Murine glioma GL261 and fibrosarcoma MCA205 cells were cultured at 37 °C under 5% CO₂ in DMEM and RPMI, respectively, containing 4.5 g/L glucose and supplemented with 2 mM glutamine, 100 μM sodium pyruvate, 100 units/ml penicillin, 100 μg/L streptomycin and 10% fetal bovine serum (FBS, Fisher Scientific, 10,082,147).

The following photosensitizers were used: photosens (PS, a mixture of di-, tri- and tetrasubstituted fractions of aluminum phthalocyanine, the number of sulfo groups is 3.4; NIOPIK, Russia) and photodithazine (PD, bis-N-methylglucamine salt of chlorin e6; Veta-grand, Russia). Spectra of absorption and fluorescence emission of PS and PD were registered using Synergy MX Microplate Reader (BioTek, USA) in black 96-well microplates with a clear glass bottom (Falcon Imaging; Corning, USA). Photosensitizer solutions were prepared in distilled water at 10 μg/ml. Absorption spectra were obtained in the range of 320–850 nm for PS and from 300 to 700 for PD. Fluorescence was excited at 405 nm and recorded in the range of 655–850 nm for PS and 600–850 nm for PD.

Cell death was induced by PS- or PD-based PDT. For this, GL261 and MCA205 cells were first incubated with 1.4 μM PS or 1.2 μM PD and 1.5 μM PS or 1.8 μM PD, respectively, in serum-free medium for 4 h. Then the cells were irradiated with a light dose of 20 J/cm² using a LED light source (λex 615–635 nm) in photosensitizer-free media. Cells loaded with photosensitizers were handled either in the dark or subdued light. After PDT, the cells were cultured in complete medium for the indicated period of time and cell death was analyzed by MTT or flow cytometry. Control cells were cultured in the same conditions but without photosensitizers or PDT.

The following blockers were used to inhibit cell death: the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk, 25 μM, Sigma-Aldrich), RIPK1 inhibitor necrostatin-1 s (Nec-1 s, 20 μM, Abcam), the inhibitor of ROS and lipid peroxidation ferrostatin-1 (Fer-1, 1 μM, Sigma-Aldrich) and the iron chelator, deferoxamine (DFO, 10 μM, Sigma-Aldrich). The cell death inhibitors were added together with the corresponding photosensitizer or DMSO and the cells were incubated for 4 h in serum-free conditions. Before PDT, the medium was replaced with complete medium containing the respective cell death inhibitor, the cells were irradiated with light at 20 J/cm², and then they were incubated for 13 h.

The cells were washed in Annexin V binding buffer and stained with SYTOX Blue Nucleic Acid Stain (Molecular Probes) and FITC Annexin V (Invitrogen). The assay was run on a BD FACSCanto flow cytometer. The data were analyzed using FlowJo software. MTT assay (AlfaAesar) was performed according to the manufacturer’s instructions and the optical density was measured at 570 nm.

Intracellular distribution of PS and PD was studied by using the LSM 710 Axio Obzerver Z1 DUO NLO laser scanning microscope (Carl Zeiss, Germany). The images were obtained using a LD C-Apochromat water immersion objective lens 40×/1.1. The GL261 cells were seeded in 96-well glass-bottom plates (Corning, USA) at 10⁴ cells per well and grown overnight. The cells were then incubated with 10 μM photosensitizers in serum-free culture medium for 1–4 h, followed by washing with PBS and confocal image acquisition. The fluorescence of PS and PD was excited at 633 nm and recorded in the range of 650–735 nm.

For colocalization analysis of PS and PD after 3.5 h of incubation of GL261 cells with the respective photosensitizer, the following dyes were added for 30 min (ThermoFisherScientific): 0.5 μM LysoTracker Green DND-26 for lysosomes, 0.5 μM ER-Tracker for endoplasmic reiculum, 0.5 μM MitoTracker Green FM for mitochondria, 5 μM BODIPY FL C5-ceramide complexed to BSA for Golgi apparatus. Dyes were added to living cells that had been incubated with the photosensitizers. Staining was performed according to the manufacturer’s instructions. Fluorescence of stained organelles was excited by an argon laser at 488 nm and registered in the range of 500–560 nm.

