TCs with untreated CAFs was able to induce vimentin expression, however, this effect was partially abolished when the incubation was done with irradiated CAFs (Fig. 3 C-D). Vimentin induction by CAFs was not observed on A549 cells in any condition (Fig. 3 C-D). Radiotherapy-induced changes in intratumoral CAF levels in preclinical tumor models The effects of radiotherapy on intratumoral CAF levels were studied in three different subcutaneous tumor models. The experimental strategy included the LLC lung adenocarcinoma model, the CT26 colon carcinoma model and the 4662PDA pancreatic adenocarcinoma model. Tumors were irradiated with a single dose of 12Gy using a high-precision image-guided irradiator. Tumor growth curves showed prominent responses to radiation from the LLC and CT26 models and a bit more modest response to radiation from the 4662 pancreatic model (Fig. 4 A). One week after radiation exposure, tumors were harvested and the content of CAFs was determined by both flow cytometry and in situ immunohistochemistry. Results from flow cytometry analyses showed no significant changes in numbers of CAFs in any of the tumor models used, determined as percentage of α-SMA + cells from the total amount of living cells (Fig. 4 B). On the other hand, outcomes from IHC determinations on fixed tissues revealed no significant changes of α-SMA + cells in the LLC and CT26 models whereas a significant reduction of aSMA+ CAFs was observed in the 4662 pancreatic model, (Fig. 4 C). In the latter approach, necrotic areas and peripheral tumor regions were excluded from analyses. Enhanced radioresistance in pirfenidone-treated animals Pirfenidone is an anti-fibrotic medication primarily administered to treat idiopathic pulmonary fibrosis [ 22 ]. Emerging evidence suggests that pirfenidone has promising anti-cancer effects through targeting and reprogramming CAFs [ 23 ]. In this study, we investigate the influence of CAFs on tumor responses to radiotherapy (RT) by pre-treating animals with pirfenidone. More specifically, we assess whether modulating CAF activity through pirfenidone administration could enhance the therapeutic efficacy of RT. Histological analyses of tumors revealed that the LLC and CT26 tumor models are associated with poorly developed stroma (Fig. 4 B). For this reason, the more stromatic 4662PDA pancreatic cancer model was chosen to run experiments with pirfenidone. Tisue analyses demonstrated higher baseline levels of α-SMA+ cells and intratumoral collagen deposition than in LLC and CT26 tumors (Fig. 4 B and 5 B). Pirfenidone treatment did not show any effect on tumor growth kinetics (Fig. 5 C-D). Moreover, pirfenidone treatment did not have observable effects on the levels of aSMA+ CAFs, however, it provoked a significant reduction of intratumoral collagen deposition (Fig. 5 B). Upon radiation, tumors in pirfenidone-naive animals showed good response to 1x12Gy treatment, however, tumor growth response to RT was partially abrogated in pirfenidone-treated animals, with tumor growth rates similar to non-irradiated tumors (Fig. 5 D-E) and survival rates significantly decreased when compared to the pirfenidone-naïve RT-treated group (Fig. 5 F). Baseline expression of CAF markers does not predict radiotherapy responses in clinical settings The prognostic impact of the CAF markers FAP-1, α-SMA, PDGFRα and PDGFRβ was studied in a cohort of tumor specimens from NSCLC patients who received radiotherapy in an adjuvant setting (n = 76). The data are summarized in table S3 and Fig. 6 . As shown in Fig. 6 A-B, neither FAP-1 nor α-SMA were correlated with disease-specific survival. However, a non-significant trend to adverse outcome for patients with high expression of PDGFRα or PDGFRβ was observed (Fig. 6 C-D). Due to a limited number of patients in the cohort, analyses within histological subtypes (adenocarcinoma and squamous cell carcinoma) were not feasible. Discussion Besides its tumor cell killing potential, radiation is able to induce substantial changes in the tumor microenvironment (TME), including effects on tumor compartments such as the vasculature, the stroma as well as immunological components. Accordingly, RT-mediated reprogramming of the TME is considered to play a major role in therapy outcomes. The impact of RT on CAFs varies among studies, and the potential mechanisms behind the influence of CAFs on tumor radioresistance remain controversial [ 24 , 25 ]. In this work, we have aimed to explore the impact of radiation on CAF-mediated tumorigenic functions and the potential role that CAFs may play in RT outcomes. Jointly considered, results indicate that upon irradiation, CAFs may lose some of their protumorigenic properties and that therapeutic strategies for reprogramming CAFs may indeed have detrimental effects in the context of radiotherapy. As shown by us and others previously, radiation given in single-high or medium-high fractionated regimens is able to induce cell senescence in primary cultures of CAFs prepared from NSCLC tumors [ 17 ]. Senescent cells are characterized by possessing an anti-fibrotic and pro-inflammatory phenotype [ 26 ]. In the context of cancer, numerous reports have demonstrated pro-tumorigenic properties of senescent cells via the release of a myriad of bioactive factors collectively termed the senescent-associated secretory phenotype (SASP) [ 27 ]. However, a deeper look at the role of senescent cells in cancer progression unveils a more balanced scenario in which both pro-tumorigenic and anti-tumorigenic functions may take place in a context-dependent manner [ 28 ]. In the context of radiotherapy, some authors have achieved tumor radiosensitization after elimination of senescent fibroblasts [ 29 , 30 ]. On the contrary, other reports have shown “normalization” of tumor stromal fibroblasts exposed to radiation with consequent loss of tumor-promoting effects [ 15 , 16 ]. In this study, we demonstrate that radiation-induced senescent CAFs maintain the expression of CAF activation markers such as α-SMA, FAP-1, PDGFRα, or podoplanin. We also observe that CAFs, whether irradiated or not, do not affect the proliferative or migratory properties of lung tumor cells. These results align well to outcomes we previously observed using irradiated CAF-CM on different lung cancer cell lines [ 31 ]. On the other hand, in co-culture settings, radiation seems to counteract CAF-mediated EMT effects on tumor cells. This is in opposition to observations from other groups reporting EMT induction from irradiated CAFs via expression of IL-6 in a model of esophageal cancer [ 32 ], via secretion of HGF in a breast cancer model [ 33 ] or via secretion of SDF-1 in a pancreatic cancer model [ 14 ]. Disparities in outcomes may come from the use of different cancer models, different cell lines, or different experimental settings. In the above-mentioned studies, tumor cells have been exposed to CAF-CM from one or two CAF donors. In our study, we perform pre-conditioning of tumor cells by incubating them with CAFs in direct cell contact co-cultures, in addition to the incubation of cells with CAF-CM during the assay. Additionally, we have run experiments in parallel with multiple CAF donors. The effects of local radiation on the dynamics of intratumoral CAFs in in vivo preclinical models have been scarcely investigated [ 24 ]. The majority of published studies exploring the role of CAFs (or irradiated CAFs) on RT responses have used co-injections of admixed CAFs and tumor cells pre-irradiated or pre-conditioned during in vitro cultures [ 15 , 29 , 34 , 35 ]. In this study, we explore the effects of external beam radiotherapy on intratumoral CAF dynamics using different subcutaneous cancer models. Quantitative analyses from flow cytometry and immunohistochemistry of α-SMA + fibroblasts in tumors reveal that there are no significant changes in the abundance of CAFs after 1x12Gy tumor irradiation, and the same outcomes were observed in the three tumor models. A limitation in our approach is that only one radiation regimen and only one-time point was studied. It remains to be investigated if things turn out differently when using different radiation regimens or if measured at different time points. For comparisons with human tumors in the clinics, Verset L et al. reported increased αSMA/epithelial ratios after neoadjuvant (chemo)radiotherapy in a colorectal cancer cohort [ 36 ]. In an ongoing study by us, performed on a cervix cancer cohort, we observed enhanced levels of fibroblast gene signatures after 5x2Gy radiotherapy cycles compared to baseline, however, the levels of α-SMA-positive cells in tissue remained unchanged in the majority of RT-treated patients (unpublished results). Given the overwhelming amount of scientific studies demonstrating the multifaceted pro-tumorigenic and therapy resistance effects of CAFs, large amounts of endeavors are now put into testing alternative therapies to target CAFs or CAF-mediated tumor supportive pathways [ 37 , 38 ]. Among the different strategies for targeting CAFs, one appealing and frequently pursued alternative is the use of drugs that promote CAF normalization or reprogramming [ 39 ]. Pirfenidone is an oral antifibrotic medication primarily administered for the treatment of idiopathic pulmonary fibrosis, with demonstrated anti-cancer effects manifested through CAFs deactivation [ 40 ]. Among other effects, pirfenidone has been shown to disrupt tumor-stroma interactions via deregulation of CAF-driven pro-tumorigenic signaling including α-SMA, collagen I, PDGFRβ, TGF-β, PD-L1 or CCL17 [ 41 – 44 ]. In our study, we have tested the effects of different anti-fibrotic and/or CAF-reprogramming agents including tranilast, calcipotriol (Vitamin D), and pirfenidone. Observable reduction in intratumoral collagen deposition was achieved only with pirfenidone (unshown results). Unexpectedly, animals in the pirfenidone-treated group showed enhanced radioresistance compared to the untreated group. Such outcomes indicate that the presence of “intact” CAFs, at least in this particular tumor model, is associated with better tumor responses to RT. Intriguingly, in a recently published study, authors observe enhanced FAP + stromal