Introduction

Anticancer dendritic cell (DC) vaccination is an immunotherapeutic approach that harnesses the power of the body’s own immune system to fight tumors.1–3 By presenting tumor antigens (TAs) to T cells, DCs activate and educate immune cells to recognize and attack cancer cells.4 The most common vaccination protocol is based on DC harvesting from the cancer patient and consecutive in vitro loading with TAs before reintroduction into the patient.5 Response rates of DC vaccination, however, rarely surpass 15%,6 and only one such product, Sipuleucel-T, has received FDA (Food and Drug Administration) approval to date.7 While these modalities offer the possibility of tailoring DC vaccination to individual patients, there are constraints regarding the identity and amount of TAs presented by DCs that will ultimately support an efficient T cell response on transfer back to the patient. Another hurdle is the cost of both DC engineering in GMP facilities and molecular assays required for patient stratification (to identify those most likely to benefit from vaccination).8

A more straightforward strategy might, therefore, consist in the use of DCs that would be exposed in situ to tumors prechallenged to acutely release a maximum of antigens covering the large proteomic diversity of cancer cells, also called anonymous antigens.9 10 Interestingly, while the endocytotic process is downregulated in mature DCs (to avoid the presentation of self-antigens encountered before activation), immature DCs (iDCs) have the potential to avidly capture antigens through a variety of mechanisms, including macropinocytosis and receptor-mediated paths.11 We, therefore, reasoned that imposing a spatial constraint to iDCs during the exposure to TAs might curb tolerance development and be favorable to a consecutive anticancer T cell response. Induction of immunogenic cell death (ICD) could meet these criteria since it combines the local release of antigens by killed cancer cells and the creation of an environment prone to an immune response. The peritoneal cavity could represent such a spatial delineation to target local tumors with an ICD-inducing modality and to introduce highly endocytotic iDCs without any requirements to mature or engineer them. This site is actually of major therapeutic importance, as peritoneal carcinomatosis has a significant negative impact on the progression of various abdominal cancers and currently represents an unmet medical need.

Photodynamic therapy (PDT) is among the most potent inducers of ICD.12–15 We have previously reported that potent vaccine may be generated by exposing in vitro iDCs to PDT-killed cancer cells.16 17 PDT involves the use of a photosensitizer (PS) that is activated on light exposure. While most porphyrinic PSs are activated by specific wavelengths of light, others respond to the full spectrum of white light, including OR141, a non-porphyrinic PS that we previously identified.18 Although this may appear as a significant hurdle in terms of penetration of light into tissues, we have shown that following systemic administration of OR141 in tumor-bearing mice,18 substantial growth inhibitory effects are observed when subcutaneous tumors are exposed to an external LED lamp placed a few centimeters away from the skin of the animal.13 19 This observation prompted us to explore, here, the possibility to use the white light of an endoscope inserted during laparoscopy to deliver PDT into the peritoneal cavity and thereby exert in situ cytotoxic effects toward a local tumor but also preparing the soil for subsequent iDC administration.

Interestingly, PDT was recently reported to promote ferroptosis in response to the high pro-oxidant stimulus resulting from the generation of singlet oxygen and more conventional ROS.15 20–22 These data led us and other investigators to hypothesize that ferroptotic cell death triggered by PDT is immunogenic, thereby offering an explanation to anterior studies supporting the potential of combining PDT and DC vaccination.23 24 This hypothesis was further supported by breakthrough studies demonstrating that ferroptosis directly participates in the cytotoxic effects of activated T cells.25 26 By contrast, evaluation of the immunogenicity of ferroptosis itself led to contrasted results with a study documenting that ferroptotic cancer cells were poorly engulfed, dampening antigen cross-presentation,27 while others investigators reported a strong immune response although possibly dependent on the biological context.28–30 In addition to our objective of implementing a laparoscopic methodology of in situ PDT protocol in combination with iDC vaccination, we, therefore, added ferroptosis as a second point of attention in the mechanistic dissection of DC-based immune response. This led us to identify ferroptosis as a necessary but not sufficient component of PDT involved in the priming of peritoneal carcinomatosis before iDC transfer and to validate the use of the endoscope white light as an efficient modality to locally photoactivate PS OR141 and induce a consecutive DC-mediated anti-cancer T cell response.

