Radiation-induced effects on TGF-ß and PDGF receptor signaling in cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) consist of heterogeneous connective tissue cells and are often constituting the most abundant cell type in the tumor stroma. Radiation effects on tumor stromal components like CAFs in the context of radiation treatment is not well-described. AIM: This study explores potential changes induced by ionizing radiation (IR) on platelet-derived growth factor (PDGF)/PDGFRs and transforming growth factor-beta (TGF-ß)/TGFßRs signaling systems in CAFs. METHODS AND RESULTS: Experiments were carried out by employing primary cultures of human CAFs isolated from freshly resected non-small cell lung carcinoma tumor tissues. CAF cultures from nine donors were treated with one high (1 × 18 Gy) or three fractionated (3 × 6 Gy) radiation doses. Alterations in expression levels of TGFßRII and PDGFRα/ß induced by IR were analyzed by western blots and flow cytometry. In the presence or absence of cognate ligands, receptor activation was studied in nonirradiated and irradiated CAFs. Radiation exposure did not exert changes in expression of PDGF or TGF-ß receptors in CAFs. Additionally, IR alone was unable to trigger activation of either receptor. The radiation regimens tested did not affect PDGFRß signaling in the presence of PDGF-BB. In contrast, signaling via pSmad2/3 and pSmad1/5/8 appeared to be down-regulated in irradiated CAFs after stimulation with TGF-ß, as compared with controls. CONCLUSION: Our data demonstrate that IR by itself is insufficient to induce measurable changes in PDGF or TGF-ß receptor expression levels or to induce receptor activation in CAFs. However, in the presence of their respective ligands, exposure to radiation at certain doses appear to interfere with TGF-ß receptor signaling.

Cancer-associated fibroblasts in radiotherapy: Bystanders or protagonists?

BACKGROUND: The primary goal of radiotherapy (RT) is to induce cellular damage on malignant cells; however, it is becoming increasingly recognized the important role played by the tumor microenvironment (TME) in therapy outcomes. Therapeutic irradiation of tumor lesions provokes profound cellular and biological reconfigurations within the TME that ultimately may influence the fate of the therapy. MAIN CONTENT: Cancer-associated fibroblasts (CAFs) are known to participate in all stages of cancer progression and are increasingly acknowledged to contribute to therapy resistance. Accumulated evidence suggests that, upon radiation, fibroblasts/CAFs avoid cell death but instead enter a permanent senescent state, which in turn may influence the behavior of tumor cells and other components of the TME. Despite the proposed participation of senescent fibroblasts on tumor radioprotection, it is still incompletely understood the impact that RT has on CAFs and the ultimate role that irradiated CAFs have on therapy outcomes. Some of the current controversies may emerge from generalizing observations obtained using normal fibroblasts and CAFs, which are different cell entities that may respond differently to radiation exposure. CONCLUSION: In this review we present current knowledge on the field of CAFs role in radiotherapy; we discuss the potential tumorigenic functions of radiation-induced senescent fibroblasts and CAFs and we make an effort to integrate the knowledge emerging from preclinical experimentation with observations from the clinics. Video Abstract.

Radiotherapy (RT) is currently used to treat more than 50% of all diagnosed cancer cases and counts for around 40% of all cure rates. The primary goal of RT has always been to induce damage on tumor cells; thus, it has been traditionally believed that the therapeutic efficacy of RT is mediated exclusively by its capacity to directly kill malignant cells. However, tumors are complex tissues composed of multiple cellular and acellular elements often referred as the tumor microenvironment (TME) or tumor stroma. Both, the malignant and the non-malignant cells in tumors receive the same prescribed radiation dose during treatment. In recent years, it has become more and more evident the fundamental role played by elements of the TME on therapy outcomes. One of the most abundant and influential elements of the reactive stroma in tumors are cancer-associated fibroblasts (CAFs). CAFs are also exposed to the full prescribed radiation dose during the course of radiotherapy. Accumulated evidence suggests that, upon radiation, fibroblasts/CAFs avoid cell death but instead enter a permanent senescent (growth arrest) state, which in turn may influence the behavior of tumor cells and other components of the TME. Despite the proposed participation of senescent fibroblasts on tumor radioprotection, it is still incompletely understood the impact that RT has on CAFs and the ultimate role that irradiated CAFs have on therapy outcomes. Further research using appropriate experimental models to study CAFs, and more information from clinical research is needed to unveil the ultimate role played by CAFs on radiotherapy.

