A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

Abstract

Article17 January 2012free access A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death Abhishek D Garg Abhishek D Garg Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Dmitri V Krysko Dmitri V Krysko Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tom Verfaillie Tom Verfaillie Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Agnieszka Kaczmarek Agnieszka Kaczmarek Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Gabriela B Ferreira Gabriela B Ferreira Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Thierry Marysael Thierry Marysael Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Noemi Rubio Noemi Rubio Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Malgorzata Firczuk Malgorzata Firczuk Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Chantal Mathieu Chantal Mathieu Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Anton J M Roebroek Anton J M Roebroek Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Wim Annaert Wim Annaert Laboratory for Membrane Trafficking, Department of Human Genetics, KU Leuven and VIB-Center for the Biology of Disease, Leuven, Belgium Search for more papers by this author Jakub Golab Jakub Golab Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Peter de Witte Peter de Witte Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Peter Vandenabeele Peter Vandenabeele Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Patrizia Agostinis Corresponding Author Patrizia Agostinis Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Abhishek D Garg Abhishek D Garg Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Dmitri V Krysko Dmitri V Krysko Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tom Verfaillie Tom Verfaillie Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Agnieszka Kaczmarek Agnieszka Kaczmarek Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Gabriela B Ferreira Gabriela B Ferreira Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Thierry Marysael Thierry Marysael Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Noemi Rubio Noemi Rubio Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Malgorzata Firczuk Malgorzata Firczuk Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Chantal Mathieu Chantal Mathieu Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Anton J M Roebroek Anton J M Roebroek Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium Search for more papers by this author Wim Annaert Wim Annaert Laboratory for Membrane Trafficking, Department of Human Genetics, KU Leuven and VIB-Center for the Biology of Disease, Leuven, Belgium Search for more papers by this author Jakub Golab Jakub Golab Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Peter de Witte Peter de Witte Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium Search for more papers by this author Peter Vandenabeele Peter Vandenabeele Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Patrizia Agostinis Corresponding Author Patrizia Agostinis Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium Search for more papers by this author Author Information Abhishek D Garg1, Dmitri V Krysko2,3, Tom Verfaillie1, Agnieszka Kaczmarek2,3, Gabriela B Ferreira4, Thierry Marysael5, Noemi Rubio1, Malgorzata Firczuk6,7, Chantal Mathieu4, Anton J M Roebroek8, Wim Annaert9, Jakub Golab6,7, Peter de Witte5, Peter Vandenabeele2,3 and Patrizia Agostinis 1 1Cell Death Research and Therapy Unit, Department of Cellular and Molecular Medicine KU Leuven, KU Leuven, Leuven, Belgium 2Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium 3Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 4Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium 5Laboratory for Pharmaceutical Biology, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium 6Department of Immunology, Centre of Biostructure Research, Medical University of Warsaw, Warsaw, Poland 7Department 3, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland 8Experimental Mouse Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium 9Laboratory for Membrane Trafficking, Department of Human Genetics, KU Leuven and VIB-Center for the Biology of Disease, Leuven, Belgium *Corresponding author. Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Leuven (KU Leuven), Campus Gasthuisberg O&N1, Herestraat 49, Box 901, 3000 Leuven, Belgium. Tel.: +32 16 345715; Fax: +32 16 345995; E-mail: [email protected] The EMBO Journal (2012)31:1062-1079https://doi.org/10.1038/emboj.2011.497 There is a Have you seen? (March 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80high, CD83high, CD86high, MHC-IIhigh) and functional stimulation (NOhigh, IL-10absent, IL-1βhigh) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress. Introduction Current anticancer regimens mediate killing of tumour cells mainly by activating apoptosis, an immunosuppressive or even tolerogenic cell death process. However, it has recently emerged that a selected class of cytotoxic agents (e.g., anthracyclines) can cause tumour cells to undergo an immunogenic form of apoptosis and these dying tumour cells can induce an effective antitumour immune response (Locher et al, 2010). Immunogenic apoptosis of cancer cells displays the main biochemical hallmarks of ‘tolerogenic’ apoptosis: phosphatidylserine exposure, caspase activation, and mitochondrial depolarization. However, this type of cell death also seems to have two other important properties: (1) surface exposure or secretion of critical ‘immunogenic signals’ that fall in the category of damage-associated molecular patterns (DAMPs; Zitvogel et al, 2010a) and (2) the ability to elicit a protective immune response against tumour cells (Obeid et al, 2007; Green et al, 2009; Garg et al, 2010b; Zitvogel et al, 2010b). Several DAMPs have recently been identified as crucial for immunogenic apoptosis. These include surface calreticulin (ecto-CRT), surface HSP90 (ecto-HSP90), and secreted ATP (Spisek et al, 2007; Kepp et al, 2009). Ecto-CRT has been shown to act primarily as an ‘eat me’ signal (Gardai et al, 2005), presumably essential for priming the innate immune system, since depletion of CRT by siRNA knockdown averts the immunogenicity of cancer cell death (Obeid et al, 2007). Similarly, bortezomib-induced ecto-HSP90 exposure is crucial for immunogenic death of tumour cells and their subsequent contact with dendritic cells (DCs; Spisek et al, 2007). On the other hand, secreted ATP acts either as a ‘find me’ signal or as an activator of the NLRP3 inflammasome (Elliott et al, 2009; Ghiringhelli et al, 2009). However, while the signalling pathways governing surface exposure of CRT have been delineated to some extent (Panaretakis et al, 2009), insufficient information exists on the molecular pathway behind ATP secretion. Finally, immunogenic apoptosis is sometimes associated with disappearance of certain surface-associated molecules, for example CD47, which are referred to as ‘do not eat me’ signals (Chao et al, 2010). One common feature of all immunogenic apoptosis-inducing stimuli so far identified is induction of endoplasmic reticulum (ER) stress (Panaretakis et al, 2009; Garg et al, 2010b; Zitvogel et al, 2010b). Importantly, in the case of ecto-CRT triggered by anthracyclines, both ER stress and reactive oxygen species (ROS) production have been found to be mandatory (Panaretakis et al, 2009). However, anthracyclines suffer from dose-limiting side effects (Minotti et al, 2004; Vergely et al, 2007). Moreover, ROS production is neither a primary effect of anthracyclines nor predominantly ER directed, which makes the anthracycline-induced ‘ROS-based’ ER stress less effective and secondary in nature (Minotti et al, 2004; Vergely et al, 2007). Thus, we envisaged that one way of improving the immunogenicity of dying cancer cells is by using a therapeutic approach that can generate strong ROS-dependent ER stress as a primary effect (Garg et al, 2011). We hypothesized that photodynamic therapy (PDT; Agostinis et al, 2011) might fit the criterion of primary ER-directed ROS production. PDT can induce oxidative stress at certain subcellular sites by activating organelle-associated photosensitizers (Castano et al, 2006; Buytaert et al, 2007). Once excited by visible light and in the presence of oxygen, photosensitizers can generate organelle-localized ROS that can cause lethal damage to the cells (Agostinis et al, 2002). Additionally, this ROS-based anticancer therapy can also cause ‘emission’ of DAMPs and activate the host immune system (Korbelik et al, 2005; Garg et al, 2010a). To test this hypothesis, we used the ER-associated photosensitizer, hypericin. When it is activated by light, it causes a ROS-mediated loss-of-function of SERCA2 with consequent disruption of ER-Ca2+ homeostasis, followed by BAX/BAK-based mitochondrial apoptosis (Buytaert et al, 2006). This photo-oxidative ER stress (phox-ER stress) is accompanied by transcriptional upregulation of components of the unfolded protein response (UPR) and by changes in the expression of various genes coding for immunomodulatory proteins (Buytaert et al, 2008; Garg et al, 2010a). We report here that phox-ER stress induces immunogenic apoptosis in treated cancer cells. Early after phox-ER stress and largely preceding phosphatidylserine externalization, cancer cells mobilize CRT at the surface and secrete ATP through an overlapping PERK- and phosphoinositide 3-kinase (PI3K)-mediated mechanism, which is dissociated from caspase signalling. Intriguingly, we found that LRP1 is required for the docking of ecto-CRT. Results Phox-ER stress causes cancer cells to undergo immunogenic apoptosis At the outset, we decided to investigate whether cancer cells dying in response to phox-ER