ResultsPDT exerts cytotoxic effects on three-dimensional tumor spheroids and cancer cells through ferroptosis

To integrate the possible influence of the microenvironment on the type of cell death induced by non-porphyrinic PS OR141,18 we first worked with three-dimensional (3D) spheroids to mimic biological gradients in tumors (such as nutrients, oxygen, and metabolic wastes) and address the issues of PS diffusion and light penetration. We first used 3D spheroids made of colorectal CT26 cancer cells, treated or not with one of the following drugs (used at the maximal non-toxic concentration when used alone): a pan-caspase inhibitor of apoptosis (zVAD-fmk), a kinase RIP1 inhibitor of necroptosis (necrostatin-1), or ferrostatin-1, a radical-trapping antioxidant inhibiting ferroptosis.31 Ferroptosis was the major contributor to cell death induced by photoactivation of OR141, as ferrostatin-1 considerably decreased cytotoxicity over time at the different PS concentrations while necroptosis and apoptosis inhibitors failed to do so (figure 1A). A shift to the right of the dose-response curve was also observed in the presence of ferrostatin-1 confirming the role of lipid ROS in the PDT-induced cell death (figure 1B). Similar results were obtained with spheroids made of AB12 mesothelioma cells (another cancer type prone to peritoneal carcinomatosis) that were slightly more sensitive to PDT effects than CT26 spheroids (figure 1B). We repeated these experiments in CT26 and AB12 cancer cells cultured as 2D monolayers and found the same profile of response and sensitivity to ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 (figure 1C and online supplemental figure 1A). By contrast, as observed in 3D spheroids, necroptosis and apoptosis inhibitors failed to dampen PDT-induced cytotoxicity (online supplemental figure 1B). Annexin V/7-AAD flow cytometry cell death analysis further confirmed that activated PS dose-dependently induces cell death in a ferrostatin-dependent manner (see upper right quadrants in online supplemental figure 1C). Necrostatin-1 and Z-VAD-FMK did not reduce the extent of double positive dead cells (and even increased it a bit). A modest, dose-dependent increase in Annexin V-positive cells (lower right quadrant), slightly inhibited by Z-VAD-FMK, was observed, suggesting a marginal proapoptotic effect of PDT in addition to its prominent proferroptotic mode of cell death (online supplemental figure 1C). As further support of photoactivated PS-induced ferroptosis, we found that PDT triggered induction of COX2, considered as a biomarker of ferroptosis, and heat shock protein 70, an oxidative stress marker, along with an increase in malondialheyde (MDA) signal, a secondary product of lipid peroxidation (figure 1D); the PDT-induced upregulation of these markers was prevented in the presence of ferrostatin-1 (figure 1D). Besides Hsp70, we also looked for other markers of ICD, including the release of ATP, a well-known danger signal. We found that ATP release in the extracellular medium was dose-dependently increased in response to PDT (figure 1E). We were unable to detect calreticulin translocation (an “eat-me” signal) due to the extended time required for dissociation and staining of cells from 3D spheroids. However, it is worth noting that we had previously characterized its translocation in the same cancer cells cultured as monolayers and exposed to PDT.17 In parallel, we also performed LegendPlex analysis following photoactivation of PS in spheroids. We found a notable upregulation of multiple chemokines, most of them known to be involved in immune cell migration and recruitment (figure 1F). While assay sensitivity limited detection at the highest PS concentration, the increase in all the chemokines (except one) was prevented with ferrostatin-1 (figure 1F). Further evidence of PDT’s potential to induce an anticancer immune response was obtained by documenting prophylactic vaccination in mice exposed to PDT-killed cancer cells, an approach regarded as the gold standard for demonstrating the ability of a therapeutic modality to induce ICD. Significant tumor growth delay and improved mouse survival were observed after challenging subcutaneously vaccinated mice with intraperitoneal (i.p.) injections of live cancer cells (figure 1G,H).

