This study was undertaken to shed light on the biological responses of cells from the stroma of lung tumours exposed to high radiation doses, proven to be successful in the treatment of medically inoperable NSCLCs. We show that ablative radiation doses exert therapeutically beneficial inhibitory effects on the proliferative, migratory and invasive capacity of CAFs, effects which are associated with increased focal adhesions, cell surface expression of integrins and modulation of MMP secretion.
Our initial experiments showed that radiation had a donor-independent inhibitory effect on the proliferative capacity of CAFs that was likely to be a consequence of radiation-induced senescence in a high proportion of cells. Ionizing radiation is well known to induce the same phenotype as replicative senescence, and is often referred to as stress-induced premature senescence (SIPS) . In fibroblasts  and mesenchymal stem cells  a senescent phenotype typically develops over several days after exposure to IR. Such radiation-induced senescent fibroblasts have been postulated to retain metabolic function and to display tumour promoting effects through paracrine secretion of pro-inflammatory signals . Furthermore, persistent DNA damage signalling has been linked to the establishment of a an irreversible senescent phenotype [13, 25, 26], and is also suggested as an indicator of lethal DNA damage . On these premises we aimed to characterize the senescent phenotype of human CAFs and measure the kinetics of DNA damage foci several days post-irradiation. In our study, CAFs showed a progressive increase of β-galactosidase staining indicative of senescence, up to 5 days after AIR. Concomitantly, nuclear foci containing DNA-damage response elements were robustly activated by AIR and lasted several days, thus supporting the notion that a significant proportion of lung CAFs enter permanent senescence after exposure to a tumour ablative radiation dose.
Stromal fibroblasts are considered to be the master regulators of matrix remodelling [5, 6]. We explored the influence of AIR on secretion by CAFs of key matrix regulators including several MMPs and their endogenous inhibitors; TIMPs. Our data show that only MMP-1 (collagenase), MMP-2 (gelatinase-A) and MMP-3 (stromelysin-1) are substantially secreted by lung CAFs. On the contrary, MMP-7, MMP-8, MMP-9 and MMP-13 were undetectable within the limits of the assay. We have observed by various means that MMP-9 is not significantly expressed by neither irradiated nor control lung CAFs, contributing to the notion that MMP-9 is primarily expressed by tumour-infiltrating inflammatory cells, rather than by CAFs .
A clear variation in expression among donors could be observed for MMP-1 and MMP-3 at both early and late time points after radiation. It remains to be explored if inter-individual variation in the inherent expression of these MMPs by CAFs has any impact on the overall tumour response to radiation among patients. Radiation was associated with changes in expression of MMP-1 and MMP-3, when examined 4 to 6 days after treatment. Secreted MMP-1 protein-levels were significantly reduced in 4 out of 5 cases, whereas MMP-3 levels were enhanced in irradiated CAFs from all donors included in the experiments. Patients with tumours expressing MMP-1 at the primary site are reported to have a significantly worse prognosis than MMP-1 negative patients . The AIR-mediated reduction of MMP-1 expression could, in part, explain the repressed invasiveness of CAFs. To rule out this hypothesis we tested the invasive capacity of CAFs in the presence of an MMP inhibitor, GM1489  (Additional file 1: figure S1). Our data show that at a concentration of 1 nM of GM1489, an amount that would inhibit over 90% of MMP-1 activity and less than 1% of other MMPs, the rate of invasion is reduced only 18%. These results indicate that MMP-1 only play a modest role for CAF invasion, at least as observed in our in vitro assay, and its reduced expression may not account for the reduced invasion observed.
On the contrary, enhanced levels of MMP-3 might represent a negative impact of AIR-based therapy, since secreted MMP-3 has been reported to correlate with tumorigenicity and invasiveness . However, recent studies indicate that MMP-3, as well as other MMPs, may also have tumour-suppressive effects . When considering the catalytic activity and quantities of MMPs, it must be taken into account that these proteases are highly regulated at multiple levels, including transcription, secretion, activation of the inactive pro-enzymes, and finally, the counterbalancing effect mediated by TIMPs . We also found that TIMP-1, TIMP-2 and to a lesser extent TIMP-3 are actively secreted by lung CAFs. However, radiation exposure did not mediate consistent stimulatory or inhibitory effects on the TIMP levels.
CAF motility is a fundamental function supporting tumour growth, invasiveness and angiogenesis. Cell adhesion to ECM and locomotion is mediated by cell surface receptors called integrins, whose ECM ligand specificity is determined by combinations of α and β integrin subunits [37, 38]. Integrins expressed on stromal fibroblasts contribute significantly in the regulation of tumour development and metastasis by affecting the migratory capacity of fibroblasts, by regulating cell proliferation and survival, and by modifying growth factor signalling . In our study, we show a dramatic redistribution of focal contacts upon AIR that appears concomitantly with the acquirement of the senescent phenotype. On the other hand, flow cytometric analysis clearly demonstrated a radiation-induced increase in surface expression of the integrin subunits examined (α2, β1, α5). Expression levels in whole cell lysates further revealed that the total protein pool of integrin α2 was unaffected after exposure to 18 Gy, suggesting that the surface accumulation was likely to be a consequence of reduced internalization and/or enhanced recycling of integrins from the intracellular pool . Interestingly, a similar enhancement (2-fold) of β1-integrin surface levels was recently demonstrated in cancer cells with defective endocytic machinery .
Overall, our findings support the view that stabilization of focal contacts (via integrins) increases attachment and impairs migration of CAFs . Radiation-induced enhancement of cell adhesion has also been demonstrated by Cordes and co-workers in various cell types, and was reflected by increased cell-surface expression of β1-integrin . In fact, augmentation of cell surface expression of integrins, in particular β1-integrin, has been postulated as a cellular mechanism to potentiate anchorage-dependent pro-survival anti-apoptotic pathways through binding to ECM components . Furthermore, inhibition of β1-integrin reportedly mediate enhanced radioresponse, and has been suggested as a therapeutic target .