Immunobiology of cancer-associated fibroblasts in the context of radiotherapy.

Radiotherapy (RT) still represents a mainstay of treatment in clinical oncology. Traditionally, the effectiveness of radiotherapy has been attributed to the killing potential of ionizing radiation (IR) over malignant cells, however, it has become clear that therapeutic efficacy of RT also involves activation of innate and adaptive anti-tumor immune responses. Therapeutic irradiation of the tumor microenvironment (TME) provokes profound cellular and biological reconfigurations which ultimately may influence immune recognition. As one of the major constituents of the TME, cancer-associated fibroblasts (CAFs) play central roles in cancer development at all stages and are recognized contributors of tumor immune evasion. While some studies argue that RT affects CAFs negatively through growth arrest and impaired motility, others claim that exposure of fibroblasts to RT promotes their conversion into a more activated phenotype. Nevertheless, despite the well-described immunoregulatory functions assigned to CAFs, little is known about the interplay between CAFs and immune cells in the context of RT. In this review, we go over current literature on the effects of radiation on CAFs and the influence that CAFs have on radiotherapy outcomes, and we summarize present knowledge on the transformed cellular crosstalk between CAFs and immune cells after radiation.

Ionizing Radiation Curtails Immunosuppressive Effects From Cancer-Associated Fibroblasts on Dendritic Cells.

Cancer-associated fibroblasts (CAFs) participate actively in tumor development and affect treatment responses, by among other mechanisms, promoting an immunosuppressive tumor microenvironment. In contrast to normal fibroblasts, reactive CAFs secrete a myriad of immunomodulatory soluble factors at high levels, i.e. growth factors, cytokines, and chemokines, which directly influence tumor immunity and inflammation. CAFs have been identified as important players in tumor radioresistance. However, knowledge on the immunomodulatory functions of CAFs during/after radiotherapy is still lacking. In this study, we investigated the effects of ionizing radiation on CAF-mediated regulation of dendritic cells (DCs). CAFs were obtained from freshly operated lung cancer tissues, while DCs were procured from peripheral blood of healthy donors. Experimental settings comprised both co-cultures and incubations with conditioned medium from control and irradiated CAFs. Functional assays to study DC differentiation/activation consisted on cytokine release, expression of cell-surface markers, antigen uptake, migration rates, T cell priming, and DC-signaling analysis. We demonstrate that CAFs induce a tolerogenic phenotype in DCs by promoting down-regulation of: i) signature DC markers (CD14, CD1a, CD209); ii) activation markers (CD80, CD86, CD40, and HLA-DR) and iii) functional properties (migration, antigen uptake, and CD4+ T cell priming). Notably, some of these effects were lost in conditioned medium from CAFs irradiated at fractionated medium-dose regimens (3x6 Gy). However, the expression of relevant CAF-derived regulatory agents like thymic stromal lymphopoietin (TSLP) or tryptophan 2,3-dioxygenase (TDO2) was unchanged upon irradiation. This study demonstrates that CAFs interfere with DC immune functions and unveil that certain radiation regimens may reverse CAF-mediated immunosuppressive effects.

Secretion rates and protein composition of extracellular vesicles released by cancer-associated fibroblasts after radiation.

Reciprocal communication between the malignant and non-malignant cellular elements in tumors is essential for cancer sustainability and plays an important role in the response of cancers to treatments. Some of this cellular crosstalk takes place via secretion of vesicles that are actively released into the extracellular space by most cell types in tumors. Recent studies have demonstrated radiation-induced changes in the secretion rate and composition of extracellular vesicles (EVs), with impact on radiation-related cellular communication. However, little is known about the effects of different radiation regimens on the release of EVs by cells of the tumor microenvironment. In this study, we provide a comprehensive molecular characterization of EVs released by cultured primary lung tumor fibroblasts. We explore the quantitative and morphological changes triggered by ionizing radiation (IR), delivered as a single dose of 18 Gy or three consecutive daily medium-doses of 6 Gy. Cancer-associated fibroblasts (CAFs) secrete EVs with sizes ranging from 80 to 200 nm, expressing some of the classical exosome markers. Exposing CAFs to a single-high radiation dose (1 × 18 Gy) or fractionated medium-dose did not alter the release of CAF-EVs. The protein composition of CAF-EVs was analyzed by LC-MS/MS proteomics and revealed that CAF-EVs are enriched with heat shock proteins, integrins, tetraspanins, proteinases, collagens, growth factors and an array of molecules involved in the regulation of cell migration and the immune system. Quantitative proteomic analyses revealed minor changes in the protein composition of CAF-EVs after radiation exposure. Taken together, this study presents original data on lung tumor CAF-EV composition and reveals that release and protein cargo of CAF-EVs are largely unaltered after exposing CAFs to IR.