Figure 1

PDT induces ferroptosis and immunogenic cell death. (A–F) CT26 colorectal carcinoma and AB12 mesothelioma two-dimensional (2D) cell cultures and 3D spheroids were treated with the following drugs: ferrostatin-1 (10 µM), liproxstatin-1 (10 µM), necrostatin-1 (20 µM for 2D and 30 µM for 3D), Zvad-fmk (25 µM for 2D or 40 µM for 3D). After 48 hours pretreatment, cell cultures or spheroids were incubated with PS-OR141 in the dark for 1 hour (2D) or 4 hours (3D) at the indicated concentrations, followed by a washing step. Photoactivation was performed using an LED light source for 60 min (2D) or 90 min (3D) and incubation was continued after the renewal of cell death inhibitors. (A) PDT-induced cytotoxicity over time in CT26 spheroids (cytotox green dye, Incucyte) in the presence of different cell death inhibitors. (B, C) Dose-response curves depicting the viability of CT26 and AB12 (B) 3D spheroid (propidium iodide measurement by flow cytometry) and (C) 2D cell monolayers (PrestoBlue) 48 hours and 24 hours post-PDT, respectively, in the presence of 10 µM ferrostatin-1. (D) Representative COX2, HSP70, MDA and β-actin immunoblots from CT26 spheroids collected 20 hours post-PDT. (E) Extracellular ATP release from CT26 spheroids (expressed as fold change) was determined 30 min post-PDT. (F) Heat map of LEGENDplex proinflammatory chemokines (13-plex) released by CT26 spheroids 3 hours post-PDT. N=2. (G, H) Prophylactic vaccination model. Mice were injected s.c. with 1×106 luc+ AB1 2D cells exposed in vitro to photoactivated 100 nM PS-OR141 (or PBS as control) and challenged 1 week later with i.p. injection of live 1×106 luc+ AB1 cells. (G) Tumor burden monitored by bioluminescence (expressed as photons/s/cm2/sr) and (H) corresponding survival curves. (**p≤0.01, N=8 mice per group). In vitro data (B, C, E) are plotted as the means±SEM from 3 independent experiments performed with ≥3 technical replicates (***p≤0.001; ****p≤0.0001; ns, p>0.05); when spheroids are involved, minimum of 6 spheroids were pooled together per condition. Significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Log-rank (Mantel-Cox) test was used for survival curves. ANOVA, analysis of variance; i.p., intraperitoneal; PDT, photodynamic therapy.

PDT-induced ferroptosis reduces spheroid outgrowth while increasing central cell death

Since the effects of bona fide ferroptosis inducers were reported to be poorly immunogenic,27 we next compared the fate of spheroids exposed to either PDT or RSL3, a well-known ferroptosis inducer. We first validated the capacity of both strategies to induce lipid peroxidation through the detection of Bodipy 581/591 C11 green fluorescent signal (online supplemental figure 2A,B). A net difference was, however, observed in the spheroid growth with PDT exerting a rapid and profound increase in cytotoxicity together with a reduction in the spheroid volume whereas RSL3 cytotoxic effects were less pronounced and developed slower (figure 2A,B). We then compared the capacity of photoactivated PS and RSL3 to induce cell death in 3D spheroids by evaluating the distribution of propidium-iodide labeling across the cell layers (figure 2C,D). This led us to reveal that as soon as 6 hours post-PDT administration (figure 2C and online supplemental movie 1A,B), the external diameter of the spheroid was dose-dependently reduced (figure 2E for quantification) while the internal dead cell core was increased (figure 2F for quantification). The latter was determined based on the central area of propidium iodide exclusion, a paradoxical but common observation in spheroids thought to result from chromatin condensation and/or fragmentation and further leakage of damaged nuclear contents (see figure 2C). Both the PDT-induced reduction in spheroid size and expansion of the central dead cell area were prevented by ferroptosis inhibitor ferrostatin-1 (figure 2C,E,F and online supplemental movie 1C). By contrast, ferroptosis inducer RSL3 reduced the spheroid area to a much lesser extent and failed to increase the death cell core (figure 2D–F for quantification). Rapid PDT-induced inhibition of 3D spheroid growth and the progressive detachment of dead cancer cells were confirmed in phase contrast pictures depicting spheroids exposed to increasing doses of PS (vs RSL3 exposure) (online supplemental figure 2C). Interestingly, spheroid staining with the hypoxia marker pimonidazole led us to identify the central hypoxic core of the spheroid as the preferred location where PDT-induced cell death is initiated within the first few hours post-photoactivation; this is particularly visible at low PS dosage (30 nM OR141) (online supplemental figure 2D). At the higher doses, PI staining appeared excluded from the central core of the spheroids (as in figure 2C) and expanded over the hypoxic zone, progressively reaching the more external layers of the entire spheroid (online supplemental figure 2D). Altogether these results indicate that even if ferroptosis represents the major mode of cell death triggered by PDT, a non-canonical cell death mode leads to the preferred induction of cytotoxicity in the depth of 3D spheroids.