GL261 and MCA205 cells were stimulated by either PS-PDT or PD-PDT as described above. After 1.5 h and 3 h of incubation, the cells were collected and then washed with ice-cold FACS buffer (PBS, BSA 1%, FBS 2%). After centrifugation (1500 rpm 4 °C 5 min), they were resuspended in ice-cold FACS buffer with anti-calreticulin antibody (ab210431; 0.5 mg/ml) or isotype control rabbit IgG (Ab208150; 0,5 mg/ml). The cells were incubated for 40 min at 4 °C and then resuspended in 200 μL of ice-cold FACS buffer and stained with 0.8 μM Sytox Green (Molecular Probes, S7020). Finally, the samples were analyzed by flow cytometry on a BD FACS Canto II. Analysis was performed using FlowJo software (v.10.0.8). The surface exposure of CRT was determined in Sytox Green negative cells.

After the indicated time points, supernatant was collected and cleared from dying tumor cells by centrifugation, frozen at − 20 °C for later HMGB1 quantification by an ELISA kit (IBL-Hamburg). All assays were performed in accordance with the respective manufacturers’ instructions and HMGB1 was quantified using Tecan Spark® 20 M microplate multimode reader. The data were analyzed with a Four Parameter Logistic Curve Fit.

GL261 and MCA205 cells were treated with PS-PDT or PD-PDT as described above and incubated for 24 h in a medium with 2% FBS. Then supernatants were collected and centrifuged at 15,000 rpm at 4 °C for 3 min. The supernatants were either stored at − 80 °C or used immediately for ATP measurements. ATP analysis was done by using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571) as described by the manufacturer. The luminescence was measured on a Tecan Spark® 20 M microplate multimode reader.

Over 10 days, bone-marrow derived dendritic cells (BMDCs) were differentiated from the femurs and tibias of C57BL/6 J mice at the age of 7–9 weeks using RPMI medium (GIBCO) supplemented with 5% heat-inactivated fetal calf serum, 20 ng/ml mGM-CSF, 1% L-glutamine and 50 μM 2-mercapthoethanol, 1 mM pyruvate. Fresh culture medium was added on day 3, and on day 6 and 9 the medium was refreshed.

Target GL261 and MCA205 cells were labeled with 1 μM CellTracker Green CMFDA (Molecular Probes) in serum-free media for 30 min and then either left untreated or induced to die by PS-PDT or PD-PDT, as described above. The cells were collected, washed, and co-cultured with BMDCs in ratios of 1:1, 1:5 or 1:10 for 2 h. Next, the co-cultured cells were harvested, incubated with a mouse Fc block (ThermoFisherScientific), immunostained with PE-Cy-anti-CD11c (BD PharMingen, 561,022), and finally analyzed by flow cytometry on a BD FACSCanto. Analysis was performed using FlowJo software (v.10.0.8). True uptake of CMFDA-labeled dead cell material by BMDCs was determined using a gating strategy that allows analysis of only single cells and was determined as CD11c CMFDA double-positive cells.

Immature murine BMDCs were isolated and cultured as described previously. Then BMDCs were co-incubated with dying GL261 or MCA205 cells stimulated with PS-PDT or PD-PDT as described above in ratios 1:1, 1:5 or 1:10 for 18 h. As a positive control, BMDCs were stimulated in parallel with 100 ng/ml of E. coli lipopolysaccharide (LPS). After co-culture for 18 h, the cells were collected, spun down (400×g, 6 min, 4 °C), and washed once in phosphate buffered saline (PBS, Life Technologies). Dead cells were excluded from the flow cytometry analysis by staining with SYTOX Blue (Molecular Probes, S11348). Maturation of BMDCs was analyzed by immunostaining with anti-CD11c PE-Cy7 (BD PharMingen), anti-CD86-eFluor 450 or -APC (eBioscience), anti-CD40 Pacific Blue (Biolegend), eFluor 45–anti-CD80-eFluor 450 (Thermo Fisher Scientific) and mouse Fc block (Thermo Fisher Scientific). After co-culturing BMDCs with the MCA205 cancer cells, the supernatants were collected and IL-6 was measured by ELISA (BioLegend).

Female C57BL/6 J mice (7–8 weeks old) were housed in specific pathogen-free conditions. All experiments were performed in accordance with the guidelines of the local Ethics Committee of Ghent University (ECD19/35).

Cell death in MCA205 cells was induced in vitro by PS-PDT, PD-PDT as described above. Next, the cells were collected, washed once in PBS, and re-suspended at the desired cell density in PBS. Mice were inoculated subcutaneously with 5 × 10⁵ dying MCA205 cells or with PBS on the left flank. On day 8 after vaccination, the mice were challenged subcutaneously on the opposite flank with 1 × 10⁵ live MCA205 cells. Tumor growth at the challenge site was monitored using a caliper for up to 4 weeks after the challenge. Mice were sacrificed when the tumors became necrotic or exceeded 2 cm³.