cells after RT in both clinical and preclinical settings, and better responses to (chemo)radiotherapy are observed in patients with therapy-induced FAP + CAFs [ 45 ]. Experimental approaches that specifically target CAFs such as FAP-targeting immunotherapeutics, or transgenic animal models in which CAFs can be selectively depleted may represent alternative experimental approaches to investigate the role of CAFs in radiotherapy. Conclusion Data generated in this study contribute significantly to the understanding of the effects of RT on CAFs and the potential role that CAFs may play in RT outcomes. Collectively, our results indicate that radiation exposure to CAFs triggers cellular senescence, however many of their phenotypic and functional traits are still preserved after irradiation. Importantly, some of the CAFs pro-tumorigenic properties such as EMT promotion become partially reduced after irradiation. In in vivo models, intratumoral CAFs levels remain nearly unchanged after a single high radiation dose and CAFs treatment by pirfenidone turns tumors more radioresistant. Observations made in our preclinical models need to be confirmed in clinical settings, where CAF dynamics (including numeral and phenotypical changes) are compared between baseline and post-treatment tissue samples. In light of our observations, therapeutic strategies involving CAF reprogramming in the context of radiotherapy should be carefully considered as they may negatively influence treatment outcomes. Declarations Ethics approval and consent to participate. All animal experiments were approved by the Norwegian Food Safety Authority (FOTS ID 18956 and 27939). Experiments involving human biological products were approved by Regional Ethical Committee of Northern Norway (REK Nord 2016/2307). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Consent for publication Not applicable Data availability declaration The original contributions presented in the study are included in the article or supplementary material. Further inquiries can be directed to the corresponding author. Competing interest Authors declare no competing interests. The FAPI-74-NOTA precursor was provided free of charge by SOFIE Biosciences without further influence on study design and data analysis. Acknowledgment The authors acknowledge the Cancer Biobank at UNN for providing human lung cancer samples. Publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway. Funding Statement The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Norwegian Regional Health Authorities under Grant (HNF1373-17 to RB and HNF1423-18 to TH); The Norwegian Cancer Society, the Aakre Foundation at UiT, and the Tromsø forskningsstiftelse (TFS) grant 180ºN/Norwegian-Nuclear-Medicine-consortium. Publication charges for this article have been funded by a grant from the publication fund at UiT, The Arctic University of Norway. Author contributions RB : Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. TH : Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing – review & editing. KL, VM and IR : Data curation, Formal analysis, Investigation, Methodology. LR : Data curation & Formal analysis. TK : Data curation & Formal analysis. IM-Z : Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest References Sharma RA, et al. Clinical development of new drug-radiotherapy combinations. Nat Rev Clin Oncol. 2016;13(10):627–42. Koka K, et al. Technological Advancements in External Beam Radiation Therapy (EBRT): An Indispensable Tool for Cancer Treatment. Cancer Manag Res. 2022;14:1421–9. Alsaihaty Z, et al. Hybrid Treatment Planning for Chest Wall Irradiation Utilizing Three-Dimensional Conformal Radiotherapy (3DCRT), Intensity-Modulated Radiation Therapy (IMRT), and Volumetric Modulated Arc Therapy (VMAT): A Systematic Review. Cureus. 2024;16(5):e59583. Hellevik T, Martinez-Zubiaurre I. Radiotherapy and the Tumor Stroma: The Importance of Dose and Fractionation. Front Oncol. 2014;4:1. Barker HE, et al. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat Rev Cancer. 2015;15(7):409–25. Sahai E, et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer. 2020;20(3):174–86. Guo T, Xu J. Cancer-associated fibroblasts: a versatile mediator in tumor progression, metastasis, and targeted therapy. Cancer Metastasis Rev. 2024;43(3):1095–116. Strell C, et al. Prognostic and predictive impact of stroma cells defined by PDGFRb expression in early breast cancer: results from the randomized SweBCG91RT trial. Breast Cancer Res Treat. 2021;187(1):45–55. Zhang R, Liu F. Cancer-associated fibroblast-derived gene signatures predict radiotherapeutic survival in prostate cancer patients. J Transl Med. 2022;20(1):453. Chu TY, et al. Crosstalk with cancer-associated fibroblasts increases the growth and radiation survival of cervical cancer cells. Radiat Res. 2014;181(5):540–7. Hwang RF, et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res. 2008;68(3):918–26. Ji X, et al. Cancer-associated fibroblasts from NSCLC promote the radioresistance in lung cancer cell lines. Int J Clin Exp Med. 2015;8(5):7002–8. Tommelein J, et al. Radiotherapy-Activated Cancer-Associated Fibroblasts Promote Tumor Progression through Paracrine IGF1R Activation. Cancer Res. 2018;78(3):659–70. Li D, et al. Radiation promotes epithelial-to-mesenchymal transition and invasion of pancreatic cancer cell by activating carcinoma-associated fibroblasts. Am J Cancer Res. 2016;6(10):2192–206. Grinde MT, et al. Ionizing radiation abrogates the pro-tumorigenic capacity of cancer-associated fibroblasts co-implanted in xenografts. Sci Rep. 2017;7:46714. Aboussekhra A, et al. Ionizing radiation normalizes the features of active breast cancer stromal fibroblasts and suppresses their paracrine pro-carcinogenic effects. Transl Oncol. 2023;37:101780. Hellevik T, et al. Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced. Radiat Oncol. 2012;7:59. Berzaghi R, Gundersen K, Pedersen BD, Utne R, Yang N, Hellevik T, Martinez I. Immunological signatures from irradiated cancer-associated fibroblasts. Frontiers in Immunology; 2024. Hald SM, et al. LAG-3 in Non-Small-cell Lung Cancer: Expression in Primary Tumors and Metastatic Lymph Nodes Is Associated With Improved Survival. Clin Lung Cancer. 2018;19(3):249–59. e2. Kilvaer TK, et al. Cancer Associated Fibroblasts in Stage I-IIIA NSCLC: Prognostic Impact and Their Correlations with Tumor Molecular Markers. PLoS ONE. 2015;10(8):e0134965. Kilvaer TK, et al. Differential prognostic impact of platelet-derived growth factor receptor expression in NSCLC. Sci Rep. 2019;9(1):10163. Ruwanpura SM, Thomas BJ, Bardin PG. Pirfenidone: Molecular Mechanisms and Potential Clinical Applications in Lung Disease. Am J Respir Cell Mol Biol. 2020;62(4):413–22. Rastegar-Pouyani N, et al. Targeting cancer-associated fibroblasts with pirfenidone: A novel approach for cancer therapy. Tissue Cell. 2024;91:102624. Martinez-Zubiaurre I, Hellevik T. Cancer-associated fibroblasts in radiotherapy: Bystanders or protagonists? Cell Commun Signal. 2023;21(1):108. Raaijmakers K, et al. Cancer-associated fibroblasts, tumor and radiotherapy: interactions in the tumor micro-environment. J Exp Clin Cancer Res. 2024;43(1):323. Burton DG, Krizhanovsky V. Physiological and pathological consequences of cellular senescence. Cell Mol Life Sci. 2014;71(22):4373–86. Davalos AR, et al. Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev. 2010;29(2):273–83. Lecot P, et al. Context-dependent effects of cellular senescence in cancer development. Br J Cancer. 2016;114(11):1180–4. Meng J et al. Targeting senescence-like fibroblasts radiosensitizes non-small cell lung cancer and reduces radiation-induced pulmonary fibrosis. JCI Insight, 2021. 6(23). Jian C, et al. Biomimetic Nanoplatform for Dual-Targeted Clearance of Activated and Senescent Cancer-Associated Fibroblasts to Improve Radiation Resistance in Breast Cancer. Small. 2024;20(25):e2309279. Hellevik T, et al. Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth. Transl Oncol. 2013;6(1):66–74. Ebbing EA, et al. Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma. Proc Natl Acad Sci U S A. 2019;116(6):2237–42. Wang B, et al. Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway. Int J Radiat Oncol Biol Phys. 2023;116(3):640–54. Pereira PMR, et al. iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy. Cancer Res. 2020;80(8):1681–92. Wang Y, et al. Cancer-associated Fibroblasts Promote Irradiat Cancer Cell Recovery Through Autophagy EBioMedicine. 2017;17:45–56. Verset L, et al. Impact of neoadjuvant therapy on cancer-associated fibroblasts in rectal cancer. Radiother Oncol. 2015;116(3):449–54. Valkenburg KC, de Groot AE, Pienta KJ. Targeting the tumour stroma to improve cancer therapy. Nat Rev Clin Oncol. 2018;15(6):366–81. Ganguly D et al. Cancer-Associated Fibroblasts: Versatile Players in the Tumor Microenvironment. Cancers (Basel), 2020. 12(9). Chen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov. 2019;18(2):99–115. Paliogiannis P, et al. Repurposing Anticancer Drugs for the Treatment of Idiopathic Pulmonary Fibrosis and Antifibrotic Drugs for the Treatment of Cancer: State of the Art. Curr Med Chem. 2021;28(11):2234–47. Fujiwara A, et al. Effects of pirfenidone targeting the tumor microenvironment and tumor-stroma interaction as a novel treatment for non-small cell lung cancer. Sci Rep. 2020;10(1):10900. Aoto K, Ito K, Aoki S. Complex formation between platelet-derived growth factor receptor beta and transforming growth factor beta receptor regulates the differentiation of mesenchymal stem cells into cancer-associated fibroblasts. Oncotarget. 2018;9(75):34090–102. Aboulkheyr Es H, et al. Pirfenidone reduces immune-suppressive capacity of cancer-associated fibroblasts through targeting CCL17 and TNF-beta. Integr Biol (Camb). 2020;12(7):188–97. Es HA et al. Pirfenidone Reduces Epithelial-Mesenchymal Transition and Spheroid Formation in Breast Carcinoma through Targeting Cancer-Associated Fibroblasts (CAFs). Cancers (Basel), 2021. 13(20). Garate-Soraluze E et al. 4-1BB agonist targeted to fibroblast activation protein alpha synergizes with radiotherapy to treat murine breast tumor models. J Immunother Cancer, 2025. 