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. 2014 Jun;20(2):359-65.
doi: 10.1016/j.intimp.2014.03.016. Epub 2014 Apr 5.

Ceramide and sphingosine-1-phosphate act as photodynamic therapy-elicited damage-associated molecular patterns: cell surface exposure

Mladen Korbelik  1 , Judit Banáth  2 , Jinghai Sun  2 , Daniel Canals  3 , Yusuf A Hannun  3 , Duska Separovic  4
Affiliations

Ceramide and sphingosine-1-phosphate act as photodynamic therapy-elicited damage-associated molecular patterns: cell surface exposure

Mladen Korbelik et al. Int Immunopharmacol. 2014 Jun.

Abstract

Molecules that appear on the surface of tumor cells after their therapy treatment may have important roles either as damage-associated molecular patterns (DAMPs) or signals for phagocytes influencing the disposal of these cells. Treatment of SCCVII and CAL27 cells, models of mouse and human squamous cell carcinoma respectively, by photodynamic therapy (PDT) resulted in the presentation of ceramide and sphingosine-1-phosphate (S1P) on the cell surface. This was documented by anti-ceramide and anti-S1P antibody staining followed by flow cytometry. The exposure of these key sphingolipid molecules on PDT-treated tumor cells was PDT dose-dependent and it varied in intensity with different photosensitizers used for PDT. The above results, together with the finding that both ceramide and S1P can activate NFκB signaling in macrophages co-incubated with PDT-treated tumor cells, establish that these two sphingolipids can act as DAMPs stimulating inflammatory/immune reactions critical for tumor therapy response.

Keywords: Ceramide; Damage-associated molecular patterns; Photodynamic therapy; Sphingosine-1-phosphate; Squamous cell carcinoma.

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Conflict of interest statement

Competing interests: The authors declare no existence of competing interests.

Figures

Figure 1
Figure 1. Cell surface expression of calreticulin, ceramide, and S1P on SCCVII cells treated by mitoxantrone or Photofrin-PDT
After mitoxantrone treatment (1 μg/ml for 16 hours) or PDT treatment (Photofrin 20 μg/ml for 18 hours followed by 1 J/cm2 of 630±10 nm light), cells were left in culture for 30 minutes before they were collected for 7-AAD and surface antibody staining. For the latter, cells were either directly stained with PE-conjugated anti-calreticulin or isotype control rabbit polyclonal antibody, while in other cases anti-ceramide, anti-S1P and or isotype control IgM exposure was followed by PE-conjugated secondary antibody. The results of flow cytometry analysis are presented for 7-AAD negative cells and shown as fluorescence (in arbitrary units per cell) directly corresponding to the levels of calreticulin, ceramide or S1P on the surface of cells. The bars denote SD, n = 4; * = statistically significant difference from untreated group levels.
Figure 2
Figure 2. Ceramide and S1P levels in SCCVII cells treated by Photofrin-PDT determined by surface and intracellular antibody staining
Shown are changes in ceramide (a) and S1P levels (b) after light alone, photosensitizer alone, or PDT treatment followed by a post-incubation of 15 min (ceramide) or either 15 min or 3 hours (S1P). Cells were treated with Photofrin-PDT as described for Fig. 1, with the 1 J/cm2 and 20 μg/ml doses used also for the light alone and Photofrin alone treatments. Ceramide and S1P levels determination was either as described for Fig. 1 (surface staining) or with fixed and permeabilized cells using the same antibody (intracellular staining). The results are presented either as relative values compared to those obtained with corresponding untreated control samples (a) or the extent of S1P-associated fluorescence per cell (b). The bars denote SD, n = 4; * = statistically significant difference from corresponding untreated group levels, ** = statistically significant difference from values obtained with corresponding light only and Photofrin only groups.
Figure 3
Figure 3. Ceramide levels in SCCVII and CAL-27 cells treated by Photofrin-PDT with dependence on PDT dose or time interval after treatment
To determine the changes in ceramide levels dependent on PDT-dose (a), cells were treated with Photofrin-PDT as described for Fig. 1, except for additional light doses (0.5 and 1.5 J/cm2). For post-treatment time-dependence analysis (b), cells were after PDT light treatment left in the 37°C incubator for either 15 min, 3 hours, or 18 hours before collected for antibody staining and flow cytometry. Using CAL-27 instead of SCCVII cells (c) served to demonstrate the PDT-induced surface ceramide and S1P induction in a different cell line. The post-incubation time interval used with these cells was 3 hours. Ceramide and S1P levels determination was as described for Figs. 1 and 2. The bars denote SD, n = 4; * = statistically significant difference from corresponding untreated group levels, ** = statistically significant difference from 15-min group.
Figure 4
Figure 4. The effect of sphingomyelinase and ceramidase treatment on cell surface ceramide expression following PDT
SCCVII cells were treated by Photofrin-PDT as described for Fig. 1. For Temoporfin-PDT, photosensitizer exposure (0.2 μg/ml for 18 hours) was followed by 1 J/cm2 light dose. After PDT, cells were then kept in culture for 15 minutes (Photofrin-PDT) or 18 hours (Temoporfin-PDT). During the last 15 minutes of this post-incubation, the cells were exposed to either sphingomyelinase (a) or ceramidase (b), both at 250 mU/ml, before their collection. Subsequent cell surface ceramide staining and flow cytometry analysis were as described for Fig. 1. The results are presented as surface ceramide-associated fluorescence intensity per cell. The bars denote SD, n = 4; * = statistically significant difference from untreated group levels, ** = statistically significant difference from corresponding sphingomyelinase/ceramidase untreated group levels.
Figure 5
Figure 5. Cell surface ceramide levels induced by PDT mediated by different photosensitizers
SCCVII cells were treated either Photofrin-PDT as described for Fig. 1 or by PDT mediated by Temoporfin (0.2 μg/ml – 24 hrs), chlorin e6 (1.5 μg/ml – 30 min), or Pc4 (1 μg/ml – 18 hrs), with light dose 1 J/cm2 in all cases. These PDT treatments produced a similar level of cell killing, as determined by the colony formation assay (insert; ● = Photofrin-PDT, ▲ = Temoporfin-PDT, ■ = Pc4-PDT, ◆ = ce6-PDT). For surface ceramide analysis, cells were in all cases collected after 30 min post-PDT incubation for antibody staining as described for Fig. 1. The results are presented as means of surface ceramide-associated fluorescence intensity per cell. The bars denote SD, n = 4; * = statistically significant difference from untreated group levels.
Figure 6
Figure 6. Activation of NFκB in TAMs induced by co-incubation with PDT-treated SCCVII cells is blocked by ceramide- or S1P-neutralizing antibodies
Cultures of TAMs obtained from SCCVII tumors were incubated alone or co-incubated with cultured SCCVII cells treated by PDT as described for Fig. 1. Some samples contained anti-ceramide or anti-S1P antibody (20 μg/ml) in the co-incubation medium. After 3 hours, TAMs were collected and their NFκB activity measured using PathScan® Phospho-NF-κB p65 (Ser536) ELISA. The results are shown as colorimetric readouts (absorbance at 450 nm) reflecting levels of phospho-NFκB p56 protein at Ser536. The bars denote SD; * = statistically significant difference from the value in TAMs incubated alone, ** = statistically significant difference from the value in TAMs co-incubated with PDT-treated SCCVII cells in the absence of corresponding antibody.
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