Most tumours are characterized by an inflammatory microenvironment with migration of leukocytes and the release of cytokines and other inflammatory markers [
The distinct correlations between inflammation and cancer progression are known. An increased presence of inflammatory cells and soluble inflammatory markers in a primary tumour is associated with a poor prognosis, e.g., due to metastasis [
Endothelial cells (ECs) as part of the inflammatory tumour microenvironment play a critical role in the inflammatory processes. ECs express a variety of cytokines and growth factors, and they are able to recruit leukocytes from peripheral blood [
About half of the tumour patients receive radiation treatment during their therapy. Owing to their capability to regulate tumour cell functions like proliferation, invasiveness, and response to and elaboration of inflammatory mediators as well as tumour outgrowth, angiogenesis, and metastasis, ECs may be critical targets of response to tumour therapy like irradiation (IR). Recent studies have clearly demonstrated that IR affects ECs not only after high doses but also after low doses of radiation exposure. ECs are considered important regulators of tumour response after high radiation doses [
In a publication by Rombouts et al., it was demonstrated that acute low doses of X-rays induce DNA damage and apoptosis in endothelial cells after exposure to 0.1, 0.5, and 5 Gy (X-ray) [
It is known that irradiation of the endothelium and its related proinflammatory signalling cascades result in micro- and macrovascular effects [
Moreover, IR with low doses affects EC by modulating inflammatory reactions [
The experiments were performed with human adenocarcinoma epithelial cells A549 and EA.hy926 cells. The EA.hy926 cells, derived from the fusion of human umbilical vein endothelial cells (HUVECs) with a thioguanine-resistant clone of A549, were used as the permanent human endothelial cell line. Both cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). EA.hy926 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Cologne, Germany) supplemented with 10% fetal bovine serum (FBS, Merck Millipore, Darmstadt, Germany), 2% sodium pyruvate, 100 U/mL penicillin, and 100
The cells were supplemented with TNF-
Cells were irradiated 26 hours after seeding at room temperature utilizing a Siemens ONCOR Expression linear accelerator (Siemens, Erlangen, Germany) with a dose rate used in clinical applications of 3.75 Gy/min (photons 6 MV). The irradiation setup ensures a homogeneous dose distribution within the whole cell culture flasks. Cells were transported in their respective cell culture dishes in a sterile box to the linear accelerator and were placed on the disinfected irradiation table and irradiated directly from below. The irradiation was carried out with the following doses: 0.1 Gy, 0.5 Gy, 2 Gy, 4 Gy, and 6 Gy. Sham-irradiated samples (0 Gy) were kept at room temperature in the control room during irradiation.
The water-soluble tetrazolium 1 (WST-1) assay (Roche Deutschland GmbH, Mannheim, Germany) was used to detect the metabolic activity of the cells 2 hours, 24 hours, and 48 hours after IR. Therefore, cells were seeded in a density of 1,000 cells/well in triplicates in 96-well plates, grown under standard conditions, and activated with TNF-
Cells were seeded in a density of
Cells were seeded in a density of
Accumulated levels of IL-8, MCP-1, E-selectin, P-selectin, sICAM-1, VCAM-1, and VEGF were quantified in supernatants harvested from EC by using a Luminex® Magnetic Screening Assay from R&D Systems (Minneapolis, MN, USA) according to the manufacturer’s protocol. Data were acquired using the Bio-Plex® 200 suspension array system and analysed with the Bio-Plex Manager™ Software (version 4.1).