Figure 2

PDT exhibits distinct cell death kinetics and potency compared with the ferroptosis inducer RSL3. (A, B) CT26 three-dimensional (3D) spheroids were exposed to PS OR141 before 90 min LED light irradiation or to the ferroptosis inducer RSL3. (A) Cytotoxicity (cytotox green dye, Incucyte) and (B) growth (spheroid volume) (B) were followed over time. (C–F) CT26 3D spheroids were pretreated with 10 µM ferrostatin-1 and incubated 48 hours later with PS OR141 in the dark for 4 hours, followed by a washing step. Photoactivation was performed using an LED light source for 90 min and incubation was continued after the renewal of ferrostatin-1. A similar protocol was applied for RSL3 treatment with 48 hours pretreatment and renewal for final incubation. Representative light sheet microscopy pictures depicting propidium iodide-positive dead cells (red) and counterstained nuclei (blue) 6 hours after (C) PDT or (D) 5 µM RSL3 treatment. Scale bar: 100 µm. Quantification of (E) spheroid volumes (µm3) and (F) necrotic core volume (µm3) determined using Zen software (see white arrows in the top left panels). Data are plotted as the means±SEM (*p≤0.05; **p≤0.01; ****p≤0.0001; ns, p>0.05) from 3 independent experiments performed with ≥3 technical replicates; when spheroids are involved, minimum of 6 spheroids were pooled together per condition. Significance was determined by one-way ANOVA with Tukey’s multiple comparison test. ANOVA, analysis of variance; ns, noi significant; PDT, photodynamic therapy.

PDT promotes the recruitment of iDCs in 3D spheroids and an increase in their cell volume

To evaluate the response of exogenous iDCs facing PDT-induced cell death, we first tracked their fate in 3D spheroids previously exposed or not to photoactivated PS OR141 (figure 3A). The resulting heterotypic 3D spheroids revealed a PS dose-dependent infiltration of CD11c+ cells as determined in equatorial sections of 3D spheroids (figure 3B,D) whole spheroids analyzed using light sheet microscopy (LSM) (figure 3C,F for quantification; see also online supplemental movie 2A–C). Of note, the polarization of the entry of iDCs into spheroids corresponds to the bottom of the well where iDCs accumulate to a larger extent on addition in the culture wells. PDT induced a parallel reduction in spheroid size (figure 3B,C,G for quantification, also online supplemental movie 2C). Interestingly, LSM-based 3D cell reconstruction modeling strongly suggested that the enhanced DC infiltration was associated with an increase in DC volume (ie, DC size), indicating a progression toward a matured state (figure 3C and online supplemental movie 2C). Both iDC infiltration and reduction in spheroid size were prevented in the presence of ferrostatin-1 (figure 3B,F,G, online supplemental movie 2D). Interestingly, ferroptosis inducer RSL3 failed to induce such increased DC infiltration (figure 3D–F). We further validated by flow cytometry the PDT-triggered increased infiltration of CD11c+ cells inside 3D spheroids, and the failure of RSL3 to do so (figure 3H and online supplemental figure 3A for gating strategy). Flow cytometry allowed us to confirm that the volume of CD11c+ cells dose-dependently increased on exposure to activated PS OR141 (figure 3I), together with a significant increase in the number of MHCI-positive and MHCII-positive CD11c+ cells in PDT-exposed spheroids (figure 3J,K). The expression level of costimulatory molecules CD40, CD80, CD86 and CCR7 was, however, not increased in CD11c+ MHCII+ cells 48 hours after spheroid infiltration, nor was the relative number of DCs positive for these markers at that time (online supplemental figure 3B–E). The need to pool approximately 20 spheroids per condition to extract DCs limited the ability to conduct extensive time course studies to identify a more optimal detection window using flow cytometry. Finally, it is worth to mention that independently of PDT exposure, ferroptosis inhibitor ferrostatin-1 showed a small increase in the expression in maturation markers (online supplemental figure 3B–E and figure 3J,K) that was, however, not associated with an increase in DC volume (figure 3I). Altogether, these data indicate that PDT-induced cell death in spheroids (but not exposure to ferroptosis inducer RSL3) drives a major recruitment of DCs, along with an increase in cell volume and the absolute numbers of MHCI+ and MHCII+ CD11c+ cells, suggesting an enhanced capacity of DCs to capture and present antigens.