13(2). Additional Declarations No competing interests reported. Supplementary Files supplementaryF1uncroppedWB1.pptx SupplementaryF2uncroppedWB2.pptx SupplementaryF3Collagen.pptx SupplementaryTable1.docx SupplementaryTable2antibodies.docx Supplementarytable3clinicaldataTMA.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8680198","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590899944,"identity":"84633ad7-bab0-40be-ae49-e3cee6199c5a","order_by":0,"name":"Rodrigo Berzaghi","email":"","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Berzaghi","suffix":""},{"id":590899945,"identity":"11aa419b-a3c3-4f85-822a-be308385c31b","order_by":1,"name":"Kristin Lode","email":"","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Kristin","middleName":"","lastName":"Lode","suffix":""},{"id":590899946,"identity":"5dad4f41-ac53-4bcb-a9f4-4826ed1bfd1d","order_by":2,"name":"Vera Maia","email":"","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Vera","middleName":"","lastName":"Maia","suffix":""},{"id":590899947,"identity":"c10fa180-257a-483b-a8bb-043392e33515","order_by":3,"name":"Indusmita Routray","email":"","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Indusmita","middleName":"","lastName":"Routray","suffix":""},{"id":590899948,"identity":"7f270148-f104-4c77-a88f-8719a3513aec","order_by":4,"name":"Lorenzo Ragazzi","email":"","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Ragazzi","suffix":""},{"id":590899949,"identity":"a5a245c7-f4d5-4cc3-93f3-ea062467864b","order_by":5,"name":"Thomas Kilvaer","email":"","orcid":"","institution":"University Hospital of North Norway","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Kilvaer","suffix":""},{"id":590899950,"identity":"991ffd4e-d850-43dd-9fc7-a6c38adc809b","order_by":6,"name":"Turid Hellevik","email":"","orcid":"","institution":"University Hospital of North Norway","correspondingAuthor":false,"prefix":"","firstName":"Turid","middleName":"","lastName":"Hellevik","suffix":""},{"id":590899951,"identity":"c636b958-2615-4cce-b46b-efb7ed76750d","order_by":7,"name":"Inigo Martinez-Zubiaurre","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACNgbGBigTzLABihCthQ3MSGNgI6gHSTMIHGYgaA2fdHPziw8M2+TN5ze3Pfjw53xin3wD24MP+MyWOdhmOYPhtuGcY4zthjN4bie2sTGwG87Ap0Uisc2Yh+E24ww2xjZpHgmwFjZpHiK02IO1/DE4R5SW5sdALYlgLQwJB4izhXGGwe3kGWyJbZI9B5KN20AMfH6Rn5H++MOHitu2M5iPP5P48cdOdn7z4WMS+EIMbBGDAYoAPD3gBMwEjBwFo2AUjIIRDwDfoEQIR8x8BgAAAABJRU5ErkJggg==","orcid":"","institution":"UiT The Arctic University of Norway","correspondingAuthor":true,"prefix":"","firstName":"Inigo","middleName":"","lastName":"Martinez-Zubiaurre","suffix":""}],"badges":[],"createdAt":"2026-01-23 14:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8680198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8680198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102940409,"identity":"24b26014-5cb0-4c76-9435-010d0441dcec","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":291944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiation-induced phenotypic changes in CAFs.\u003c/strong\u003e A) Induction of premature cellular senescence in CAFs exposed to ionizing radiation. B) CAF-resistance to radiation-induced apoptosis. C) Western blot analysis, using antibodies against FAP-1, FSP1, α-SMA, PDGFR-α and Podoplanin on whole cell lysates from irradiated and non-irradiated CAFs. Results were normalized against β-actin expression. In D), relative intensity of the bands corresponding to panels in C, determined by densitometry. Data represents mean (± SD) values from three different CAF donors. Two-way ANOVA test and \u003cem\u003ep-\u003c/em\u003evalueswere determined between non-irradiated CAFs and the two irradiated-CAF groups individually.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/32ec9bea01726f2c60a0f2ef.png"},{"id":102940411,"identity":"e3710f8f-acd5-4a74-bfa4-4410dc1eed0c","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAF effects on tumor cell proliferation, migration and radioresistance\u003c/strong\u003e. A \u0026amp; B) Clonogenic survival of tumor cells A549 and donor-derived tumor cells (Donor-TCs) exposed to ionizing radiation as monocultures or in co-cultures with CAFs. C) Proliferation rates of tumor cells A549, HCC829, and donor-TCs \u0026nbsp;after co-culturing with irradiated or sham-irradiated CAFs. D) Migration rates of tumor cells after co-culturing with CAFs compared to rates of monocultures. Migration rates were measured 24 h after generation of a scratch. Two-way ANOVA test and \u003cem\u003ep-\u003c/em\u003evalues were determined between non-irradiated CAFs and the two irradiated CAF groups individually.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/6bff5f16158a74b0020f9734.png"},{"id":102940416,"identity":"24c147ca-20f9-40bb-9d78-95bd9be49cd6","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":220837,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAF-mediated regulation of EMT phenotype in tumor cells\u003c/strong\u003e. A \u0026amp; B) Western blot analysis showing baseline expression of E-cadherin, β-catenin and Vimentin on tumor cell lysates (A549, HCC827, and Donor-TC) and fibroblast cell lysates (normal skin fibroblasts and CAFs). D \u0026amp; D) Expression of EMT markers measured on tumor cells lysates after co-culturing with irradiated or non-irradiated CAFs from three unrelated donors. In (B) and (D), average values of relative intensity of the bands corresponding to panels A and C, respectively, is shown as a bar graph. Results were normalized against β-actin expression. Data represents mean (± SD) values from 3 different CAF donors. Two-way ANOVA test and \u003cem\u003ep-\u003c/em\u003evalueswere determined between tumor cells co-cultured with non-irradiated CAFs or with one of the two irradiated CAF groups, individually.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/c7e61962393a6e1ecbf759a3.png"},{"id":102940427,"identity":"2083a6d3-ff53-424d-b549-225c391090c3","added_by":"auto","created_at":"2026-02-18 17:06:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadiation-induced changes on intratumoral CAFs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e tumor models.\u003c/strong\u003e (A) Growth curves of LL/2-Luc2, CT26 and 4662PDA tumors corresponding to non-treated group (squares, n = 8) and irradiated (1x12Gy) group (circles, n = 8). X-ray tumor irradiation (arrows) was performed when tumors reached approx. 5-6 mm in diameter (Day 9 for LL/2-Luc2 and CT26 tumors and Day14 for 4662 tumors), (B) Flow cytometry analysis of tumor-associated α-SMA+ cells presented as % of total viable cells. Bar graphs represent mean (±SD) values from flow cytometry analysis of 8 tumors for each experimental group, measured independently. Welch ANOVA test and \u003cem\u003ep-\u003c/em\u003evalues were determined between control and irradiated tumors individually. (C) Comparative expression of αSMA in untreated and (1x12Gy) irradiated tumors in three different tumor models.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/c46507d20a2018abd9e7ccc7.png"},{"id":102964451,"identity":"aa075984-d54a-4bcd-9d82-193bb459d431","added_by":"auto","created_at":"2026-02-19 04:22:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":198643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of pirfenidone treatment on tumor responses to radiotherapy\u003c/strong\u003e. A) Chronological view of animal treatments, tumor collection and tissue analyses. B) Effects of pirfenidone treatment on α-SMA+CAFs expression and collagen deposition in non-irradiated and irradiated tumors retrieved at day 21 post-cell implantation. C) Average values corresponding to percentage of aSMA positive areas in tumors from the different experimental groups. D) Individual tumor-volumes in untreated (±RT) and pirfenidone (±RT) treated animals. E) Tumor volumes over time (mm\u003csup\u003e3\u003c/sup\u003e) for the four experimental groups (n=6) are shown as means ± SD and statistical comparisons (Two-way ANOVA, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). F) Percentage of survival over time, in which individual experimental groups are compared (n=6) (statistical significance calculated by Mantel-Cox test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/32e9b7cddfa4e1b583d7c55d.png"},{"id":102964489,"identity":"dcfd3fce-0afa-46db-873b-e3228dda01a7","added_by":"auto","created_at":"2026-02-19 04:22:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":173936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival curves for CAF markers expression in NSCLC patients\u003c/strong\u003e. Associations of high vs low FAP-1 (A), high vs low α-SMA (B), high vs low PDGFRα (C) and high vs low PDGFRβ (D) expression in primary NSCLC tumor tissue as prognostic markers in patients that received adjuvant radiotherapy after primary resection for non-small cell lung cancer (n = 76, log-rank tests\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/f5cea9fcd09cfee388146d24.png"},{"id":105106526,"identity":"8b32460e-b3e3-4674-942f-b7241dec78dc","added_by":"auto","created_at":"2026-03-21 09:25:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2039609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/ed911be2-67cc-4cd2-bff1-83b2e7d94043.pdf"},{"id":102964302,"identity":"62c2bbef-9440-4e70-a348-d4d43319c856","added_by":"auto","created_at":"2026-02-19 04:22:02","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":891953,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryF1uncroppedWB1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/10a0914c92f4ef747a64512b.pptx"},{"id":102940418,"identity":"f2fdda64-7146-4448-b0b6-8086639e7a9b","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":717415,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryF2uncroppedWB2.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/788811860af2aea70104fd97.pptx"},{"id":102940412,"identity":"3f9e0cd9-e365-4952-86bc-bd21a8aacead","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1935404,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryF3Collagen.