GARP promotes the proliferation and therapeutic resistance of bone sarcoma cancer cells through the activation of TGF-ß.

Sarcomas are mesenchymal cancers with poor prognosis, representing about 20% of all solid malignancies in children, adolescents, and young adults. Radio- and chemoresistance are common features of sarcomas warranting the search for novel prognostic and predictive markers. GARP/LRRC32 is a TGF-ß-activating protein that promotes immune escape and dissemination in various cancers. However, if GARP affects the tumorigenicity and treatment resistance of sarcomas is not known. We show that GARP is expressed by human osteo-, chondro-, and undifferentiated pleomorphic sarcomas and is associated with a significantly worse clinical prognosis. Silencing of GARP in bone sarcoma cell lines blocked their proliferation and induced apoptosis. In contrast, overexpression of GARP promoted their growth in vitro and in vivo and increased their resistance to DNA damage and cell death induced by etoposide, doxorubicin, and irradiation. Our data suggest that GARP could serve as a marker with therapeutic, prognostic, and predictive value in sarcoma. We propose that targeting GARP in bone sarcomas could reduce tumour burden while simultaneously improving the efficacy of chemo- and radiotherapy.

Irradiated Tumor Fibroblasts Avoid Immune Recognition and Retain Immunosuppressive Functions Over Natural Killer Cells.

Recent studies have demonstrated that radiotherapy is able to induce anti-tumor immune responses in addition to mediating direct cytotoxic effects. Cancer-associated fibroblasts (CAFs) are central constituents of the tumor stroma and participate actively in tumor immunoregulation. However, the capacity of CAFs to influence immune responses in the context of radiotherapy is still poorly understood. This study was undertaken to determine whether ionizing radiation alters the CAF-mediated immunoregulatory effects on natural killer (NK) cells. CAFs were isolated from freshly resected non-small cell lung cancer tissues, while NK cells were prepared from peripheral blood of healthy donors. Functional assays to study NK cell immune activation included proliferation rates, expression of cell surface markers, secretion of immunomodulators, cytotoxic assays, as well as production of intracellular activation markers such as perforin and granzyme B. Our data show that CAFs inhibit NK cell activation by reducing their proliferation rates, the cytotoxic capacity, the extent of degranulation, and the surface expression of stimulatory receptors, while concomitantly enhancing surface expression of inhibitory receptors. Radiation delivered as single high-dose or in fractioned regimens did not reverse the immunosuppressive features exerted by CAFs over NK cells in vitro, despite triggering enhanced surface expression of several checkpoint ligands on irradiated CAFs. In summary, CAFs mediate noticeable immune inhibitory effects on cytokine-activated NK cells during co-culture in a donor-independent manner. However, ionizing radiation does not interfere with the CAF-mediated immunosuppressive effects.

Differential prognostic impact of platelet-derived growth factor receptor expression in NSCLC.

Preclinical evidence suggests that stromal expression of platelet-derived growth factor receptors (PDGFRs) stimulates tumor development and diminishes intratumoral drug uptake. In non-small cell lung cancer (NSCLC), the clinical relevance of stromal PDGFR expression remains uncertain. Tumor specimens from 553 patients with primary operable stage I-IIIB NSCLC was obtained and tissue micro-arrays (TMA) were constructed (Norwegian cohort). Immunohistochemistry (IHC) was used to evaluate the expression of PDGFRα and -ß in stromal cells and to explore their impact on patient survival. Results were validated in a non-related cohort consisting of TMAs of 367 stage I (A and B) NSCLC patients (Swedish cohort). High stromal PDGFRα expression was an independent predictor of increased survival in the overall populations and SCC (squamous cell carcinoma) subgroups of both investigated cohorts. PDGFRß was an independent predictor of poor survival in the overall Norwegian cohort and an independent predictor of increased survival in the ADC (adenocarcinoma) subgroup of the Swedish cohort. Tumors displaying the combination PDGFRα-low/PDGFRß-high exhibited inferior survival according to increasing stage in the Norwegian cohort. This study confirms that high stromal expression of PDGFRα is a predictor of increased survival in NSCLC. Further exploration of the prognostic impact of PDGFRß and the relationship between PDGFRα and -ß is warranted.