Figure 3

PDT-induced ferroptosis promotes iDC infiltration in three-dimensional (3D) spheroids together with an increase in MHCI and MHCII expression. (A) Protocol of iDC transfer to CT26 spheroids prechallenged with photoactivated OR141 or RSL3. CT26 3D spheroids were pretreated with 10 µM ferrostatin-1 and incubated 48 hours later with PS OR141 in the dark for 4 hours, followed by a washing step. Photoactivation was performed using an LED light source for 90 min and incubation was continued after the renewal of ferrostatin-1. A similar protocol was applied for RSL3 treatment with 48 hours pretreatment and renewal for final incubation. Exogenous iDCs (5×103 per spheroid) were added 48 hours post-PDT or after the last RSL3 addition. (B, D) Representative IF pictures depicting the extent of CD11c+ cell infiltration after spheroid exposition to (B) PDT or (D) 5 µM RSL3. Scale bar: 100 µm. (C, E) Representative modeling of CD11c+ cell infiltration (red) derived from light sheet microscopy-based reconstruction (Imaris program). Scale bar: 50 µm. (F) Quantification of wholemount IF-based infiltrated CD11c+ cells and (G) impact on spheroid growth for the indicated conditions. (H) Flow cytometry-based quantification of infiltrated CD11c+ cells and associated changes in (I) iDC volume, (J) MHCI and (K) MHCII expression. Data are plotted as the means±SEM (*p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns, p>0.05) from 3 independent experiments performed with ≥3 technical replicates (minimum of 6 spheroids were pooled together per condition). Significance was determined by one-way ANOVA with Tukey’s multiple comparison test. ANOVA, analysis of variance; iDC, immature dendritic cell; ns, not significant; PDT, photodynamic therapy.

PDT promotes DC-mediated activation of CD4+ and CD8+ T cells and associated cytotoxic effects in 3D spheroids

We next evaluated whether PDT-dependent increase in infiltration and DC volume supported an increased capacity of T cell activation despite a mitigated stimulation of costimulatory molecules. For this purpose, we added naive T cells (CD62L+ CD44− CD25− CD3+) 48 hours after iDCs transfer to PDT-exposed spheroids (figure 4A and online supplemental figure 4A for gating strategy). After 6 days of coincubation, a significant T cell-dependent reduction in spheroid growth was observed in a PS dose-dependent manner in the experimental set-up where DCs were present (see phase contrast pictures in figure 4B at 100 nM of photoactived PS and quantification in figure 4C). Note that when PDT alone was used, spheroids had the time to regrow after 6 days, masking the cytotoxic effects observed at earlier timing in figures 1–3). Propidium iodide staining further confirmed T cell-mediated cytotoxic effects consecutive to PDT/iDC administration (see IF pictures in figure 4B). The degeneration of 3D spheroids was such that the dead cell core was sometimes expelled due to spheroid loss of integrity (online supplemental figure 4B) giving rise to PI labeling covering most of the remaining spheroids (figure 4B, middle panel) or exhibiting an asymmetrical distribution (online supplemental figure 4B). Interestingly, T cell-mediated cytotoxic effects were not observed in the presence of ferrostatin-1 (figure 4B,C) and could not be recapitulated with ferroptosis inducer RSL3 (figure 4D). Evidence for T cell activation was further obtained by flow cytometry identifying an increase in the CD62L− CD44+ and CD69+ phenotype within both CD8+ (figure 4E,G) and CD4+ T cells (figure 4F,H) in the experimental set-up including iDC addition on PDT-exposed spheroids (see also online supplemental figure 4C,D for quantification). Here again, T cell response was blunted when ferrostatin was used during the PDT prechallenge (figure 4E–H) but could not be recapitulated by ferroptosis inducer RSL3 (online supplemental figure 4E–G).