Statistical analysis was performed in GraphPad Prism (v.6.0). Cell death was analyzed by ANOVA followed by t-criteria with Bonferroni correction. The phagocytosis assay was analyzed by two-way ANOVA. The results of the BMDC activation and maturation assay were analyzed by Mann-Whitney non-parametric t-test. Kaplan-Meier survival curves showing the timeline for tumor development were analyzed by log-rank Mantel-Cox test. Differences between tumor volumes on the mice in the vaccination experiments were analyzed by a non-parametric Mann-Whitney test.

First, we analyzed the absorption and fluorescence spectra of PD belonging to the chlorins derivatives. For PS, we observed the typical absorption and fluorescence spectra (Additional file 1: Figure S1A), which is in agreement with the previously published data [19]. On the other hand, for PD, absorption peaks were present in the short-wave (Soret band) and long-wave (Q-band) regions of the spectrum (Additional file 1: Figure S1B). Although PS and PD accumulated in GL261 glioma cells during in vitro incubation, their uptake rates and intracellular localizations differed significantly. PS had a lower rate of accumulation in GL261 cells than PD because it is a hydrophilic compound that enters cells by active endocytosis (Additional file 1: Figure S1C, S1D). Notably, incubation for 4 h was enough for both photosensitizers to accumulate to a significant extent in GL261 cells. Therefore, this incubation time was chosen for analysis of their photodynamic activities.

It is known that the capacity to induce ICD is associated with localization of the photosensitizers or drugs in the ER and their ability to induce ER stress [7, 11, 27]. Therefore, we next analyzed sub-cellular localization of PS and PD in glioma GL261 cells. PS and PD differed significantly not only in the rate of internalization but also in subcellular localization. PS co-localized mostly with lysosomes but possibly with other intercellular vesicles as well (Fig. 1a). However, PS was not detected in organelles such as mitochondria, endoplasmic reticulum (ER), Golgi apparatus and nucleus (Fig. 1a). This localization pattern is typical for hydrophilic phthalocyanines due to the lysosome-tropic effect [28] and is in agreement with previous reports, including ours [29, 30].Fig1Fig. 1

Subcellular distribution of photosens (PS) and photodithazine (PD) in cancer cells. The subcellular localization of PS (a) and PD (b) differ significantly as studied by confocal microscopy after 4 h of incubation (both at 10 μM) with GL261 cells. PS is mostly co-localized with lysosomes and, potentially, other intercellular vesicles (a). PS was not detected in mitochondria, ER, Golgi apparatus and nuclei. In contrast, PD accumulated mostly in the ER and Golgi apparatus (b). Fluorescence signal profiles along the lines indicated by the white arrow on the images with superimposed fluorescence channels. I_fl: fluorescence intensity; D: distance along the specified segment. The following dyes were used: LysoTracker Green for lysosomes; MitoTracker Green for mitochondria; ER-Tracker for ER; BODIPY FL С5-ceramide for Golgi apparatus. Scale bars, 20 μm

Next, we analyzed the possibility of inducing cell death in glioma GL261 cells by treatment with PS or PD followed by irradiation with a light dose of 20 J/cm². The control GL261 cells were incubated in the dark with the same doses of photosensitizers for 4 h and then incubated further. Cell death was not induced by PS in concentrations of up to 100 μM in the dark (Fig. 2a) but PD at > 30 μM significantly reduced cell viability (Fig. 2a). Irradiation with a light dose of 20 J/cm² resulted in cell death at concentrations of photosensitizers not exceeding ~ 1 μM (Fig. 2a, b). PS and PD had IC₅₀ of 0.96 μM and 0.8 μM, respectively, after irradiation of GL261 cells with a light dose of 20 J/cm².Fig2Fig. 2