Fibroblast-Mediated Immunoregulation of Macrophage Function Is Maintained after Irradiation.

The abilities of cancer-associated fibroblasts (CAFs) to regulate immune responses in the context of radiotherapy remain largely unknown. This study was undertaken to determine whether ionizing radiation alters the CAF-mediated immunoregulatory effects on macrophages. CAFs were isolated from freshly-resected non-small cell lung cancer tumors, while monocyte-derived macrophages were prepared from peripheral blood of healthy donors. Experimental settings included both (CAF-macrophage) co-cultures and incubations of M0 and M1-macrophages in the presence of CAF-conditioned medium (CAF-CM). Functional assays to study macrophage polarization/activation included the expression of cell surface markers, production of nitric oxide, secretion of inflammatory cytokines and migratory capacity. We show that CAFs promote changes in M0-macrophages that harmonize with both M1-and M2-phenotypes. Additionally, CAFs inhibit pro-inflammatory features of M1-macrophages by reducing nitric oxide production, pro-inflammatory cytokines, migration, and M1-surface markers expression. Radiation delivered as single-high dose or in fractioned regimens did not modify the immunoregulatory features exerted by CAFs over macrophages in vitro. Protein expression analyses of CAF supernatants showed that irradiated and non-irradiated CAFs produce approximately the same protein levels of immunoregulators. Thus, CAF-derived soluble factors mediate measurable changes on uncommitted macrophages and down-regulate pro-inflammatory features of M1-polarized macrophages. Notably, ionizing radiation does not curtail the CAF-mediated immunosuppressive effects.

Radiation-Induced Transformation of Immunoregulatory Networks in the Tumor Stroma.

The implementation of novel cancer immunotherapies in the form of immune checkpoint blockers represents a major advancement in the treatment of cancer, and has renewed enthusiasm for identifying new ways to induce antitumor immune responses in patients. Despite the proven efficacy of neutralizing antibodies that target immune checkpoints in some refractory cancers, many patients do not experience therapeutic benefit, possibly owing to a lack of antitumor immune recognition, or to the presence of dominant immunosuppressive mechanisms in the tumor microenvironment (TME). Recent developments in this field have revealed that local radiotherapy (RT) can transform tumors into in situ vaccines, and may help to overcome some of the barriers to tumor-specific immune rejection. RT has the potential to ignite tumor immune recognition by generating immunogenic signals and releasing neoantigens, but the multiple immunosuppressive forces in the TME continue to represent important barriers to successful tumor rejection. In this article, we review the radiation-induced changes in the stromal compartments of tumors that could have an impact on tumor immune attack. Since different RT regimens are known to mediate strikingly different effects on the multifarious elements of the tumor stroma, special emphasis is given to different RT schedules, and the time after treatment at which the effects are measured. A better understanding of TME remodeling following specific RT regimens and the window of opportunity offered by RT will enable optimization of the design of novel treatment combinations.

Tissue analyses reveal a potential immune-adjuvant function of FAP-1 positive fibroblasts in non-small cell lung cancer.

OBJECTIVES: Selective targeting of cancer-associated fibroblasts (CAFs) has been proposed to synergize with immune-checkpoint inhibitors. While the roles of CAFs in cancer development are well described, their immune-regulatory properties remain incompletely understood. This study investigates correlations between CAF and immune-markers in tumor stroma from non-small cell lung cancer (NSCLC) patients, and examines whether a combination of CAF and immune cell scores impact patient prognosis. METHODS: Tumor specimens from 536 primary operable stage I-III NSCLC patients were organized in tissue microarrays. Expression of protein-markers was evaluated by immunohistochemistry. RESULTS: Fibroblast and stromal markers PDGFRα, PDGFRß, FAP-1 and vimentin showed weak correlations while αSMA, and Masson's trichrome did not correlate with any of the investigated markers. Hierarchical clustering indicated the existence of different CAF-subsets. No relevant correlations were found between any CAF-marker and the immune-markers CD3, CD4, CD8, CD20, CD68, CD1a, CD56, FoxP3 and CD45RO. High density of fibroblast-activation protein positive mesenchymal cells (CAFFAP) was associated with better prognosis in tumors with high infiltration of CD8 and CD3 T-lymphocytes. CONCLUSIONS: The presented data suggest that CAFs, irrespective of identity, have low influence on the degree of tumor infiltration by inflammatory- and/or immune-cells. However, CAFFAP may exert immuno-adjuvant roles in NSCLC, and targeting CAFs should be cautiously considered.