Figure 4

PDT promotes DC-mediated T cell response against three-dimensional (3D) spheroids. (A) Protocol of T cell transfer to CT26 spheroids primed by iDCs. CT26 3D spheroids were pretreated with 10 µM ferrostatin-1 and incubated 48 hours later with PS OR141 in the dark for 4 hours, followed by a washing step. Photoactivation was performed using an LED light source for 90 min and incubation was continued after the renewal of ferrostatin-1. A similar protocol was applied for RSL3 treatment with 48 hours pretreatment and renewal for final incubation. Exogenous iDCs (5×103 per spheroid) were added 48 hours post-PDT or after the last RSL3 addition, followed 48 hours later by addition of CD62L+ CD44− CD25− CD3+ lymphocytes (5×103 per spheroid); cytotoxic activity was analyzed 6 days later. (B) Representative phase-contrast (left panel, scale bar: 200 µm) and fluorescence pictures depicting propidium iodide positive (red) signal (right panel, scale bar: 100 µm) from CT26 spheroids treated as indicated, and (C) associated changes in spheroid volume (µm3). (D) Lack of T cell-induced DC-mediated cytotoxic effects when RSL3 is used instead of PDT. (E–H) Flow cytometry-based phenotyping of CD8+ (E, G) and CD4+ (F, H) T cells using CD44, CD62L and CD69 markers from 3D spheroids treated as indicated. Data are plotted as the means±SEM (*p≤0.05; ** p≤0.01; ***p≤0.001; ****p≤0.0001; ns, p>0.05) from 3 independent experiments performed with ≥3 technical replicates (minimum of 6 spheroids were pooled together per condition). Significance was determined by one-way ANOVA with Tukey’s multiple comparison test. ANOVA, analysis of variance; DC, dendritic cell; ns, not significant; PS, photosensitizer.

Combining iDC vaccination with in situ endoscope light-induced PDT achieves a robust immune memory response against peritoneal carcinomatosis

To gauge the applicability of the strategy in vivo, we next set up a model of in situ PDT administration using an endoscope LED white light to photoactivate OR141 within the inflated mouse peritoneal cavity (figure 5A). Peritoneal carcinomatosis induced by i.p. injection of luciferase-expressing AB1 cancer cells was used as an orthotopic tumor model in immunocompetent mice and adoptive transfer of iDCs was consecutively carried out through i.p. injection post-PDT administration. It is worth mentioning that for these in vivo experiments we opted to use AB1 mesothelioma cells instead of mesothelioma AB12 and colon CT26 cancer cells used above. This decision was influenced by the aggressive nature of CT26 tumors, which often reached the ethical endpoint by days 7–10 (ie, before the immune response could develop) and at the opposite, by the slow growth of AB12 tumors leading to greater variability from mouse to mouse. AB1 cells were transduced with a luciferase-expressing vector to track the development of peritoneal carcinomatosis using in vivo detection of bioluminescence after luciferin administration (online supplemental figure 5A). Also, in a pilot study, we found that photoactivation of 40 mg/kg OR141 gave rise to lethal gastrointestinal toxicity in half of the mice whereas mice treated with 4 mg/kg OR141 dosage fully recovered from the procedure. As a proof of principle of the capacity of PDT alone to affect peritoneal carcinomatosis and induce ICD, we performed histological analysis of tumor samples collected 6 days after in vivo PDT. HE staining revealed extensive hypereosinophilic regions (online supplemental figure 5B) in comparison to peritoneal tumors from sham-operated mice, indicating extensive cellular damage resulting from PDT administration. Furthermore, hypereosinophilic areas were infiltrated by native T cells (online supplemental figure 5B). Multiplex immunohistofluorescence further confirmed an enhanced immune infiltration, demonstrating increases in both native CD11c+ DCs, and CD3+ and CD8+ T cells in PDT-treated tumors, while these immune populations were largely absent in tumors from sham mice (figure 5B). We next examined the effects of this PS dosage and a yet smaller OR141 concentration (0.4 mg/kg) on the capacity of iDCs to influence tumor growth and mouse survival (see protocol in figure 5C). Follow-up of tumor growth revealed that iDCs alone delayed tumor growth (figure 5D) and that the combination with a PDT prechallenge further increased this delay and even completely prevented tumor regrowth in some mice (figure 5E,F) without signs of gross toxicity or weight loss (online supplemental figure 5C). Mouse survival was eventually not influenced by iDC transfer alone (figure 5G), while the combination of PDT and iDC transfer significantly increased mouse survival (ie, 25% and 50% mice still alive at day 80 in response to 0.4 and 4 mg/kg OR141, respectively) (figure 5H,I and online supplemental figure 5D for complete curve overlapping). In the latter condition, rechallenge with i.p. injection of cancer cells at day 69 did not give rise to detectable tumors (figure 5I). Absence of relapse was confirmed up to day 130, confirming a strong anticancer memory immune response. In a parallel experiment, we also documented that iDC administration in mice bearing larger tumors (PDT administered at day 15 instead of day 7) also led to tumor growth delay and mouse survival (ie, 20% mice) (online supplemental figure 5E,F).