stress (Hyp-PDT based; unless otherwise mentioned) can activate human immature DCs (hu-iDCs). We used phox-ER stress (Supplementary Figure S1) mediated apoptosis-inducing conditions reported in our previous studies (Hendrickx et al, 2003; Buytaert et al, 2006) generating ∼87% of cell death of the human bladder carcinoma T24 cells within 24 h (Supplementary Figure S2). T24 cells subjected to Hyp-PDT underwent phagocytic interactions with hu-iDCs (Figure 1A). They were also phagocytosed by Mf4/4 phagocytes preferentially over untreated T24 cells (Supplementary Figure S3). Moreover, these Hyp-PDT-treated dying T24 cells induced phenotypic maturation of hu-iDCs, as indicated by surface upregulation of MHC class II (HLA-DR) and co-stimulatory CD80, CD83 and CD86 molecules (Figure 1B; Supplementary Figure S4A and B). The significant surface expression of these molecules was similar to that induced by lipopolysaccharide (LPS), a known pathogen-associated molecular pattern (PAMP) (Figure 1B; Supplementary Figure S4A and B). In contrast, freeze-thawed T24 cells undergoing accidental necrosis (AN) did not strongly stimulate DC maturation (Figure 1B; Supplementary Figure S4A and B). These findings rule out the possibility that AN might be responsible for the increased DC maturation seen against phox-ER stressed cells. Figure 1.Tumour cells dying under phox-ER stress conditions induce DC maturation and activate the adaptive immune system. (A) In-vitro phagocytosis of T24 cells treated with Hyp-PDT (red) by human immature dendritic cells (hu-iDCs) (green). The confocal fluorescence images show various phagocytic interactions between dying T24 cells and hu-iDCs, such as tethering (a), initiation of engulfment by extending the pseudopodia (b), and final stages of engulfment (c); scale bar=20 μm. (B) Human DC maturation analysis. T24 cells were left untreated (CNTR), freeze/thawed (accidental necrosis=AN), or treated with a high PDT dose. They were then co-incubated with hu-iDCs. As a positive control, hu-iDCs were stimulated with LPS for 24 h. After co-incubation, the cells were immunostained in two separate groups for CD80/CD83 positivity and CD86/HLA-DR positivity and scored by FACS analysis. Data have been normalized to the ‘CNTR T24 + hu-iDCs’ values. Fold change values are means of two independent experiments (two replicate determinations in each)±s.e.m. (*P<0.05, versus ‘CNTR T24+hu-iDCs’). (C, D) Cytokine and respiratory burst patterns exhibited by human DCs. The T24-hu-iDC co-incubation conditioned media obtained during the experiments detailed in (B) were recuperated followed by analysis for concentrations of nitrite (solubilized form of nitric oxide or NO) (C), and IL-10 (D). Absolute concentrations are the means of two independent experiments (four replicate determinations in each)±s.d. (*P<0.05 versus hu-iDC only). (E) Priming of adaptive immune system by dead/dying CT26 cells. Following immunization with PBS (CNTR) or with CT26 cells treated with tunicamycin (TUN), mitoxantrone (MTX) and the highest PDT dose, the mice were rechallenged with live CT26 tumour cells. Subsequently, the percentage of mice with tumour-free rechallenge site was determined (n represents the number of mice). Download figure Download PowerPoint To get further insight into the functional status of DCs, we evaluated the pattern of certain cytokines including the generation of nitric oxide (NO) as a marker for respiratory burst (Stafford et al, 2002). We compared DCs exposed to Hyp-PDT-treated T24 cells with those exposed to LPS or T24 cells dying following AN. We found that hu-iDCs exposed to Hyp-PDT-treated cancer cells displayed a distinguished pattern of functional activation characterized by NOhigh, IL-10absent (Figure 1C and D). This was clearly different from that induced by accidental necrotic cells (NOhigh, IL-10high) or by LPS (NOlow, IL-10low) (Figure 1C and D). Interestingly, LPS and especially accidental necrotic cells stimulated the production of IL-10 (Figure 1D), whereas Hyp-PDT-treated cells failed to stimulate the production of this immunosuppressive cytokine (Kim et al, 2006; Zitvogel et al, 2006) by hu-iDCs. To investigate the ability of cancer cells undergoing phox-ER stress to activate the adaptive immune system, we carried out in-vivo experiments in immunocompetent BALB/c mice. Before initiating the in-vivo experiments, we optimized the mouse colon carcinoma CT26 cell line for Hyp-PDT-induced apoptosis (Supplementary Figure S5) and ER stress (Supplementary Figure S1). As observed previously in other cells (Hendrickx et al, 2003; Buytaert et al, 2006), hypericin colocalized strongly with ER Tracker (Supplementary Figure S5A) and upon light irradiation induced not only appreciable cell killing (Supplementary Figure S5B) but also the main hallmarks of apoptosis, including caspase-3 and PARP cleavage (Supplementary Figure S5C). Furthermore, the CT26 cells exposed to Hyp-PDT were preferentially phagocytosed over untreated CT26 cells by murine JAWSII DCs (Supplementary Figure S6). Then, in the in-vivo study, we immunized BALB/c mice with Hyp-PDT-treated dying/dead CT26 cells. As positive and negative controls for immunogenic cell death, respectively, we used CT26 cells treated with the anthracycline, mitoxantrone (MTX) or tunicamycin (TN, an inhibitor of N-linked glycosylation) (Obeid et al, 2007). The immunized mice were then rechallenged with live CT26 tumour cells. Protection against tumour growth at the rechallenge site was interpreted as a sign of successful priming of the adaptive immune system (Figure 1E). Mice immunized with CT26 cells treated with MTX or Hyp-PDT showed robust signs of activation of the adaptive immune system: both procedures strongly prevented the tumour growth seen in the non-immunized mice. By contrast, most of the mice immunized with tunicamycin-treated CT26 cells experienced tumour growth after rechallenge (Figure 1E), which confirms the poor immunogenic properties of cancer cell death induced by this ER stress agent (Obeid et al, 2007). These data suggest that apoptotic cancer cells dying from phox-ER stress induced by Hyp-PDT activate the immune system, which is one of the important properties of immunogenic apoptosis. Cancer cells exposed to phox-ER stress surface expose or secrete/release immunogenic DAMPs We next analysed the surface exposure/release of CRT, secreted ATP and extracellular heat-shock proteins (i.e., HSP90 and HSP70) following phox-ER stress using three different Hyp-PDT doses—low, medium, and high PDT. Moreover, because of the reported effects of anthracyclines, MTX, and doxorubicin (DOXO) on immunogenic cell death (Obeid et al, 2007), we used them throughout the study for comparison. Ecto-CRT surface exposure, detected by immunofluorescence staining of T24 cells treated with Hyp-PDT or MTX, showed the characteristic surface ‘patches’ reported previously (Gardai et al, 2005; Obeid et al, 2007; Figure 2A). Cell surface biotinylation followed by immunoblot analysis of the isolated plasma membrane proteins derived from T24 cancer cells treated with Hyp-PDT revealed that phox-ER stress (Supplementary Figure S1) induced enhanced surface exposure of CRT (Figure 2B). This ecto-CRT preceded apoptosis-associated phosphatidylserine exposure (Supplementary Figure S2) under plasma membrane non-permeabilizing conditions (Figure 2C). On-cell western assay (Gonzalez-Gronow et al, 2007) confirmed these results (Supplementary Figure S7). In general, Hyp-PDT was observed to be superior to DOXO and MTX (Figure 2D and E), in terms of mobilizing CRT to the surface of cancer cells. Moreover, ecto-CRT was detectable as early as 30 min after Hyp-PDT and increased with time (Figure 2E). The 30-min threshold is much earlier than reported for anthracyclines (Obeid et al, 2007). This induction of ecto-CRT by Hyp-PDT was diminished in the presence of the 1O2 quencher L-histidine, thus revealing its ROS dependence (Buytaert et al, 2006; Supplementary Figure S8A). In contrast to anthracycline-induced ecto-CRT exposure (Panaretakis et al, 2008), ecto-CRT exposure following Hyp-PDT was not accompanied by co-translocation of ERp57 to the surface (Figure 2B). Figure 2.Phox-ER-stressed cancer cells expose calreticulin on the surface (ecto-CRT). (A) Immunofluorescence analysis of ecto-CRT. T24 cells were treated with MTX (1 μM for 4 h) and a high PDT dose (recovered 1 h post PDT) or left untreated (CNTR). Alternatively, some cells were saponin permeabilized. This was followed by staining with Sytox Green (exclusion dye), fixation, and immunostaining for CRT and counterstaining with DAPI; scale bar=20 μm. (B) Surface biotinylation analysis of ecto-CRT following phox-ER stress. T24 cells were treated with indicated doses of PDT. They were recovered at the indicated intervals after PDT treatment. Surface proteins were biotinylated followed by immunoblotting. In (B), (D), and (F), ‘+BIO' indicates controls exposed to buffer with biotin and ‘−BIO' indicates controls exposed to buffer without biotin (negative control). (C) Plasma membrane permeabilization kinetics following phox-ER stress. T24 cells were treated with PDT and the resulting conditioned media derived at the indicated times post-PDT were analysed for the presence of cytosolic LDH. Total LDH content was determined following Triton-based permeabilization of cells. Data are presented as percent LDH release; values are means of five replicate determinations±s.d. (*P<0.05, versus CNTR). (D) Phox-ER stress induces more ecto-CRT than anthracyclines. T24 cells were treated with PDT, DOXO (25 μM for 4 h), and MTX (1 μM for 4 h). They were recovered at the indicated intervals after PDT treatment. Surface proteins were biotinylated