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

Abstract

BackgroundCancer-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.MethodsCAFs 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.ResultsExposing 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, β1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.ConclusionsOur 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.

Tumorigenic Responses of Cancer-Associated Stromal Fibroblasts after Ablative Radiotherapy: A Transcriptome-Profiling Study

“…Despite the available knowledge on normal tissue fibroblast responses to RT, few reports have focused on the effects of RT on intratumoral reactive stromal fibroblast (CAFs). In a recent study from our laboratory we show that CAF survive to relative high single radiation doses, however the cellular phenotype becomes profoundly altered, characterized by the induction of permanent DNA damage responses and the acquisition of a senescent phenoltype and growth arrest [14]. Of note, in that study we show that the expression of some matrix metalloproteinases becomes altered along with overexpression of cell surface integrins.…”

“…All the data generated in this study come from cell cultures directly prepared from lung tumor specimens obtained from 8 different donors after surgical resection (Figure 1). In a previous study from our group we describe in detail the isolation and characterization of the tumor fibroblast cultures that have been used also for this study [14]. Of note, flow cytometry analyses using the fibroblast specific marker α-SMA reveal a purity above 99.5%, which gives full credibility to the data generated with this material.…”

“…Radiation-doses were confirmed to be correct within an acceptable ± 4% by Thermo-Luminescent Dosimeters (TLDs) [15]. Cell survival/death after radiation was assessed by light microscopy during the initial three weeks, by automated cell index analyses (xCelligence, Roche) and by MTT cell proliferation and viability assays [16] at different time points [14]. The extent of radiation-induced apoptosis and necrosis in CAFs was determined by staining for annexin V and propidium iodide respectively (data not shown).…”

Cyclooxygenase-2 blockade can improve efficacy of VEGF-targeting drugs

“…The cell lines were not authenticated by the authors as cell authentication testing can be conducted only on human cell lines. Primary CAFs were isolated from tumor tissue of n=2 lung cancer patients as described [70] (kindly provided by Iñigo Martinez-Zubiaurre, Arctic University of Norway, Tromsø, Norway) and cultured in DMEM. The embryonic fibroblast cell line MRC-5 was cultured in MEM supplemented with 1% sodium pyruvate.…”

Exploring the rationale for combining ionizing radiation and immune checkpoint blockade in head and neck cancer

“…50,51 Some groups have demonstrated that CAFs seem to be highly resistant to the cytotoxic effects of ionizing radiation, even at high doses. 52,53 However, Grinde et al 54 demonstrated that greater engraftment kinetics when CAFs were mixed with tumor cells before transplantation were abrogated when the CAFs were irradiated before the mixture. This effect was the same between 18 Gyx1 and 6 Gyx3 schemas.…”