Ionizing radiation abrogates the pro-tumorigenic capacity of cancer-associated fibroblasts co-implanted in xenografts.

Cancer-associated fibroblasts (CAFs) are abundantly present in solid tumors and affect tumorigenesis and therapeutic responses. In the context of clinical radiotherapy, the impact of irradiated CAFs to treatment outcomes is largely unexplored. Aiming at improving radiotherapy efficacy, we have here explored the effect of radiation on the inherent pro-tumorigenic capacity of CAFs in animals. Ionizing radiation was delivered to cultured CAFs as single-high or fractionated doses. Tumor development was compared in mice receiving A549 lung tumor cells admixed with irradiated or control CAFs. Biological mechanisms behind tumor growth regulation were investigated by quantitative histology and immunohistochemistry. Viability assessments confirmed that irradiated CAFs are fully functional prior to implantation. However, the enhanced tumorigenic effect observed in tumors co-implanted with control CAFs was abrogated in tumors established with irradiated CAFs. Experiments to ascertain fate of implanted fibroblasts showed that exogenously administered CAFs reside at the implantation site for few days, suggesting that tumor growth regulation from admixed CAFs take place during initial tumor formation. Our work demonstrate that irradiated CAFs lose their pro-tumorigenic potential in vivo, affecting angiogenesis and tumor engraftment. This finding propose a previously unknown advantageous effect induced by radiotherapy, adding to the direct cytotoxic effects on transformed epithelial cells.

Cancer Associated Fibroblasts in Stage I-IIIA NSCLC: Prognostic Impact and Their Correlations with Tumor Molecular Markers.

BACKGROUND: Cancer Associated Fibroblasts (CAFs) are thought to regulate tumor growth and metastasis. Fibroblast Activating Protein 1 (FAP-1) is a marker for fibroblast activation and by many recognized as the main marker of CAFs. Alpha Smooth Muscle Actin (α-SMA) is a general myofibroblast marker, and can be used to identify CAFs. This study investigates the prognostic impact of FAP-1 and α-SMA in non-small cell lung cancer (NSCLC) patients and correlates their expression to 105 proteins investigated in the same cohort. METHODS: Tumor specimens from 536 NSCLC patients were obtained and tissue micro-arrays were constructed. Immunohistochemistry was used to evaluate the expression of FAP-1 and α-SMA and explore their impact on survival and association with other tumor molecular markers in NSCLC patients. RESULTS: High expression of FAP-1, but not α-SMA, in squamous cell carcinoma (SCC, P = 0.043, HR = 0.63 95% CI 0.40-0.99) was significantly associated with increased disease-specific survival. FAP-1 and α-SMA were not significantly correlated to each other. Analyses of FAP-1 and α-SMA associated with other tumor-related proteins revealed histotype-specific correlation patterns. CONCLUSION: The presence of FAP-1 expressing CAFs is an indicator of positive outcome for NSCLC-SCC patients. In addition, correlation analyses suggest FAP-1 and α-SMA to label different subsets of fibroblasts and their associations with other tumor-related proteins diverge according to histological subtype.

Cancer-associated fibroblasts from lung tumors maintain their immunosuppressive abilities after high-dose irradiation.

Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-ß were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.

Radiotherapy and the tumor stroma: the importance of dose and fractionation.

Ionizing radiation is a non-specific but highly effective way to kill malignant cells. However, tumor recurrence sustained by a minor fraction of surviving tumor cells is a commonplace phenomenon caused by the activation of both cancer cell intrinsic resistance mechanisms, and also extrinsic intermediaries of therapy resistance, represented by non-malignant cells and structural components of the tumor stroma. The improved accuracy offered by advanced radiotherapy (RT)-technology permits reduced volume of healthy tissue in the irradiated field, and has been triggering an increase in the prescription of high-dose oligo-fractionated regimens in the clinics. Given the remarkable clinical success of high-dose RT and the current therapeutic shift occurring in the field, in this review we revise the existing knowledge on the effects that different radiation regimens exert on the different compartments of the tumor microenvironment, and highlight the importance of anti-tumor immunity and other tumor cell extrinsic mechanisms influencing therapeutic responses to high-dose radiation.

Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth.

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1ß, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced.

BACKGROUND: Cancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers. METHODS: CAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining. RESULTS: Exposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, ß1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed. CONCLUSIONS: Our data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.