Figure 5

Laparoscopic PDT administration promotes iDC-based (memory) immune response against peritoneal tumors. Balb/CByJ mice were i.p. injected with 1×106 luciferase-positive AB1 tumor cells and exposed 7 days later to PDT. PS-OR141 (0.4 or 4 mg/kg) was injected i.p. 90 min before laparoscopy and photo-activated for 15 min on endoscopal light exposure; sham animals were used as controls. (A) Experimental set-up depicting laparoscopy procedure to introduce the endoscope LED light source in the inflated peritoneal cavity. (B) Representative multiplex immunofluorescence pictures depicting immune cell infiltration (CD11c+ cells in red, CD3 + T cells light blue and CD8+ T cells in green) 6 days post in situ PDT. Scale bar: 200 µm. (C) Protocol of in situ PDT-based iDC vaccination against mouse peritoneal tumors. iDCs (2×106) were injected i.p. 60 min after in situ PDT. (D–F) Tumor burden monitored by bioluminescence (expressed as photons/s/cm2/sr) and (G–I) corresponding survival curves. n=6–8 mice per group. Significance was determined by comparing curves presented in G–I with log-rank (Mantel-Cox) test (*p≤0.05). Experimental endpoint was determined when mice reached the ethical limits (ie, bioluminescence signal over 50,000 ph/s/cm2/sr or euthanasia (†) on major ascite accumulation). In (I), arrow at day 69 indicates rechallenge by i.p. injection of 1×106 luciferase-positive AB1 tumor cells. (J, K) Immune cell activation analysis carried out by flow cytometry from mesenteric lymph nodes collected 10 days after treatment. Bar graphs represent (J) CD40+, CD80 +, CD86 + MHCII + and OX40L + MHCI + CD11+ cells and (K) CD62L+ CD69+ early activated CD8+ and CD4+ T cells in PDT 4 mg/kg+iDC experimental condition (vs sham mice). Data are plotted as the means±SEM (*p≤0.05; ns, p>0.05) from 3 to 4 mice per group. Significance was determined by Student’s t-test. iDC, immature dendritic cell; i.p., intraperitoneal; MLN, mesenteric lymph nodes; PDT, photodynamic therapy.

To further characterize the PDT-induced immune remodeling, we collected mesenteric lymph nodes (MLNs) and spleens (10 days after treatment initiation) to evaluate DC maturation and T cell activation. In the MLNs from PDT/iDC-treated mice, we detected a consistent increase in the frequency of mature CD40+, CD80+, CD86+ MHCII+ DCs (figure 5J), together with a significant increase in CCR7+ MHCII+ DCs in the spleen (online supplemental figure 5G). This was accompanied by a significant rise in early-activated CD62L+ CD69+ CD8+ (and CD4+) T cells within MLNs (figure 5K), suggesting that matured DCs effectively induced T cell activation. Moreover, a trend toward activated CD62L− CD69+ CD8+ T cells was observed in both the spleen and MLNs (online supplementa