as described for (B). (E) Integrated band densitometric analysis of ecto-CRT. T24 cells were treated with DOXO (25 μM for 4 h), MTX (1 μM for 4 h), and PDT (dose and recovery time points are indicated); and surface proteins were resolved as detailed in (B). Following this, the ecto-CRT protein bands were quantified for the integrated band density via Image J software. Data have been normalized to the CNTR values. Fold change values are means of three independent determinations±s.e.m. (*P<0.05, versus CNTR). (F) Surface biotinylation analysis for KDEL sequence detection following phox-ER stress. CRT WT and KO MEFs were treated with a low PDT dose and surface biotinylated as mentioned in (B). Immunoblotting was done to detect the C-terminal KDEL sequence of various ER proteins (expected molecular weights are indicated). Download figure Download PowerPoint Likewise ecto-HSP90, certain ER proteins, such as calnexin (CNX), PERK and BiP were also undetectable on the surface of the cells under the same conditions that efficiently mobilized ecto-CRT (Supplementary Figure S8B). In addition, several other ER proteins have been reported to undergo translocation to the plasma membrane (Zai et al, 1999; Korbelik et al, 2005; Zhang et al, 2010). Therefore, we used cell surface biotinylation combined with immunoblotting to screen for surface-translocated proteins containing the KDEL ‘ER retrieval’ signal sequence, in wild-type (WT) and CRT−/− MEFs. Ecto-CRT (∼63 kDa) was the only protein with the KDEL sequence recognizable on the surface of Hyp-PDT-treated cells (Figure 2F). No KDEL-containing proteins were found in the plasma membrane fraction of cells lacking CRT (Figure 2F). On the other hand, KDEL sequences of ER resident proteins, such as GRP94, GRP78, ERp72 (ER resident protein 72), and PDI, were identifiable by their molecular weights in the intracellular protein fractions of WT and CRT−/− cells (Figure 2F). Overall, these results indicate that phox-ER stress does not lead to a general surface scrambling of ER proteins (luminal or membrane associated) but rather to a selective and rapid surface exposure of CRT in pre-apoptotic conditions. We next asked whether photo-oxidative stress mediated by other photosensitizers known to localize to other subcellular sites in addition to the ER were equally capable of surface-exposing CRT. To this end, we used photofrin (PF-PDT), a photosensitizer used in the clinic and known to induce phox-ER stress (Szokalska et al, 2009). Interestingly, while phox-ER stress mediated by Hyp-PDT strongly induced ecto-CRT, it was not so for PF-PDT (Supplementary Figure S8C) under similar apoptosis-inducing conditions as reported previously (Szokalska et al, 2009). This difference between Hyp-PDT and PF-PDT in ecto-CRT induction might be due to the more pronounced ER localization of hypericin when compared with photofrin (Buytaert et al, 2007; Szokalska et al, 2009; Luo et al, 2010). These data further underline the importance of a robust ER-directed oxidative stress in inducing ecto-CRT. Next, we addressed the possibility that apart from induction of ecto-CRT, Hyp-PDT-treated T24 cancer cells can secrete ATP into the extracellular environment. Analysis of the conditioned media showed that T24 cancer cells treated with Hyp-PDT secreted ATP (Figure 3A) under non-permeabilizing plasma membrane conditions (Figure 2C). Secretion of ATP preceded apoptosis-associated phosphatidylserine exposure (Supplementary Figure S2) and downregulation of the ‘do not eat me’ signal CD47 (Supplementary Figure S8D). Interestingly, at least at medium Hyp-PDT dose, the corresponding intracellular ATP content rose considerably in the pre-apoptotic stages (Figure 3B). Figure 3.Cancer cells exposed to phox-ER stress actively secrete ATP, passively release HSP70, HSP90, CRT, and induce IL-1β production in DCs. (A, B) ATP secretion following phox-ER stress. T24 cells were treated with PDT or left untreated (CNTR), and the conditioned media derived from these cells (1 h post PDT in serum-free media) were analysed for the presence of ATP (A). Simultaneously, the corresponding cells were permeabilization with saponin (1 h post PDT) followed by determination of ATP content in the lysate (B). Absolute concentrations are mean values of six replicate determinations±s.d. (*P<0.05, versus CNTR). (C) Cancer cells subjected to phox-ER stress stimulate IL-1β production in hu-iDCs. T24 cells were treated to undergo accidental necrosis (AN), or treated with a high PDT dose (recovered 24 h post PDT), and then co-incubated with hu-iDCs for 24 h. Simultaneously hu-iDCs were stimulated with LPS. After co-incubation, the co-incubation conditioned media (CCM) were analysed for the presence of IL-1β. Cytokine concentrations (in pg/ml) are means of two independent experiments (four replicate determinations in each) ±s.e.m. (*P<0.