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/aa0f8eb574721717522cf521.pptx"},{"id":102940419,"identity":"d0a8b30f-3c21-4aaf-8514-08bbf9f36204","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16328,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/c02bf76d8dbcd3ababf28bed.docx"},{"id":102940417,"identity":"635c5377-1261-4956-b26f-a7ee185a7e19","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15481,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2antibodies.docx","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/cc4c2833c839198a2846fcf3.docx"},{"id":102940415,"identity":"98d01ef9-22f4-45b6-841c-8d1a19aec083","added_by":"auto","created_at":"2026-02-18 17:06:05","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":24085,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable3clinicaldataTMA.docx","url":"https://assets-eu.researchsquare.com/files/rs-8680198/v1/36c8de64c1167910dcc34cf1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eRadiation treatment curtails pro-tumorigenic functions from cancer-associated fibroblasts in preclinical models\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRadiotherapy (RT) is a long-established and widely applied treatment modality in oncology, used in more than half of all cancer patients and responsible for roughly 40% of curative outcomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. External beam radiotherapy (EBRT), the most common form of RT, has benefited from major technological advances in recent years, resulting in improved tumor control and enhanced patient quality of life [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Contemporary EBRT techniques\u0026mdash;such as three-dimensional conformal radiotherapy (3DCRT), intensity-modulated radiotherapy (IMRT), stereotactic body radiotherapy (SBRT), image-guided radiotherapy (IGRT), and particle therapy\u0026mdash;rely on sophisticated imaging modalities (CT, MRI, PET) to accurately define tumor volumes and optimize dose delivery while minimizing exposure to surrounding healthy tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although the primary objective of RT is to induce lethal damage in malignant cells and inhibit their proliferation, it also triggers complex biological responses in the non-malignant components of the tumor microenvironment (TME). Increasing evidence indicates that RT-induced modifications within the TME and the resulting cellular interactions are key determinants of therapeutic success [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCancer-associated fibroblasts (CAFs) represent a major stromal population within the TME and are known to influence tumor progression and resistance to therapy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Beyond establishing a supportive niche for cancer cell growth, CAFs contribute to immunosuppression and can directly promote angiogenesis and metastasis through the secretion of cytokines, growth factors, extracellular matrix (ECM) components, and matrix-modifying enzymes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In clinical settings, elevated expression of CAF-specific markers or CAF-related gene signatures has been associated with responses to (chemo)radiotherapy and is frequently linked to poor prognosis across multiple cancer types [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite these correlations, the mechanisms underlying CAF-mediated radio-resistance remain insufficiently understood.\u003c/p\u003e \u003cp\u003eCompared with other TME constituents, the effects of radiotherapy on CAFs have been less extensively investigated. Existing studies suggest that radiation can induce phenotypic and functional changes in CAFs that may enhance tumor radio-resistance. Irradiated CAFs have been shown to release factors that support cancer cell survival and proliferation [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, radiation-induced senescence in CAFs can lead to the secretion of pro-inflammatory cytokines and growth factors, further promoting tumor growth and potentially contributing to treatment resistance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Conversely, some preclinical animal studies report that irradiation may diminish the tumor-promoting properties of CAFs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we explore how irradiation influences the tumor-supportive functions of CAFs using 2D in vitro culture systems. We further examine radiation-induced changes in CAF infiltration within tumors in in vivo models. Additionally, we evaluate the impact of CAF modulation on radiotherapy outcomes by pre-treating animals with pirfenidone, a CAF-reprogramming agent. Collectively, our findings provide new insights into how RT affects CAF biology and clarify the potential role of CAFs in shaping radiotherapy responses\u003c/p\u003e"},{"header":"Material \u0026 methods","content":"\u003cp\u003eHuman material, CAF isolation, and cell cultures\u003c/p\u003e \u003cp\u003eHuman cancer-associated fibroblasts (CAFs) were derived from surgically resected non-small cell lung cancer (NSCLC) tissues obtained from patients at the University Hospital of Northern Norway (UNN), as previously described [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (clinical and patient characteristics can be found in supplementary material, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Briefly, CAFs were isolated by enzymatic digestion of tissues and the outgrowth method, followed by phenotypic characterization. Patient consent was obtained in writing, and the study was conducted in compliance with ethical guidelines approved by the Regional Ethical Committee of Northern Norway (REK Nord 2016/2307). CAFs were isolated using enzymatic digestion and the outgrowth method, followed by phenotypic characterization. Tumor samples from ten patients were processed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). CAFs were cultured in DMEM high glucose medium (Sigma-Aldrich, Cat. # D5796) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin). Experiments were conducted with low-passage cells (passages 3\u0026ndash;6). Donor-derived tumor cells (Donor-TC) were separated from mixed fibroblast cultures through differential detachment and characterized by cytokeratin expression, epithelial morphology, and colony-forming ability. Human lung adenocarcinoma cell lines A549 and HCC827 were obtained from LGC Standards AB and cultured in DMEM or RPMI-1640 medium, respectively, supplemented with 10% FBS and antibiotics.\u003c/p\u003e \u003cp\u003eIrradiation of cell cultures\u003c/p\u003e \u003cp\u003eAdherent cells were seeded in T-75 flasks or 6-well plates at 80% confluency one day prior to irradiation. High-energy photons were delivered using a clinical Varian linear accelerator, employing either a single dose of 18 Gy or a fractionated regimen of 3 \u0026times; 6 Gy at 24-hour intervals. Standard dose delivery parameters included a depth of 30 mm, beam quality of 15 MV, dose rate of 6 Gy/min, and a field size of 20 \u0026times; 20 cm.\u003c/p\u003e \u003cp\u003eWestern blots\u003c/p\u003e \u003cp\u003eWhole-cell lysates were prepared using RIPA buffer (Cell Signaling) supplemented with protease and phosphatase inhibitors (ThermoFisher, Cat. # 78440). Proteins were separated via 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 1% BSA in TBS-T for 2 hours at room temperature and incubated overnight at 4\u0026deg;C with primary antibodies (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) diluted 1:1000. After washing, membranes were incubated with HRP-conjugated secondary antibodies (1:2000; Cell Signaling, #7074) for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence and quantified with ImageJ software.\u003c/p\u003e \u003cp\u003eβ-galactosidase and apoptosis assay\u003c/p\u003e \u003cp\u003eCAFs were seeded in 6-well plates (10,000 cells/well) and irradiated the following day. Seven days post-irradiation, cells were fixed with 4% formaldehyde and stained for β-galactosidase activity using the Cellular Senescence Assay Kit (Sigma Aldrich, Cat. # KA0002). Senescent cells were identified by blue staining under a light microscope. Apoptosis was assessed in parallel using the CellEvent\u0026trade; Caspase-3/7 Green Flow Cytometry Assay Kit (ThermoFisher, Cat. # C10740) and analyzed by flow cytometry.\u003c/p\u003e \u003cp\u003eTumor cell treatments with CAFs and preparation of CAFs conditioned medium\u003c/p\u003e \u003cp\u003eIn some experimental settings, tumor cells were directly treated with CAF conditioned medium (CAF-CM), and in other settings tumor cells were incubated with CAFs in co-cultures for some phases of the experiment. Co-culture experiments were conducted by combining tumor cells (A549, HCC827, or donor-TC) with irradiated or non-irradiated CAFs at a 1:4 ratio. After three days, conditioned medium (CM) was collected, centrifuged, filtered (0.22 \u0026micro;m), and stored at -80\u0026deg;C. CAFs were selectively detached using enzyme-free dissociation solution, and tumor cells were analyzed for purity based on morphology and CD90 negativity by flow cytometry. The procedure resulted in purities\u0026thinsp;\u0026gt;\u0026thinsp;98%.\u003c/p\u003e \u003cp\u003eProliferation of tumor cells\u003c/p\u003e \u003cp\u003eProliferation of tumor cells exposed to CAFs and CAF-CM was assessed using the xCELLigence Real-Time Cell Analysis system (Agilent). Tumor cells were seeded in E-plates (A549 and HCC827: 2,000 cells/well; donor-TC: 1,000 cells/well) and cultured in a 50:50 mixture of CM and fresh medium. Cell index (CI) was recorded every 30 minutes for seven days.\u003c/p\u003e \u003cp\u003eCell migration assay\u003c/p\u003e \u003cp\u003eMigratory potential of tumor cells following co-cultures with irradiated and sham-irradiated CAFs was assessed by a scratch assay. Tumor cells were pre-treated with irradiated or untreated CAFs for three days and then replated in 6-well plates at 20.000, 30.000, and 30.000 cells/well for A549, HCC827, and donor-TCs, respectively, to ensure confluency 24 h after seeding. Each of the cultures were seeded with 3 technical replicates, and cells were maintained in the CM from the co-cultures. Twenty four hours post-seeding, a vertical scratch was made in each well using a 100 \u0026micro;L pipette-tip. Each well was photographed (100x magnification) using an Idea SPOT digital camera (Spot Imaging, Michigan, USA) immediately after the scratch-making, to designate the start of migration, and next 24 h post-scratch. Open areas from the scratch at the different time points were quantified using ImageJ Software and used to determine relative migration rates for each tumor cell type from the different CAF co-culture conditions.