EA.hy926 cells were seeded in duplicates in 6-well plates 26 hours before IR to reach a confluent cell monolayer. A549 tumour cells were then stained with CellTrace™ CFSE (cat. no. C34554, Molecular Probes, Thermo Fisher Scientific, Darmstadt, Germany) in a concentration of 1.2 mM for 10 minutes, washed twice with PBS, and seeded in a T75 flask with a density of
The medium was replaced with serum-free medium 2 hours before IR, and EA.hy926 cells were stimulated with TNF-
Tumour cells were counted 24 hours after IR, and
The ability of A549 cells to migrate into a cell-free area after IR and/or TNF-
All data are presented as
The influence of IR on metabolic activity was investigated to exclude an alteration of metabolic activity as a reason of the changes in subsequent analyses. The cellular metabolic activity was measured in EA.hy926 and A459 cells 2 hours, 24 hours, and 48 hours after different doses of IR. The metabolic activity slightly increased in EC EA.hy926 over time but remained unaffected by radiation treatment or activation of the cells with TNF-
The influence of irradiation on cytokine secretion by EC, with and without simulating an inflammatory environment (±TNF-
Interleukin-8 is a cytokine with proangiogenetic and antiapoptotic effects on a variety of cells like monocytes or ECs and promotes the migration of ECs into the ECM. The accumulation of IL-8, derived from nonactivated EA.hy926 cells, was observed in a time-dependent manner from 2 hours to 72 hours after IR with a maximum concentration of approximately 100 pg/mL IL-8 at the latest time point (Figure
Accumulated levels of (a) interleukin-8 (IL-8) and (b) monocyte chemoattractant protein-1 (MCP-1) in the supernatant of EA.hy926. The protein concentration was determined by multiplex assay at five time points after irradiation with photons. Changes in protein concentrations are presented as
IL-8
MCP-1
MCP-1, which induces a chemotactic activity in several cell types, can promote the infiltration of monocytes and macrophages into various tumours as well as enhance the proliferation and promote the migration and infiltration of tumour cells. The accumulation of MCP-1 derived from EA.hy926 without TNF-
The concentrations ranged from approximately 2,064 pg/mL to 18,973 pg/mL. The level of accumulated MCP-1 in the supernatant remained similar 72 hours after IR compared to samples at 48 hours. Dose-dependent decreases in MCP-1 were detected 48 hours after IR at all tested doses compared to the 0 Gy sample at the respective time points, but only for doses of 0.1 Gy, 0.5 Gy, 2 Gy, and 4 Gy, the decrease was statistically significant.
Selectins not only mediate the rolling of leukocytes on the surface of endothelial cells but are also involved in tumour metastasis. The accumulated levels of E- and P-selectins did not change over the examined time period of 2 hours to 72 hours after IR (See supplement Fig.
ICAM-1 is a cell surface glycoprotein typically expressed on ECs and plays a key role during inflammation and immune responses as well as transendothelial migration. The accumulation of sICAM-1 derived from nonactivated EA.hy926 remained at the same level over a time period of 2 hours to 72 hours after IR with concentrations ranging from 18,982 pg/mL to 21,768 pg/mL (Figure
Accumulated levels of (a) soluble intercellular adhesion molecule-1 (sICAM-1) and (b) vascular cell adhesion molecule-1 (VCAM-1) in the supernatant of EA.hy926. The protein concentration was determined by multiplex assay at five time points after irradiation with photons. Changes in protein concentrations are presented as
sICAM-1
VCAM-1
VCAM-1 not only mediates the adhesion of a variety of cells to the endothelium but also takes part in the regulation of immune surveillance and inflammation, besides other adhesion molecules. The accumulation of sVCAM-1 derived from unstimulated ECs on an average ranged from 6,332 pg/mL to 6,933 pg/mL (Figure
VEGF is a signalling molecule playing a pivotal role in vasculogenesis and angiogenesis with stimulatory properties on EC division and migration. The concentration of accumulated VEGF in the supernatant of irradiated ECs increased time-dependently from 2 hours to 72 hours after IR (see supplement Fig.