Cell death analysis by MTT assay in cancer cells treated with PDT-PS or PDT-PD. a Dark toxicity (black lines) was analyzed after incubating GL261 cells with the respective photosensitizer in serum-free medium for 24 h. For PDT-induced cell death (red lines), cells were first incubated with 10 μM PS or PD in serum-free medium for 4 h and then they were irradiated with a light dose of 20 J/cm² using a LED light source (615–635 nm). MTT assays were performed 24 h after irradiation. ^#IC₅₀ for PS was 0.96 μM [0.79–1.18] and for PD 0.8 μM [0.67–0.92]; the values were calculated with 95% confidence intervals (3 to 5 individual experiments with three replicates in each). b Morphology of GL261 cells before and 60 min after PDT. Cells were stained with propidium iodide (blue). Scale bars, 20 μm. c Effect of different inhibitors on cell death of GL261 cells induced by PS-PDT or PD-PDT. The following inhibitors were used: 25 μM zVAD-fmk (apoptosis), 20 μM Necrostatin-1 s (necroptosis), and 1 μM Ferrostain-1 or 10 μM DFO (ferroptosis). Cell death in GL261 cells induced by PS-PDT was significantly blocked by zVAD-fmk, ferrostatin-1, and DFO. In contrast, cell death induced by PD-PDT was inhibited only by zVAD-fmk. Cells were first incubated with 10 μM PS or PD in the presence of the respective cell death inhibitor in serum-free medium for 4 h and then the medium was replaced to photosensitizer-free medium followed by irradiation at 20 J/cm² using a LED light source (615–635 nm). After irradiation, the respective inhibitor was added again. MTT assays were performed 13 h after irradiation. Cell viability of the untreated control (no photosensitizer or inhibitor) was set as 100% (dotted line). The values are the means ±SEM. Statistical significance was calculated by using t-criteria with Bonferroni correction, *p < 0.05; ^#IC₅₀ values are given with 95% confidence interval

One of the main characteristics of ICD is the emission of DAMPs, such as surface exposure of CRT and release of HMGB1 and ATP, which have a beneficial role in anticancer therapy due to their interaction with the innate immune system [4, 37, 38]. In GL261 and MCA205 cells, double staining with Sytox Green, a plasma impermeable dye, and anti-CRT antibodies showed that CRT exposure was a rapid process detectable within 1.5–3 h after PS-PDT or PD-PDT treatment (Fig. 3a, b and Additional file 2: Figure S2A, S2B). Of note, upregulation of CRT at the surface of GL261 cells after PS-PDT or PD-PDT was more pronounced than after MTX, a positive control and a known ICD inducer [3, 39]. We also observed that GL261 and MCA205 cells induced by PS-PDT or PD-PDT release HMGB1 (Fig. 3c) and ATP (Fig. 3d) but this was associated with plasma membrane rupture (Additional file 2: Figure S2C). Thus, both cancer cell lines stimulated with PS-PDT or PD-PDT induce emission of the three crucial DAMPs (CRT, HMGB1 and ATP), which points to the immunogenic nature of cell death.Fig3Fig. 3

Cell death in cancer cells is associated with CRT exposure at the cell surface and HMGB1 and ATP release. a and b Quantification of flow cytometry analysis of CRT exposure at the cell surface of Sytox Green negative cells. GL261 (a) and MCA205 (b) cells were recovered after 1.5 h and 3 h of treatment with either PDT-PS or PDT-PD or left untreated (live). As a positive control, cells were stimulated for 24 h with the ICD inducer, MTX (2 μM). Calreticulin exposure values represent the mean values ± SEM from three independent experiments (each experiment was done in a duplicate). Statistical significance was calculated by using Mann Whitney non-parametric test, *p < 0.05. c GL261 and MCA205 cells were recovered for 24 h after PDT-PS or PDT-PD treatment or left untreated (live) and HMGB1 was measured in the supernatants. Cell death was analyzed by an MTT assay, is presented in Additional file 2: Figure S2C. HMGB1 values represent the mean values of four independent experiments. Statistical significance was calculated by Mann Whitney non-parametric test, *p < 0.01. d GL261 and MCA205 cells were recovered for 24 h after PDT-PS or PDT-PD treatment or left untreated (live) and ATP was measured in the supernatants. ATP values represent fold increase relative to untreated cells and the mean values of eight independent experiments. Statistical significance was calculated by using Mann Whitney non-parametric test, * p < 0.006

Phagocytosis of cancer GL261 and MCA205 cells killed by PS-PDT or PD-PDT by BMDCs was analyzed in vitro (Fig. 4a, d and Additional file 3: Figure S3A, S3B). After co-culturing live, untreated cancer cells or PDT-treated cells with BMDCs, only dying cancer cells were effectively engulfed by BMDCs. Increasing the ratio of BMDCs to dead GL261 or MCA205 cells from 1:1 to 1:5 proportionally increased the rate of their engulfment (Fig. 4a, d and Additional file 3: Figure S3A, S3B).Fig4Fig. 4