\u003c/p\u003e \u003cp\u003eClonogenic survival of tumor cells\u003c/p\u003e \u003cp\u003eTumor cells were plated at low densities as monocultures, and CAFs were added 24h later, in a proportion according to Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Tumor cells were plated 48 h before IR, whereas CAFs were plated 24 h before IR, to ensure that all cells were properly attached before IR exposure. Ten days post-IR, cultures were fixed (formaldehyde 4%, 10 min, 20\u0026deg;C) Cultures were then stained with crystal violet (Sigma-Aldrich, St Louis, MO, USA; Cat. #V5265) for 2h and washed with PBS. Colonies of tumor cells in the different cultures were counted and normalized according to the number of cells seeded in the well.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of cells seeded per well in 6well/plates used for the clonogenic assay.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCells/well\u003c/p\u003e \u003cp\u003e0 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCells/well\u003c/p\u003e \u003cp\u003e3 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCells/well\u003c/p\u003e \u003cp\u003e9 Gy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCells/well\u003c/p\u003e \u003cp\u003e12 Gy\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA549\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCAFs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDonor TCs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCAFs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e models\u003c/p\u003e \u003cp\u003e Female C57BL/6J and BALB/cJ mice (age 6\u0026ndash;8 weeks), were purchased from Charles River (Sulzfeld, Germany), and acclimatized in the local animal facility for a minimum of five days before experimentation. All procedures and experiments involving animals were conducted according to regulations by the Federation of European Laboratory Animal Science Association (FELASA) and approved by the National Animal Research Authority (permission ID 6373, 6942, and 7873). Maximum permitted size of tumors was 1000mm3, and animals were sacrificed if this humane endpoint was reached by cervical dislocation while being sedated. Animal sedation was achieved by short exposure to 2% isoflurane gas. The CT26 mouse colon carcinoma cells and the LL/2-Luc2 mouse luciferase-expressing Lewis lung carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM high glucose basal medium (Sigma-Aldrich, St Louis, MO, USA, Cat. # D5796) supplemented with 10% FBS, 100 U/mL penicillin/100 \u0026micro;g/mL streptomycin; plus blasticidin (10 ng/mL) for LL/2-Luc2 cells. All cells used for implantation were tested for pathogens by Idexx Bioanalytics (Mice Comprehensive test). Before transplantation, LL/2-Luc2 and CT26 cells were prepared in RPMI culture medium plus Matrigel (1:1, GelTrex, Thermo Fisher Scientific, Cat. #A1413202) and injected (5x10\u003csup\u003e5\u003c/sup\u003e cells/100 \u0026micro;L/mouse) subcutaneously into the right flank of animals. Tumors were measured three times per week using a digital caliper, and tumor volumes calculated by the modified ellipsoidal formula (V= \u0026frac12; (Length x width\u003csup\u003e2\u003c/sup\u003e). Animals were sacrificed on the day of irradiation (baseline) or 7 days post-irradiation as indicated above.\u003c/p\u003e \u003cp\u003ePirfenidone treatment\u003c/p\u003e \u003cp\u003eImmediately after tumor cell inoculation, animals received intraperitoneal injections of pirfenidone (Sigma-Aldrich, Cat# P2116) 200 mg/kg in PBS, 100 \u0026micro;L intraperitoneally) daily for 2 weeks (as indicated in scheme in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e RT treatment\u003c/p\u003e \u003cp\u003eTumor-bearing mice received tumor-specific radiation exposure by the image-guided irradiator X-RAD SmART system (Precision X-Ray irradiation, Madison, USA), as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Radiation treatments were performed when tumors reached 5\u0026ndash;6 mm in diameter (~\u0026thinsp;100 mm3, approx. 8\u0026ndash;10 days cell post-implantation). Structural imaging measurements (CT), as well as digital caliper measurements, were used for monitoring tumor growth. A single dose of 12 Gy was delivered to tumors using two opposing photon beams (maximum energy 225 kV, 13 mA), at dose-rate of 3 Gy/min. The treatment-planning system SmART-plan (version 1.3.9 Precision X-ray, North Branford, CT) was used for dose calculations of the plan with selected beam-angles and to deliver the treatment. Before delivering radiation to tumors, animals were sedated by continuous isoflurane gas anesthesia via induction in an anesthesia chamber (0.5 L/min oxygen, 4.0% isoflurane). During the imaging/radiation procedure, the animals were maintained sedated by continuous isoflurane anesthesia gas via a nose cone (0.4 L/min oxygen, 2% isoflurane).\u003c/p\u003e \u003cp\u003eFlow cytometry\u003c/p\u003e \u003cp\u003eCAF-specific markers were analyzed by flow cytometry on BD FACSAria III using the FlowJo software, Ver.7.2.4 (Tree Star, Ashland, OR, USA). Briefly, tumors were excised and minced with scalpels in 2.5 mL PBS, and the resulting cells and fragments digested in Accutase\u0026reg; solution (Sigma-Aldrich, Cat. #A6964) for 1 h at 37\u0026deg;C on a platform rocker. Digested tumors were further dissociated by mechanical disruption and sieving through 70-\u0026micro;m cell strainers (BD Biosciences, Bedford, MA, USA). Single-cell suspensions were labeled with panels of specific antibodies(Miltenyi Biotec, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). CAF-specific markers consisted of FAP-1 and α-SMA. Isotype controls consisted of REA control and IgG2a (Cat. no. 130-113-450 and 130-104-612, respectively). Data were obtained by flow cytometry using the following gating strategy: cells gated according to their scatter properties (FSC-A vs SSC-A), doublets exclusion (FSC-A vs FSC-H), and analyzed by the percentage of viable cells expressing FAP-1 and α-SMA, and also by median fluorescence intensity (Median FI).\u003c/p\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003cp\u003eFor immunohistochemistry evaluations, tumors were fixed in paraformaldehyde (4% in PBS) immediately after resection, incubated for at least 24h before embedding in paraffin blocks. Tissue sections (4\u0026micro;m thickness) were made and sections deparaffinized and rehydrated before staining. The Ventana Discovery-Ultra Research instrument (Roche 05987750001) was used for automated preparation and immunohistochemical staining of tumor tissue samples. Anti-mouse α-SMA antibody (D4K9N, cell signaling) was manually applied at a dilution of 1:100 followed by incubation at 36\u0026deg;C for 30 min. The antibody was validated for IHC-P (formalin-fixed and paraffin-embedded tissue) by the supplier. Stained slides were digitalized by an Olympus VS120 scanner. Quantification of positive areas/cells per square mm2 of intact tumor tissue was determined electronically using the QuPath Software for Bioimage Analysis (version v.0.5.1, tool: positive cell detection).\u003c/p\u003e \u003cp\u003eClinical cohort\u003c/p\u003e \u003cp\u003eSeventy-six patients who underwent adjuvant radiotherapy after curative intent surgery were identified from a larger retrospective cohort of lung-tumor specimens from NSCLC patients collected at the University Hospital of North-Norway and Nordland central hospital between 1990\u0026ndash;2010. The cohort is previously described by Hald et al. and approved by The Regional Committee for Medical and Health Research Ethics (REK-Nord Project-ID: 2016/2307/REK-Nord) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In brief, the cohort comprises a complete set of clinicopathological variables and tissue-microarrays (TMAs) with two to four replicate cores for each patient. The overall cohort was previously evaluated by us for the prognostic impact of several CAF markers including FAP1, αSMA, PDGFRα and PDGFRβ [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Comparison of data between experimental groups was analyzed using the Brown-Forsythe and Welch ANOVA test, and significance values were adjusted by Dunnett\u0026rsquo;s T3 correction for multiple comparisons. Outcomes of Western blot experiments were analyzed using the 2way ANOVA test, and significance values were adjusted by Dunnett correction for multiple comparisons. Results were presented in graphs, where each donor was plotted as an individual dot in the dataset. Survival data for the clinical cohort was visualized using the Kaplan-Meier method and assessed with the log-rank test. The chosen endpoint was DSS defined as the time from surgical resection to lung cancer specific death. For all analyses, lLevel of significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRadiation effects on CAF viability and phenotype\u003c/h2\u003e \u003cp\u003eInitial experiments were performed to study the direct effects of radiation exposure on the viability and phenotype of primary NSCLC human CAFs in culture. Analyses performed 5\u0026ndash;6 days post-IR exposure show that CAFs are surviving all the given radiation regimens. However, radiation-induced cellular damage was able to trigger cell senescence in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Of note, only marginal levels of apoptosis were detected in CAFs exposed to either 1x18Gy or 3x6Gy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, expressions of classical CAF activation markers was measured and compared between experimental groups. Results show minor (non-significant) variations in the expression of CAFs markers including FAP-1, FSP-1, α-SMA, PDGFRα and podoplanin on irradiated CAFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffects of irradiated CAFs on lung cancer cells pro-tumorigenic functions\u003c/h3\u003e\n\u003cp\u003eThe