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. 2011 Oct;60(10):1431-7.
doi: 10.1007/s00262-011-1047-x. Epub 2011 Jun 5.

Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and high-mobility group box-1 release

Mladen Korbelik  1 , Wei Zhang, Soroush Merchant
Affiliations

Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and high-mobility group box-1 release

Mladen Korbelik et al. Cancer Immunol Immunother. 2011 Oct.

Abstract

Damage-associated molecular patterns (DAMPs), danger signal molecules expressed after injury or infection, have become recognized as prerequisite for orchestrating effective anti-tumor host response. The expression of two prototypical DAMPs, calreticulin and high-mobility group box-1 (HMGB1) protein, was examined following Photofrin-photodynamic therapy (PDT) of Lewis lung carcinoma (LLC) cells in vitro and LLC tumors growing in syngeneic mice. Cell surface expression of calreticulin was found to be highly increased at 1 h after PDT treatment both in vitro and in vivo. Increased exposure of calreticulin was also detected on the surface of macrophages from PDT-treated LLC tumors. At the same time interval, a rise in serum HMGB1 was detected in host mice. Intracellular staining of macrophages co-incubated for 16 h with PDT-treated LLC cells revealed elevated levels of HMGB1 in these cells. The knowledge of the involvement of these DAMPs uncovers important mechanistic insights into the development of host response induced by PDT.

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Conflict of interest statement

The authors declare to have no conflict of interest in any form with respect to this article.

Figures

Fig. 1
Fig. 1
Expression of calreticulin on the surface of LLC tumor cell following PDT in vitro. Cultured LLC cells were incubated with Photofrin™ (20 μg/ml) for 24 h followed by exposure to 1 J/cm2 of 630 ± 10 nm light. The cells were then left in culture for 1 or 3 h before they were collected for flow cytometry. Expression of calreticulin was detected by surface staining with chicken polyclonal antibody to calreticulin followed by FITC-conjugated secondary antibody. Dying cells were identified by their decreased light scatter characteristics. The results show the extent of calreticulin-associated fluorescence (in arbitrary units per cell corrected by values obtained with the isotype control) in all cells, or separately in alive vs. dying cell populations (insert). The bars denote SE, N = 4; *statistically significant difference compared to the level in untreated group (P < 0.05); **statistically significant difference compared to the value with alive cell populations (P < 0.05)
Fig. 2
Fig. 2
Expression of calreticulin on the surface of cells from PDT-treated LLC tumors. Photofrin™ (10 mg/kg, i.v.) was administered to C57BL/6 mice bearing subcutaneous LLC tumors, and 24 h later, the tumors were exposed to light (150 J/cm2, the same wavelength as in vitro). Illumination of tumors in mice that were not injected with photosensitizer was done for light-alone group. The mice were killed 1 h after light treatment, and single-cell suspensions prepared from the excised tumors were examined by flow cytometry. Surface calreticulin expression was determined as described for Fig. 1. Two major cell populations in LLC tumors were delineated as cancer parenchymal cells (stained negatively by panleukocyte marker CD45) and tumor-associated macrophages (positively stained for CD45 and macrophage marker F4/80). The results are shown as percentage of calreticulin-positive (bright) cells, and additionally for the cancer cells as the extent of calreticulin-associated fluorescence in arbitrary units per cell (insert). The bars denote SE, N = 4; *statistically significant difference compared to the level in untreated group (P < 0.05)
Fig. 3
Fig. 3
Serum HMGB1 increase in mice bearing PDT-treated LLC tumors. Subcutaneous LLC tumors growing in C57BL/6 mice were treated by PDT as described for Fig. 2, and blood for serum samples was taken from host mice either at 1 or 4 h after therapy. The results show serum levels of HMGB1 determined by ELISA. The bars denote SE, N = 4; *statistically significant difference compared to the level in untreated group (P < 0.05)
Fig. 4
Fig. 4
Changes in intracellular HMGB1 levels in IC-21 cells co-incubated with PDT-treated LLC cells. In vitro-cultured IC-21 cells were co-incubated 16 h with LLC cells that were either untreated or treated with PDT as described for Fig. 1. Intracellular staining of IC21 cells collected after the co-incubation with antibody recognizing mouse HMGB1 was followed by flow cytometry analysis. The results show the extent of intracellular HMGB1-associated fluorescence (isotype control values deducted) in arbitrary units per cell. The bars denote SE, N = 4; *statistically significant difference compared to the level in IC-21-alone group (P < 0.05)
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