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. 2012 Feb;61(2):215-221.
doi: 10.1007/s00262-011-1184-2. Epub 2011 Dec 23.

Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin

Abhishek D Garg  1 , Dmitri V Krysko  2   3 , Peter Vandenabeele  2   3 , Patrizia Agostinis  4
Affiliations

Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin

Abhishek D Garg et al. Cancer Immunol Immunother. 2012 Feb.

Erratum in

Abstract

Surface-exposed HSP70 and calreticulin are damage-associated molecular patterns (DAMPs) crucially involved in modulating the success of cancer therapy. Photodynamic therapy (PDT) involves the administration of a photosensitising (PTS) agent followed by visible light-irradiation. The reactive oxygen species that are thus generated directly kill tumours by damaging their microvasculature and inducing a local inflammatory reaction. PDT with the PTS photofrin is associated with DAMPs exposure, but the same is not true for other PTSs. Here, we show that when cancer cells are treated with hypericin-based PDT (Hyp-PDT), they surface-expose both HSP70 and calreticulin (CRT). Induction of CRT exposure was not accompanied by co-exposure of ERp57, but this did not compromise the ability of the exposed CRT to regulate the phagocytosis of Hyp-PDT-treated cancer cells by dendritic cells. Interestingly, we found that Hyp-PDT-induced CRT exposure (in contrast to anthracycline-induced CRT exposure) was independent of the presence of ERp57. Our results indicate that Hyp-PDT is a potential anti-cancer immunogenic modality.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Hypericin-PDT-treated cancer cells surface-expose HSP70 and calreticulin but not ERp57. a T24 cells were treated with high PDT dose followed by surface protein biotinylation at 1 h post-PDT and immunoblotted. b, c T24 cells were treated with high PDT dose, fixed and surface immunostained at 1.5 h post-PDT for either CRT (b) or HSP70 (c) such that extranuclear fluorescence of residual hypericin was also visualised (scale bar = 20 μm). d T24 cells were incubated with 100 nM or 150 nM hypericin for 16 h and irradiated at a fluency of either 1.35 or 2.16 J/cm2. This was followed by surface protein biotinylation at 2 h post-PDT and immunoblotting. CNTR denotes untreated samples. Furthermore, ‘+BIO’ indicates controls exposed to buffer with biotin and ‘−BIO’ indicates controls exposed to buffer without biotin. Representative Western blots are shown for three independent experiments
Fig. 2
Fig. 2
Hypericin-PDT induces ERp57-independent ecto-CRT that regulates phagocytosis of PDT-treated cells. a MEF cells containing ERp57 (WT) or lacking it (KO) were treated with a medium PDT dose (recovered, 1 h post-PDT), MTX (1 μM for 4 h) or left untreated (CNTR). This was followed by surface protein biotinylation and immunoblotting. b CRT, HSP70 and HSP90 were analysed for the presence of homologous sequences representing the phagocytic motifs (NPxY and RGD) with respect to positive controls (MFG-E8 and Annexin-1) and negative control (Actin). In parentheses are the respective homologous sequences for each protein. c CT26 murine tumour cells were treated with high PDT dose followed by surface protein biotinylation at 1 h post-PDT and immunoblotting. d CT26 cells were treated with PDT or left untreated (CNTR) and recovered 24 h post-PDT followed by staining with annexin-V-FITC and the vital dye 4,6-diamidino-2-phenylindole (DAPI). e Hypericin-labelled CT26 cells treated with PDT were incubated with chicken anti-CRT or isotype antibody, followed by detection of phagocytosis by Cell Tracker Green (CTGr)-labelled JAWSII DCs. JAWSII cells accumulated in quadrant I (no phagocytosis) and quadrant II (phagocytic uptake) while free CT26 cells accumulated in quadrant III. Calculated % phagocytosis is mentioned for the respective pictograms. Results are representative of two independent experiments with two replicates in each. Furthermore in a and c, ‘+BIO’ indicates controls exposed to buffer with biotin and ‘−BIO’ indicates controls exposed to buffer without biotin. Representative Western blots are shown for three independent experiments
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