capacity of CAFs to confer tumor cell radioresistance was investigated in clonogenic assays performed with A549 lung adenocarcinoma and an in-house generated patient-derived tumor cell line (Donor-TC) in the presence or absence of CAFs. Outcomes from clonogenic assays show no differences between experimental groups, indicating that CAFs are not interfering with tumor cell radioresistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In separate experiments, we investigated potential effects of CAFs on tumor cell proliferative properties. Tumors cells and CAFs (irradiated or untreated) were co-cultured for three days followed by measurements of tumor cell proliferation during 7 days in the presence of CAF-CM. CAFs were eliminated from co-cultures by selective detachment as described in the method section. Results show no variations in the proliferation rates in any of the tested lung tumor cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In similar type of experiments, we analyzed tumor cells migration rates following exposure of cancer cells to irradiated and control CAFs in co-cultures in wound healing assays. Once again, migration rates of tumor cells seemed unaffected by CAFs, regardless of whether they were irradiated or not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). All experiments presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e were reproduced three times (3X) with human CAFs from three randomly selected donors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRadiation reduces CAF-mediated induction of EMT on lung cancer cells\u003c/h3\u003e\n\u003cp\u003eEpithelial-mesenchymal transition (EMT) is associated with tumor cell aggressiveness and metastatic potential. To explore potential effects of irradiated CAFs on EMT processes, tumor cells were incubated in co-cultures with irradiated or control CAFs, followed by tumor cell isolation and quantification of EMT-related markers in whole cell protein lysates. The epithelial marker E-cadherin was abundantly expressed in untreated A549 and HCC827 cells and to a lesser extent in the in-house made lung tumor cell line (donor-TC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). On the contrary, the mesenchymal marker vimentin was non-detectable in untreated A549 and HCC827 cells but detectable in the donor-derived tumor cells, indicating that tumor cells from this patient display an EMT-like phenotype at baseline. Upon incubation with irradiated and control CAFs, A549 cells lost E-cadherin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, top-left panel). This phenomenon was not observed in HCC827 cells or donor TCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-top middle and right panels). Importantly, incubation of HCC827 and donor TCs with untreated CAFs was able to induce vimentin expression, however, this effect was partially abolished when the incubation was done with irradiated CAFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Vimentin induction by CAFs was not observed on A549 cells in any condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRadiotherapy-induced changes in intratumoral CAF levels in preclinical tumor models\u003c/h2\u003e \u003cp\u003eThe effects of radiotherapy on intratumoral CAF levels were studied in three different subcutaneous tumor models. The experimental strategy included the LLC lung adenocarcinoma model, the CT26 colon carcinoma model and the 4662PDA pancreatic adenocarcinoma model. Tumors were irradiated with a single dose of 12Gy using a high-precision image-guided irradiator. Tumor growth curves showed prominent responses to radiation from the LLC and CT26 models and a bit more modest response to radiation from the 4662 pancreatic model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). One week after radiation exposure, tumors were harvested and the content of CAFs was determined by both flow cytometry and \u003cem\u003ein situ\u003c/em\u003e immunohistochemistry. Results from flow cytometry analyses showed no significant changes in numbers of CAFs in any of the tumor models used, determined as percentage of α-SMA\u003csup\u003e+\u003c/sup\u003e cells from the total amount of living cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). On the other hand, outcomes from IHC determinations on fixed tissues revealed no significant changes of α-SMA\u003csup\u003e+\u003c/sup\u003e cells in the LLC and CT26 models whereas a significant reduction of aSMA+ CAFs was observed in the 4662 pancreatic model, (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In the latter approach, necrotic areas and peripheral tumor regions were excluded from analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnhanced radioresistance in pirfenidone-treated animals\u003c/h3\u003e\n\u003cp\u003ePirfenidone is an anti-fibrotic medication primarily administered to treat idiopathic pulmonary fibrosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Emerging evidence suggests that pirfenidone has promising anti-cancer effects through targeting and reprogramming CAFs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, we investigate the influence of CAFs on tumor responses to radiotherapy (RT) by pre-treating animals with pirfenidone. More specifically, we assess whether modulating CAF activity through pirfenidone administration could enhance the therapeutic efficacy of RT. Histological analyses of tumors revealed that the LLC and CT26 tumor models are associated with poorly developed stroma (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For this reason, the more stromatic 4662PDA pancreatic cancer model was chosen to run experiments with pirfenidone. Tisue analyses demonstrated higher baseline levels of α-SMA+ cells and intratumoral collagen deposition than in LLC and CT26 tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Pirfenidone treatment did not show any effect on tumor growth kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Moreover, pirfenidone treatment did not have observable effects on the levels of aSMA+ CAFs, however, it provoked a significant reduction of intratumoral collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Upon radiation, tumors in pirfenidone-naive animals showed good response to 1x12Gy treatment, however, tumor growth response to RT was partially abrogated in pirfenidone-treated animals, with tumor growth rates similar to non-irradiated tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E) and survival rates significantly decreased when compared to the pirfenidone-na\u0026iuml;ve RT-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n\u003ch3\u003eBaseline expression of CAF markers does not predict radiotherapy responses in clinical settings\u003c/h3\u003e\n\u003cp\u003eThe prognostic impact of the CAF markers FAP-1, α-SMA, PDGFRα and PDGFRβ was studied in a cohort of tumor specimens from NSCLC patients who received radiotherapy in an adjuvant setting (n\u0026thinsp;=\u0026thinsp;76). The data are summarized in table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B, neither FAP-1 nor α-SMA were correlated with disease-specific survival. However, a non-significant trend to adverse outcome for patients with high expression of PDGFRα or PDGFRβ was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Due to a limited number of patients in the cohort, analyses within histological subtypes (adenocarcinoma and squamous cell carcinoma) were not feasible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBesides its tumor cell killing potential, radiation is able to induce substantial changes in the tumor microenvironment (TME), including effects on tumor compartments such as the vasculature, the stroma as well as immunological components. Accordingly, RT-mediated reprogramming of the TME is considered to play a major role in therapy outcomes. The impact of RT on CAFs varies among studies, and the potential mechanisms behind the influence of CAFs on tumor radioresistance remain controversial [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this work, we have aimed to explore the impact of radiation on CAF-mediated tumorigenic functions and the potential role that CAFs may play in RT outcomes. Jointly considered, results indicate that upon irradiation, CAFs may lose some of their protumorigenic properties and that therapeutic strategies for reprogramming CAFs may indeed have detrimental effects in the context of radiotherapy.\u003c/p\u003e \u003cp\u003eAs shown by us and others previously, radiation given in single-high or medium-high fractionated regimens is able to induce cell senescence in primary cultures of CAFs prepared from NSCLC tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Senescent cells are characterized by possessing an anti-fibrotic and pro-inflammatory phenotype [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the context of cancer, numerous reports have demonstrated pro-tumorigenic properties of senescent cells via the release of a myriad of bioactive factors collectively termed the senescent-associated secretory phenotype (SASP) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, a deeper look at the role of senescent cells in cancer progression unveils a more balanced scenario in which both pro-tumorigenic and anti-tumorigenic functions may take place in a context-dependent manner [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the context of radiotherapy, some authors have achieved tumor radiosensitization after elimination of senescent fibroblasts [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. On the contrary, other reports have shown \u0026ldquo;normalization\u0026rdquo; of tumor stromal fibroblasts exposed to radiation with consequent loss of tumor-promoting effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, we demonstrate that radiation-induced senescent CAFs maintain the expression of CAF activation markers such as α-SMA, FAP-1, PDGFRα, or podoplanin. We also observe that CAFs, whether irradiated or not, do not affect the proliferative or migratory properties of lung tumor cells. These results align well to outcomes we previously observed using irradiated CAF-CM on different lung cancer cell lines [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. On the other hand, in co-culture settings, radiation seems to counteract CAF-mediated EMT effects on tumor cells. This is in opposition to observations from other groups reporting EMT induction from irradiated CAFs via expression of IL-6 in a model of esophageal cancer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], via secretion of HGF in a breast cancer model [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] or via secretion of SDF-1 in a pancreatic cancer model [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Disparities in outcomes may come from the use of different cancer models, different cell lines, or different experimental settings. In the above-mentioned studies, tumor cells have been exposed to CAF-CM from one or two CAF donors. In our study, we perform pre-conditioning of tumor cells by incubating them with CAFs in direct cell contact co-cultures, in addition to the incubation of cells with CAF-CM during the assay. Additionally, we have run experiments in parallel with multiple CAF donors.