No influence of irradiation of recipient cell on the effect of conditioned medium (CM).

<p>Inhibition of colony formation by 15 Gy irradiation CM was similar on unirradiated (a) and 4 Gy irradiated (b) recipient MCF7 cells. The different CM production conditions are shown on the abcissa. The different media showed essentially the same effect on unirradiated and irradiated recipients and were in line with previous experiments (with the exception of the 15 Gy IM (irradiated medium); see text for explanation). In addition, the volume of CM during production does not seem to alter the magnitude of the BE. Mean±standard errors (n = 3) are shown.</p

The Biological Effect of Large Single Doses: A Possible Role for Non-Targeted Effects in Cell Inactivation

<div><p>Background and Purpose</p><p>Novel radiotherapy techniques increasingly use very large dose fractions. It has been argued that the biological effect of large dose fractions may differ from that of conventional fraction sizes. The purpose was to study the biological effect of large single doses.</p><p>Material and Methods</p><p>Clonogenic cell survival of MCF7 and MDA-MB-231 cells was determined after direct X-ray irradiation, irradiation of feeder cells, or transfer of conditioned medium (CM). Cell-cycle distributions and the apoptotic sub-G1 fraction were measured by flow cytometry. Cytokines in CM were quantified by a cytokine antibody array. γH2AX foci were detected by immunofluorescence microscopy.</p><p>Results</p><p>The surviving fraction of MCF7 cells irradiated in vitro with 12 Gy showed an 8.5-fold decrease (95% c.i.: 4.4–16.3; P<0.0001) when the density of irradiated cells was increased from 10 to 50×10³ cells per flask. Part of this effect was due to a dose-dependent transferrable factor as shown in CM experiments in the dose range 5–15 Gy. While no effect on apoptosis and cell cycle distribution was observed, and no differentially expressed cytokine could be identified, the transferable factor induced prolonged expression of γH2AX DNA repair foci at 1–12 h.</p><p>Conclusions</p><p>A dose-dependent non-targeted effect on clonogenic cell survival was found in the dose range 5–15 Gy. The dependence of SF on cell numbers at high doses would represent a “cohort effect” in vivo. These results support the hypothesis that non-targeted effects may contribute to the efficacy of very large dose fractions in radiotherapy.</p></div

Effect of cell density on cell survival after high-dose irradiation.

<p>(a) Influence of the number of seeded cells on the shape of the survival curve of MCF7 cells in the colony formation assay. A significant decrease (P<0.0001) in surviving fraction (SF) after 12 Gy was found when seeding 50,000 cells/T25 flask (open symbol) compared with 10,000 cells/T25 flask. This resulted in an increased downward curvature of the survival curve (n = 7). (b) SF after 12 Gy (SF12) for different numbers of seeded cells (MCF7: 5–50×10³/T25 flask) confirmed a significant inverse correlation (P<0.0001) with cell numbers (n = 5). (c) Feeder cell experiment to test the effect of high-dose (20 Gy) irradiated cell numbers on colony formation of unirradiated cells (MCF7). The experimental design is shown above the data diagram. A significant inverse correlation (P<0.0001) between the number of irradiated feeder cells per T25 flask and the normalized plating efficiency of unirradiated MCF7 cells was observed (n = 3). Mean±standard errors are shown.</p

Role of cytokines in intercellular signalling.

<p>(a) 3–10 ng/ml TGF-β1 had no effect on proliferation of MCF7 in mass culture (3 days incubation of proliferation test with vital dye alamarBlue). Inhibition of Mv1Lu cells confirmed the activity of TGF-β1. (b) No effect of 3–10 ng/ml TGF-β1 on colony formation of MCF7 cells. (c) Detection of cytokines expressed in CM from 15 Gy irradiated and unirradiated MCF7 cultures. No significant difference was observed (P = 0.46–0.95). The presence of the CM-mediated inhibitory effect on colony formation was verified in each experiment (not shown). Mean±standard errors (n = 3) are shown. None of the complete set of 36 cytokines tested showed a significant difference (P = 0.18–0.96). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084991#pone.0084991.s001" target="_blank">Figure S1</a>.</p

Conditioned medium (CM) induces prolonged γ-H2AX foci in MCF7 cells.

<p>Recipient cells were incubated with conditioned medium (CM) from 15 Gy or sham irradiated MCF7 cells, or with 15 Gy irradiated or sham irradiated medium (IM). Treatment with 15 Gy irradiated CM produced rapid induction of γH2AX foci which stayed elevated up to 6–12 h. The mean number of foci per cell receiving CM from 15 Gy irradiated cells was significantly higher than in cells receiving unirradiated CM, 15 Gy irradiated or unirradiated non-conditioned medium (IM; P<0.0001 for time = 1–12 h) Mean±standard errors (n = 3) are shown.</p

No effect of conditioned medium (CM) on cell cycle distribution and apoptosis.

<p>Flow cytometry was performed at different time points after incubation of recipient MCF7 (a) or MDA-MB-231 (b) cells with 15 Gy CM. No effect on the cell cycle distribution or apoptosis rate (sub-G1) was observed at any time point 6–48 h. Mean±standard errors (n = 3) are shown.</p

Medium transfer experiments to test effect of transferrable factor.

<p>The experimental design is show above the diagrams. The effect of incubation with conditioned medium (CM) from irradiated cells on the clonogenic growth of unirradiated recipient cells for MCF7 (a) and MDA-MB-231(b). A dose-dependent decrease was found for MCF7 (P = 0.0001) and MDA-MB-231 (P<0.0001) compared to the CM from unirradiated cells. 15 Gy IM (non-CM): medium irradiated without cells. Mean±standard errors (n = 4) are shown.</p

Additional file 3: of Biology of high single doses of IORT: RBE, 5 R’s, and other biological aspects

DSB and SLD repair data. (XLSX 26 kb

Additional file 4: of Biology of high single doses of IORT: RBE, 5 R’s, and other biological aspects

RBE and gH2AX data. (XLSX 56 kb

TGF-β1, -β2 and -β3 reduce lymphatic marker expression in LECs.

<p>Primary human LECs were treated with 10, 20 or 30 ng/ml TGF-β1, -β2 and -β3 for 72 hours (<b>a</b>) or 100 hours (<b>b</b>). Untreated cells served as a control. Lysates were prepared and analysed by Western blot using antibodies specific for Lyve-1, Prox-1, VEGFR-3 or vimentin. Vinculin served as loading control. The experiment was performed twice with equivalent results. For densitometry evaluation, protein bands were analysed using the software ImageJ. Bands for the Prox-1, Lyve-1, vimentin and VEGFR-3 proteins were normalized to the corresponding loading control and are displayed as the expression level relative to the untreated control samples.</p