To quantify the influence of IR on the adhesive capacity of tumour cells, the adhesion of A549 to EA.hy926 was measured under different conditions (Figure
Adhesion of A549 to EA.hy926 (a) without TNF-
Without TNF-
With TNF-
When only EA.hy926 cells were irradiated and not activated before IR, the adhesion of A549 to ECs was altered with a significantly lower adhesion after IR with 0.5 Gy. For higher IR doses up to 2 Gy to 6 Gy, adhesion rose significantly and dose-dependently compared to the control (Figure
The simultaneous IR exposure of A549 and EA.hy926 cells caused a dose-dependent higher adhesion of tumour cells to ECs, which was significant at doses of 2 Gy to 6 Gy (Figure
To investigate the effect of IR and inflammation on migration, A549 cells were treated with TNF-
Analysis of tumour cell migration by
ECs, as key players in “cancer-related inflammation,” regulate diverse aspects of cancer cell functions. Besides the secretion of inflammatory markers, ECs also have a crucial role in angiogenesis and may play a crucial role in the radiation response of the actual tumour. The objective of this study was to investigate the effect of IR on ECs under inflammatory conditions and their interactions with tumour cells in more detail. Therefore, nonactivated and activated human EC EA.hy926 were irradiated with doses between 0.1 Gy and 6 Gy with a linear accelerator and secretion of cytokines, adhesion of A549 tumour cells to the EC and the cell motility of tumour cells were characterized.
Owing to the results derived from the analyses of metabolic activities, an alteration in metabolic activity as a cause of the observed changes in subsequent analyses—secretion of cytokines, adhesion of tumour cells to ECs, and cell motility of tumour cells—could be excluded. It was demonstrated that IR up to single doses of 6 Gy and until 48 hours after IR did not affect the metabolic activity significantly. Moreover, no significant changes were detected in an inflammatory environment after activation with TNF-
One main goal of this study was to analyse the cytokine secretion of the EC activated with TNF-
Interleukin-8 is a cytokine with a proangiogenetic and antiapoptotic effect on a variety of cells like monocytes or ECs and promotes the migration of ECs into the extracellular matrix. The angiogenic property of IL-8 and other CXC chemokines was investigated for many years [
In experiments published by Van der Meeren et al., HUVECs secreted IL-8 time-dependently over 6 days, which is in accordance with our findings measured over 3 days as well as the higher secretion, if cells were treated with TNF-
High IL-8 concentrations are known to predominate in human malignant melanomas [
Monocyte chemoattractant protein-1 (MCP-1) can promote the infiltration of monocytes and macrophages into various tumours as well as enhance the proliferation and promote the migration and infiltration of tumour cells. High concentrations of MCP-1 could also be associated with an increased incidence of metastases in breast, colon, prostate, and neck tumours [
An increase in the proinflammatory marker, MCP-1, by HUVECs after stimulation with a higher concentration of TNF-
It has also been stated that microvascular endothelial cells from human lungs (HLEC) can secrete chemokines like MCP-1 or IL-8 generally and in higher concentrations after TNF-
Well known as mediators of cell-cell and cell-matrix interactions and essential for a variety of processes including tumour development, invasion, and metastasis are adhesion molecules. Several studies focused on the effect of IR on different members of adhesion molecules, like immunoglobulins and selectins; and their modulation by IR seems to play a role in radiation-induced tumour response, tumour inflammation, and metastasis and angiogenesis [
Whereas no altered concentrations of E- and P-selectins could be observed, both cell adhesion molecules (CAMs) were released in the supernatant in higher concentrations after stimulation with TNF-
IR-induced expressions of leukocyte adhesion molecules like ICAM and VCAM by ECs were also described in other studies [
Our experiments indicate that the exposure to various doses of IR did not alter the secretion of E- and P-selectins. A time-dependent increase or decrease of these adhesion molecules could not also be detected. The activation of EC with 10 ng/mL TNF-
Another signalling molecule playing a pivotal role in blood vessel formation and angiogenesis with stimulatory property on ECs is the vascular endothelial growth factor (VEGF).