\u003c/p\u003e \u003cp\u003eThe effects of local radiation on the dynamics of intratumoral CAFs in \u003cem\u003ein vivo\u003c/em\u003e preclinical models have been scarcely investigated [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The majority of published studies exploring the role of CAFs (or irradiated CAFs) on RT responses have used co-injections of admixed CAFs and tumor cells pre-irradiated or pre-conditioned during \u003cem\u003ein vitro\u003c/em\u003e cultures [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, we explore the effects of external beam radiotherapy on intratumoral CAF dynamics using different subcutaneous cancer models. Quantitative analyses from flow cytometry and immunohistochemistry of α-SMA\u003csup\u003e+\u003c/sup\u003e fibroblasts in tumors reveal that there are no significant changes in the abundance of CAFs after 1x12Gy tumor irradiation, and the same outcomes were observed in the three tumor models. A limitation in our approach is that only one radiation regimen and only one-time point was studied. It remains to be investigated if things turn out differently when using different radiation regimens or if measured at different time points. For comparisons with human tumors in the clinics, Verset L et al. reported increased αSMA/epithelial ratios after neoadjuvant (chemo)radiotherapy in a colorectal cancer cohort [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In an ongoing study by us, performed on a cervix cancer cohort, we observed enhanced levels of fibroblast gene signatures after 5x2Gy radiotherapy cycles compared to baseline, however, the levels of α-SMA-positive cells in tissue remained unchanged in the majority of RT-treated patients (unpublished results).\u003c/p\u003e \u003cp\u003eGiven the overwhelming amount of scientific studies demonstrating the multifaceted pro-tumorigenic and therapy resistance effects of CAFs, large amounts of endeavors are now put into testing alternative therapies to target CAFs or CAF-mediated tumor supportive pathways [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Among the different strategies for targeting CAFs, one appealing and frequently pursued alternative is the use of drugs that promote CAF normalization or reprogramming [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Pirfenidone is an oral antifibrotic medication primarily administered for the treatment of idiopathic pulmonary fibrosis, with demonstrated anti-cancer effects manifested through CAFs deactivation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Among other effects, pirfenidone has been shown to disrupt tumor-stroma interactions via deregulation of CAF-driven pro-tumorigenic signaling including α-SMA, collagen I, PDGFRβ, TGF-β, PD-L1 or CCL17 [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our study, we have tested the effects of different anti-fibrotic and/or CAF-reprogramming agents including tranilast, calcipotriol (Vitamin D), and pirfenidone. Observable reduction in intratumoral collagen deposition was achieved only with pirfenidone (unshown results). Unexpectedly, animals in the pirfenidone-treated group showed enhanced radioresistance compared to the untreated group. Such outcomes indicate that the presence of \u0026ldquo;intact\u0026rdquo; CAFs, at least in this particular tumor model, is associated with better tumor responses to RT. Intriguingly, in a recently published study, authors observe enhanced FAP\u003csup\u003e+\u003c/sup\u003e stromal cells after RT in both clinical and preclinical settings, and better responses to (chemo)radiotherapy are observed in patients with therapy-induced FAP\u003csup\u003e+\u003c/sup\u003e CAFs [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Experimental approaches that specifically target CAFs such as FAP-targeting immunotherapeutics, or transgenic animal models in which CAFs can be selectively depleted may represent alternative experimental approaches to investigate the role of CAFs in radiotherapy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eData generated in this study contribute significantly to the understanding of the effects of RT on CAFs and the potential role that CAFs may play in RT outcomes. Collectively, our results indicate that radiation exposure to CAFs triggers cellular senescence, however many of their phenotypic and functional traits are still preserved after irradiation. Importantly, some of the CAFs pro-tumorigenic properties such as EMT promotion become partially reduced after irradiation. In \u003cem\u003ein vivo\u003c/em\u003e models, intratumoral CAFs levels remain nearly unchanged after a single high radiation dose and CAFs treatment by pirfenidone turns tumors more radioresistant. Observations made in our preclinical models need to be confirmed in clinical settings, where CAF dynamics (including numeral and phenotypical changes) are compared between baseline and post-treatment tissue samples. In light of our observations, therapeutic strategies involving CAF reprogramming in the context of radiotherapy should be carefully considered as they may negatively influence treatment outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Norwegian Food Safety Authority (FOTS ID 18956 and 27939). Experiments involving human biological products were approved by Regional Ethical Committee of Northern Norway (REK Nord 2016/2307). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article or supplementary material. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests. The FAPI-74-NOTA precursor was provided free of charge by SOFIE Biosciences without further influence on study design and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Cancer Biobank at UNN for providing human lung cancer samples. Publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Norwegian Regional Health Authorities under Grant (HNF1373-17 to RB and HNF1423-18 to TH); The Norwegian Cancer Society, the Aakre Foundation at UiT, and the Troms\u0026oslash; forskningsstiftelse (TFS) grant 180\u0026ordm;N/Norwegian-Nuclear-Medicine-consortium. Publication charges for this article have been funded by a grant from the publication fund at UiT, The Arctic University of Norway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRB\u003c/strong\u003e: Data curation, Formal analysis, Investigation, Methodology, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eTH\u003c/strong\u003e: Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eKL, VM and IR\u003c/strong\u003e: Data curation, Formal analysis, Investigation, Methodology. \u003cstrong\u003eLR\u003c/strong\u003e: Data curation \u0026amp; Formal analysis. \u003cstrong\u003eTK\u003c/strong\u003e: Data curation \u0026amp; Formal analysis. \u003cstrong\u003eIM-Z\u003c/strong\u003e: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma RA, et al. Clinical development of new drug-radiotherapy combinations. Nat Rev Clin Oncol. 2016;13(10):627\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoka K, et al. Technological Advancements in External Beam Radiation Therapy (EBRT): An Indispensable Tool for Cancer Treatment. Cancer Manag Res. 2022;14:1421\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlsaihaty Z, et al. Hybrid Treatment Planning for Chest Wall Irradiation Utilizing Three-Dimensional Conformal Radiotherapy (3DCRT), Intensity-Modulated Radiation Therapy (IMRT), and Volumetric Modulated Arc Therapy (VMAT): A Systematic Review. Cureus. 2024;16(5):e59583.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellevik T, Martinez-Zubiaurre I. Radiotherapy and the Tumor Stroma: The Importance of Dose and Fractionation. Front Oncol. 2014;4:1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarker HE, et al. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat Rev Cancer. 2015;15(7):409\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahai E, et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer. 2020;20(3):174\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo T, Xu J. Cancer-associated fibroblasts: a versatile mediator in tumor progression, metastasis, and targeted therapy. Cancer Metastasis Rev. 2024;43(3):1095\u0026ndash;116.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrell C, et al. Prognostic and predictive impact of stroma cells defined by PDGFRb expression in early breast cancer: results from the randomized SweBCG91RT trial. Breast Cancer Res Treat. 2021;187(1):45\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang R, Liu F. Cancer-associated fibroblast-derived gene signatures predict radiotherapeutic survival in prostate cancer patients. J Transl Med. 2022;20(1):453.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu TY, et al. Crosstalk with cancer-associated fibroblasts increases the growth and radiation survival of cervical cancer cells. Radiat Res. 2014;181(5):540\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang RF, et al. Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res. 2008;68(3):918\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi X, et al. Cancer-associated fibroblasts from NSCLC promote the radioresistance in lung cancer cell lines. Int J Clin Exp Med. 2015;8(5):7002\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTommelein J, et al. Radiotherapy-Activated Cancer-Associated Fibroblasts Promote Tumor Progression through Paracrine IGF1R Activation. Cancer Res. 2018;78(3):659\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi D, et al. Radiation promotes epithelial-to-mesenchymal transition and invasion of pancreatic cancer cell by activating carcinoma-associated fibroblasts. Am J Cancer Res. 2016;6(10):2192\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrinde MT, et al. Ionizing radiation abrogates the pro-tumorigenic capacity of cancer-associated fibroblasts co-implanted in xenografts. Sci Rep. 2017;7:46714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAboussekhra A, et al. Ionizing radiation normalizes the features of active breast cancer stromal fibroblasts and suppresses their paracrine pro-carcinogenic effects. Transl Oncol. 2023;37:101780.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellevik T, et al. Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced. Radiat Oncol. 2012;7:59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerzaghi R, Gundersen K, Pedersen BD, Utne R, Yang N, Hellevik T, Martinez I. Immunological signatures from irradiated cancer-associated fibroblasts. Frontiers in Immunology; 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHald SM, et al. LAG-3 in Non-Small-cell Lung Cancer: Expression in Primary Tumors and Metastatic Lymph Nodes Is Associated With Improved Survival. Clin Lung Cancer. 2018;19(3):249\u0026ndash;59. e2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKilvaer TK, et al. Cancer Associated Fibroblasts in Stage I-IIIA NSCLC: Prognostic Impact and Their Correlations with Tumor Molecular Markers. PLoS ONE. 2015;10(8):e0134965.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKilvaer TK, et al. Differential prognostic impact of platelet-derived growth factor receptor expression in NSCLC. Sci Rep. 2019;9(1):10163.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuwanpura SM, Thomas BJ, Bardin PG. Pirfenidone: Molecular Mechanisms and Potential Clinical Applications in Lung Disease. Am J Respir Cell Mol Biol. 2020;62(4):413\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRastegar-Pouyani N, et al. Targeting cancer-associated fibroblasts with pirfenidone: A novel approach for cancer therapy. Tissue Cell. 2024;91:102624.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez-Zubiaurre I, Hellevik T. Cancer-associated fibroblasts in radiotherapy: Bystanders or protagonists? Cell Commun Signal. 2023;21(1):108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaaijmakers K, et al. Cancer-associated fibroblasts, tumor and radiotherapy: interactions in the tumor micro-environment. J Exp Clin Cancer Res. 2024;43(1):323.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurton DG, Krizhanovsky V. Physiological and pathological consequences of cellular senescence. Cell Mol Life Sci. 2014;71(22):4373\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavalos AR, et al. Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev. 2010;29(2):273\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecot P, et al. Context-dependent effects of cellular senescence in cancer development. Br J Cancer. 2016;114(11):1180\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng J et al. Targeting senescence-like fibroblasts radiosensitizes non-small cell lung cancer and reduces radiation-induced pulmonary fibrosis. JCI Insight, 2021. 6(23).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJian C, et al. Biomimetic Nanoplatform for Dual-Targeted Clearance of Activated and Senescent Cancer-Associated Fibroblasts to Improve Radiation Resistance in Breast Cancer. Small. 2024;20(25):e2309279.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellevik T, et al. Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth. Transl Oncol. 2013;6(1):66\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbbing EA, et al. Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma. Proc Natl Acad Sci U S A. 2019;116(6):2237\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, et al. Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway. Int J Radiat Oncol Biol Phys. 2023;116(3):640\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePereira PMR, et al. iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy. Cancer Res. 2020;80(8):1681\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, et al. Cancer-associated Fibroblasts Promote Irradiat Cancer Cell Recovery Through Autophagy EBioMedicine. 2017;17:45\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerset L, et al. Impact of neoadjuvant therapy on cancer-associated fibroblasts in rectal cancer. Radiother Oncol. 2015;116(3):449\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValkenburg KC, de Groot AE, Pienta KJ. Targeting the tumour stroma to improve cancer therapy. Nat Rev Clin Oncol. 2018;15(6):366\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanguly D et al. Cancer-Associated Fibroblasts: Versatile Players in the Tumor Microenvironment. Cancers (Basel), 2020. 12(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Discov. 2019;18(2):99\u0026ndash;115.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaliogiannis P, et al. Repurposing Anticancer Drugs for the Treatment of Idiopathic Pulmonary Fibrosis and Antifibrotic Drugs for the Treatment of Cancer: State of the Art. Curr Med Chem. 2021;28(11):2234\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujiwara A, et al. Effects of pirfenidone targeting the tumor microenvironment and tumor-stroma interaction as a novel treatment for non-small cell lung cancer. Sci Rep. 2020;10(1):10900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAoto K, Ito K, Aoki S. Complex formation between platelet-derived growth factor receptor beta and transforming growth factor beta receptor regulates the differentiation of mesenchymal stem cells into cancer-associated fibroblasts. Oncotarget. 2018;9(75):34090\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAboulkheyr Es H, et al. Pirfenidone reduces immune-suppressive capacity of cancer-associated fibroblasts through targeting CCL17 and TNF-beta. Integr Biol (Camb). 2020;12(7):188\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEs HA et al. Pirfenidone Reduces Epithelial-Mesenchymal Transition and Spheroid Formation in Breast Carcinoma through Targeting Cancer-Associated Fibroblasts (CAFs). Cancers (Basel), 2021. 13(20).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarate-Soraluze E et al. 4-1BB agonist targeted to fibroblast activation protein alpha synergizes with radiotherapy to treat murine breast tumor models. J Immunother Cancer, 2025. 13(2).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Radiotherapy, cancer-associate fibroblasts, epithelial-mesenchymal transition, in vivo models","lastPublishedDoi":"10.21203/rs.3.rs-8680198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8680198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and active drivers of tumor progression, metastasis, and resistance to therapy. However, the effects of radiotherapy (RT) on CAFs and the consequent impact on tumor cell behavior remain controversial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Primary cultures of human CAFs isolated from non-small cell lung cancer (NSCLC) tumor samples were exposed to ionizing radiation by a single-high dose or a hypofractionated regimen. Different lung tumor cell lines were exposed to control or irradiated CAFs in direct co-cultures or to CAFs conditioned medium (CM), and \u0026nbsp;changes in tumor cell proliferation, migration, epithelial-mesenchymal transition (EMT) and clonogenic survival were measured. The impact of RT on intratumoral CAF levels in different subcutaneous tumor models was measured by α-SMA expression in excised tumor specimens. In addition, pirfenidone, a CAF-reprogramming agent, was investigated for its capacity to influence tumor responses to radiation. Finally, correlations of CAF markers with treatment outcomes was studied in a cohort of RT-treated non-small cell lung cancer (NSCLC) patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Ionizing radiation induced premature senescence in CAFs; however, the expression of established CAF activation markers remained unchanged. Exposure of lung tumor cell lines to CM from irradiated CAFs did not alter their proliferation, migratory capacity, or clonogenic survival. Conversely, CAF-driven epithelial–mesenchymal transition (EMT) was attenuated in all tumor cell lines after incubations with CM from irradiated CAFs.\u003c/p\u003e\n\u003cp\u003eRadiation delivered to subcutaneously transplanted tumors (1x12 Gy) did not change intra-tumoral CAF abundance in the LLC and CT26 murine models, whereas CAF numbers were significantly reduced in the stroma-rich 4662PDA pancreatic model. Pirfenidone-treated animals exhibited enhanced tumor radio-resistance, along with decreased collagen levels within tumors, and unchanged numbers of aSMA+CAFs.\u003c/p\u003e\n\u003cp\u003eIn clinical specimens, baseline tissue expression levels of the CAF markers FAP and aSMA were not associated with disease-specific survival.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur findings suggest that ionizing radiation may lessen certain pro-tumorigenic CAF functions—such as EMT induction—, whereas pharmacologic CAF reprogramming with pirfenidone paradoxically confers increased tumor radio-resistance, highlighting potential negative implications from CAF-targeted therapies.\u003c/p\u003e","manuscriptTitle":"Radiation treatment curtails pro-tumorigenic functions from cancer-associated fibroblasts in preclinical models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 17:06:00","doi":"10.21203/rs.3.rs-8680198/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"13a7ac3b-b198-4a63-bb6c-765e946f99e6","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-21T09:24:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 17:06:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8680198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8680198","identity":"rs-8680198","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}