Phagocytosis assay and analysis of BMDCs maturation in vitro. Tumor cells dying after treatment with PS-PDT or PD-PDT were efficiently engulfed by BMDCs in vitro (a and d). The data for the uptake of GL261 (a) and MCA205 (d) cells treated with PS-PDT or PD-PDT represent the mean values ± SEM of the duplicates from three independent experiments The rate of phagocytosis increased with the increase in the number of dying/dead cells (1:1 versus 1:5). Statistical significance was calculated by two-way ANOVA, *p < 0.01. Representative flow cytometry dot plots show the uptake of CMFDA-labeled dead GL261 and MCA205 cell material by BMDCs (CD11c⁺CMFDA⁺ double-positive cells) are shown in the Additional file 3: Figure S3A, S3B. b-f Tumor cells dying after PS-PDT or PD-PDT treatment induce BMDC maturation in vitro. Co-culture of BMDCs with dying GL261 (b) and MCA205 (e) cells in two different ratios (1:1 and 1:5) and the percentage of CD11c⁺CD86⁺ BMDCs is expressed as the mean value ±SEM. Statistical significance was calculated by a Mann-Whitney non parametric t-test, *p < 0.01. Co-culture of BMDCs with dying GL261 (c) and MCA205 (f) cells after treatment with PS-PDT or PD-PDT in two different ratios (1:1 and 1:5) and the percentage of CD11c⁺CD40⁺ BMDCs is expressed as the mean value ±SEM of five independent experiments for PS-PDT and four independent experiments for PD-PDT; each experiment was done in the duplicate. In all figure panels, BMDCs stimulated with LPS served as a positive control. MCA205 cells subjected to the several rounds of freeze-thaw (F/T) cycles were used as a negative control in (e and f). Statistical significance was calculated by a Mann-Whitney non parametric t-test, p < 0.05. g Absolute concentrations of IL-6 are the mean values ± SEM from three independent experiments in the co-cultures of BMDCs with the respective target MCA205 cells at three different ratios (1:1, 1:5 and 1:10). LPS treated BMDCs were used as a positive control. Statistical significance was calculated by a Mann-Whitney non parametric t-test. The differences are shown by comparing the respective group with BMDCs co-cultured with either *live MCA205 or ^#F/T MCA205 cells. p < 0.03

To investigate the ability of cancer cells treated with PS-PDT or PD-PDT to activate the adaptive immune system, we carried out a well-established in vivo mouse fibrosarcoma MCA205 cancer vaccination experiment in immunocompetent C57BL/6 J mice (Fig. 5a) [42]. The experimental conditions for inducing cell death by PS-PDT and PD-PDT were optimized for the MCA205 cell line, which is conventionally used in this experimental model (data not shown). Next, we immunized C57BL/6 J mice with MCA205 cells that were dying after PS-PDT or PD-PDT treatment (Fig. 5b). Negative control mice were injected with PBS [42] or with MCA205 cells undergoing accidental necrosis. The immunized mice were then challenged with live MCA205 tumor cells. Protection against tumor growth at the challenge site was interpreted as a sign of successful priming of the adaptive immune system. Mice immunized with MCA205 cells treated with PS-PDT or PD-PDT showed signs of robust activation of the adaptive immune system and protection against tumor growth. By contrast, there was tumor growth in most of the mice immunized with PBS (Fig. 5c), which confirms our in vitro findings and points to the strong immunogenic properties of cancer cells treated with PS-PDT or PD-PDT. Moreover, the tumors growing at the challenge site of the PBS-vaccinated mice was large in size and occurred earlier (Fig. 5d), confirming that dying cancer cells are strongly immunogenic in vivo. Notably, the mice that were vaccinated with the same number of F/T cells developed more tumors at the challenge site (Fig. 5c and d), confirming the previously published findings that accidental necrotic cells are less immunogenic [39]. These data indicate that induction of death in cancer cells by PS-PDT or PD-PDT activates an adaptive immune response, which is one of the important properties of ICD.Fig5Fig. 5

Tumor cells dying after PS-PDT or PD-PDT treatment induce anti-tumor immunogenicity in vivo. a In vivo prophylactic tumor vaccination model. b Cell death measured by flow cytometry of the cells used for immunization of the mice in (c). The cells used for immunization were stimulated with PS-PDT or PD-PDT and re-suspended in PBS before injection. c shows the evolution of tumor incidence over time as a Kaplan–Meier curve. MCA205 cells treated with PS-PDT or PD-PDT were used to vaccinate C57BL/6 J mice, which were challenged 1 week later with living cells of the same type. Dying MCA205 cells induced by PS-PDT or PD-PDT triggered an anti-tumor immune response when mice were immunized with 5 × 10⁵ cells. The statistical difference from PBS immunization (negative control) was calculated by a long-rank Manel-Cox test, *p < 0.01. d The size of the tumors growing at the challenge site of the mice in the prophylactic tumor vaccination experiments used in (c). The statistical differences from PBS immunization or immunization with accidental necrotic cells (F/T) are shown for each vaccination group and was calculated by a Mann-Whitney non parametric t-test, *p < 0.05. *Different from PBS group; ^#different from F/T group