Critical steps involved in haematogenous metastasis of tumour cells are the adhesion to the endothelium, the extravasation through the endothelial layer, and the invasion into the extracellular matrix. In the first two steps, tumour cells interact with endothelial cells in order to invade through the matrix. Therefore, the influence of IR under inflammatory conditions on adhesion of tumour cells to endothelial cells as well as the tumour cell migration was the purpose of our further investigation.
The influence of IR on the adhesion of A549 tumour cells to EA.hy926 cells was measured under different conditions. IR of only A549 cells led to a significant, dose-dependent reduced adhesion of the tumour cells to ECs after 0.5 Gy to 6 Gy. This was in contrast to the effect described by Kiani et al., where A549 cell adhesion to ECs increased after IR. But in their study, HUVECs were used as ECs [
In our study, the tumour cell adhesion to ECs was altered only when the EA.hy926 cells were irradiated, with reduced adhesion after low doses of IR (0.1 Gy and 0.5 Gy) and rose dose-dependently for higher IR doses (2 Gy to 6 Gy). It is well known that high doses of IR result in proinflammatory reactions. But low doses can also cause anti-inflammatory reactions [
In the present study, when cells were additionally stimulated with TNF-
Besides the adhesion to the endothelium, further critical steps involved in haematogenous metastasis of tumour cells are the extravasation through the endothelial layer and the invasion into the extracellular matrix. Against this background, the effect of IR and inflammation on A549 tumour cell migration was investigated. Tumour cells responded to IR with a significant dose-dependent reduced migration 24 hours and 48 hours after IR. Interestingly, if A549 cells were in an inflammatory environment, the migration was significantly enhanced at low IR doses but reduced at higher doses. As described above for the tumour cell adhesion, for tumour cell migration, too, proinflammatory reactions at high doses of IR and anti-inflammatory response at low doses of IR could be demonstrated.
In addition, only tumour cells that were activated with TNF-
Endothelial cells (EC), as part of the tumour microenvironment, play a critical role in the inflammatory processes of “cancer-related inflammation.” They express a variety of cytokines and growth factors and are essential for blood vessel functions. This importance suggests that ECs are critical targets of response to irradiation during tumour therapy. In our study, we demonstrated that IR of ECs results in the modified release of chemokines (IL-8, MCP-1) as well as adhesion molecules (sICAM-1, sVCAM-1) in the human EC. The adhesion capability of A549 tumour cells to ECs was also affected by IR; the nature of the effect was dependent on the IR-treated cell type. An inflammatory milieu of TNF-
Cell adhesion molecules
Dulbecco’s modified Eagle’s medium
Endothelial cells
Extracellular matrix
Epithelial-mesenchymal transition
Gray
Human microvascular endothelial cells
Human umbilical vein endothelial cells
Interleukin
Irradiation
Keratinocyte-derived chemokine
Monocyte chemoattractant protein-1
Nuclear factor kappa B
Peripheral blood mononuclear cells
Phosphate-buffered saline
Tumour necrosis factor alpha
Vascular endothelial growth factor
Water-soluble tetrazolium 1.
The data supporting this study are provided in Results or as supplementary information accompanying this paper. Further datasets used and/or analysed during the current study are available and are stored by the authors at the University Medical Center Rostock.
The authors declare that there are no conflicts of interest.
The research was performed as part of the employment of the authors by University Medical Center Rostock, Germany.
Supplement Figure 1: influence of low-dose irradiation on metabolic activity of (A) EA.hy926 and (B) A549. The cells were irradiated with different doses; a WST-1 assay was performed at three time points after irradiation. The extinctions were normalized to samples of 0 Gy/2 hours. Error bars present the standard deviation (±SD) from four independent experiments; wells were assayed in triplicates in each of the experiments. Supplement Figure 2: influence of irradiation on cellular vitality of EA.hy926 cells. The cells were irradiated with different doses; the number of live and dead cells was counted at four time points after irradiation. Error bars present the standard deviation (±SD) from four independent experiments; wells were assayed in duplicates in each of the experiments; asterisks illustrate significance: