Vaccination with early ferroptotic cancer cells induces efficient antitumor immunity

Abstract

Background: Immunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term survival of patients. Induction of immunogenic apoptosis or necroptosis in cancer cells does activate antitumor immunity, but resistance to these cell death modalities is common. Therefore, it is of great importance to find other ways to kill tumor cells. Recently, ferroptosis has been identified as a novel, iron-dependent form of regulated cell death but whether ferroptotic cancer cells are immunogenic is unknown.

Methods: Ferroptotic cell death in murine fibrosarcoma MCA205 or glioma GL261 cells was induced by RAS-selective lethal 3 and ferroptosis was analyzed by flow cytometry, atomic force and confocal microscopy. ATP and high-mobility group box 1 (HMGB1) release were detected by luminescence and ELISA assays, respectively. Immunogenicity in vitro was analyzed by coculturing of ferroptotic cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and activation/maturation of BMDCs (CD11c⁺CD86⁺, CD11c⁺CD40⁺, CD11c⁺MHCII⁺, IL-6, RNAseq analysis). The tumor prophylactic vaccination model in immune-competent and immune compromised (Rag-2^-/-) mice was used to analyze ferroptosis immunogenicity.

Results: Ferroptosis can be induced in cancer cells by inhibition of glutathione peroxidase 4, as evidenced by confocal and atomic force microscopy and inhibitors' analysis. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2^-/- mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells.

Conclusions: These results pave the way for the development of new therapeutic strategies for cancers based on induction of ferroptosis, and thus broadens the current concept of immunogenic cell death and opens the door for the development of new strategies in cancer immunotherapy.

Keywords: alarmins; cytotoxicity; immunogenicity; immunologic; immunotherapy; phagocytosis; vaccine.

Figure 1

Ferroptotic cell death induction in cancer MCA205 cells. (A) Ferroptotic cell death analysis by staining with Sytox Green (% of dead cells). Cell death was significantly reduced by ferroptosis inhibitors (DFO, Fer-1 and α-toc) whereas inhibitors of apoptosis (zVAD-fmk) and necroptosis (Nec-1s) were ineffective. The following concentrations of inhibitors were used: 25 µM zVAD-fmk, 20 µM Nec-1s, 1 µM FER-1, 10 µM DFO and 100 µM α-toc. The values are the means±SEM and represent four independent experiments. Statistical significance was calculated by two-way ANOVA followed by Dunnett’s multiple comparisons test: ****p<0.0001. (B) Morphological analysis of ferroptotic MCA205 cells by fluorescence microscopy showing swelling and rounding of MCA205 cells undergoing ferroptosis. MCA205 cells were stained with propidium iodide (red) and Hoechst33342 (blue). The images were derived from online supplemental movies S1. (C) Characterization of ferroptotic MCA205 cells by AFM revealing the formation of membrane protrusions (white arrows) on the surface of ferroptotic MCA205 cells. Each panel is divided into two images: left: 3D AFM image; right: whole cell 2D AFM image. (A–C) MCA205 cells were stimulated with 2.5 µM RSL3. DFO, deferoxamine; Fer-1, ferrostatin-1; RSL3, RAS-selectivelethal 3; α-toc, α-tocopherol.

Figure 2

Phagocytosis assay showing effective uptake of early ferroptotic cancer cells by BMDCs. (A) Ferroptotic cell death measured by flow cytometry of the MCA205 cells used in the phagocytosis assay shown in figure 2B, C. Quantification was done by Annexin-V (AnV) and Sytox Blue (Sytox) staining. The values are the means±SEM and represent four independent experiments. (B) Ferroptotic MCA205 cancer cells are efficiently engulfed by BMDCs. Of note, ferroptotic MCA205 cells are efficiently engulfed after 1 hour stimulation with RSL3 even in the absence of Annexin-V/Sytox Blue staining (figure 2A). MCA205 cells were stimulated with RSL3 for 1 hour or 24 hours. Data represent mean values±SEM from four independent experiments. Statistical significance was calculated by two-way ANOVA followed by Tukey’s multiple comparisons test: **p<0.01, ***p<0.001, ****p<0.0001. (C) The flow cytometry dot plots show the uptake of pHrodo-labeled ferroptotic cells by BMDCs (CD11c⁺pHrodo⁺ double positive cells). The rate of phagocytosis is proportionally increased by increasing the ratio of BMDCs: MCA205 (1:1 to 1:5). BMDCs, bone-marrow derived dendritic cells; RSL3, RAS-selectivelethal 3.

Figure 3

Ferroptotic cells induce maturation of bone-marrowderived dendritic cells (BMDCs) in a manner depending on the cell-death stage. (A) Representative flow cytometry dot plots demonstrating the gating and percentage of CD11c⁺MHCII⁺, CD11c⁺CD80⁺ and CD11c⁺CD86⁺ BMDCs cocultured in a 1:5 ratio with ferroptotic (RAS-selectivelethal 3 (RSL3) 1 hour, 3 hours, or 24 hours), untreated (viable), and mitoxantrone-treated (MTX) MCA205 cells. As a positive control BMDCs stimulated with lipopolysaccharide (LPS) were used. (B) Ferroptotic MCA205 cancer cells induce maturation of BMDCs in a manner that depends on the cell-death stage: early ferroptotic cells (after 1 hour or 3 hours of RSL3 stimulation) are more potent inducers of BMDCs maturation than late ferroptotic cells (after 24 hours of RSL3 stimulation). Notably, early ferroptotic cells (ie, after 1 hour of RSL3 stimulation) were as immunogenic as cancer cells treated with mitoxantrone (2 µM MTX, 24 hours), a positive control. BMDCs were cocultured with ferroptotic MCA205 cells in a 1:5 ratio. Dashed lines specify percentages of the CD11c⁺MHCII⁺, CD11c⁺CD80⁺ and CD11c⁺CD86⁺ populations, respectively, for BMDCs cocultured with viable MCA205 cells (control). BMDCs stimulated with LPS served as an additional positive control. Percentages of CD11c⁺MHCII⁺, CD11c⁺CD80⁺ and CD11c⁺CD86⁺ cells shown as mean values±SEM of four independent experiments for RSL3 1 hour, and seven independent experiments for other conditions. Two-way ANOVA was used to calculate the statistical significance: **p<0.01, ***p<0.001, ****p<0.0001. (C) Linear correlation of AnV⁺Sytox⁺ ferroptotic cells (figure 3E) and CD11c⁺MHCII⁺, CD11c⁺CD80⁺ or CD11c⁺CD86⁺ cells, respectively. (D) Absolute concentrations of IL-6 in cocultures of BMDCs with the respective target MCA205 cells at two different ratios (1:1, 1:5). LPS-treated BMDCs and coculture of BMDCs with MTX-treated MCA205 cells were used as a positive control. The values are the means±SEM of eight (for BMDCs alone, BMDCs cocultured with viable MCA205 cells, BMDCs cocultured with RSL3-treated MCA205 cells for 24 hours groups), seven (for BMDCs co-cultured with MTX-treated MCA205 cells, BMDCs co-cultured with RSL3-treated MCA205 cells for 3 hour groups) and six (for BMDCs treated with LPS, BMDCs cocultured with RSL-treated MCA205 cells for 1 hour groups) independent measurements performed in duplicates. Statistical significance was calculated by a Mann-Whitney nonparametric t-test: *p<0.05, ***p<0.001. (E) Ferroptotic cell death measured by flow cytometry of the MCA205 cells used in the maturation assay shown in figure 3A–C.

Figure 4

Early ferroptotic cells are immunogenic in vivo. (A) The in vivo prophylactic tumor vaccination model. The MCA205 cells used for immunization were stimulated with RSL3 for 3 hours or 24 hours and resuspended in PBS before injection. (B) Kaplan-Meier curve of the progression of tumor development over time. C57BL/6 J mice were vaccinated with 5 × 10⁵ MCA205 cells treated with RSL3. Then 1 week late the mice were challenged with viable MCA205 cells. MCA205 cells induced by RSL3 for 3 hours triggered an anti-tumor immune response in mice immunized with 5 × 10⁵ cells. Negative control mice were injected with PBS or with MCA205 cells undergoing accidental necrosis (F/T, freeze and thaw group). (C) Kaplan–Meier curve of the progression of tumor development over time. Rag-2^-/- mice were vaccinated with 5 × 10⁵ MCA205 cells treated with RSL3 for 3 hours. after 1 week, the mice were challenged with viable MCA205 cells. No antitumor immune response was triggered in mice Rag-2^−/− immunized with ferroptotic MCA205 cells. The statistical differences were calculated by a log-rank (Mantel-Cox) test. Survival curves comparison: *p<0.05, ****p<0.0001. PBS, phosphate-buffered saline; RSL3, RAS-selectivelethal 3.

Figure 5

Release of HMGB1 and ATP from ferroptotic cancer cells. (A) Cell death progression in cancer MCA205 cells on stimulation with RSL3 measured by Sytox Green fluorescence. Pool of three independent experiments. (B) Time-dependent ATP release from ferroptotic cancer MCA205 cells stimulated with RSL3. Note that the peak of ATP release corresponds to 6 hours of RSL3 stimulation but for 24 hours of stimulation ATP release is minimal. The values are the means±SEM of four independent measurements performed in triplicates. Two-way ANOVA was used to calculate the statistical significance: ***p<0.001, ****p<0.0001. (C) Release of HMBG1 from ferroptotic MCA205 cancer cells stimulated with RSL3. The peak of HMGB1 release was at 24 hours of ferroptosis induction with RSL3. This is in reverse to the release of ATP: release of HMGB1 was minimal for early ferroptotic time-points (1 and 3 hours). The values are the means±SEM of four independent measurements performed in duplicates. One-way ANOVA with Tukey’s multiple comparison test was used to calculate the statistical significance: *p<0.05, ****p<0.0001. (D) Ferroptotic cell death measured by flow cytometry of the MCA205 cells used for HMGB1 release. The values are the means±SEM of four independent measurements performed in duplicates. HMGB1, high-mobility group box 1; MTX, mitoxantrone; RSL3, RAS-selective lethal 3.

Figure 6

Early but not late ferroptotic cancer cells induce immunogenic cell death in vitro. (A) Cell-death recovery was measured by Sytox Green fluorescence in cancer MCA205 cells on stimulation with RSL3. After 1 hour, 3 hours and 6 hours of RSL3 stimulation, the cells were washed in PBS and reseeded in fresh culture medium (without RSL3) and cultured further for 23 hours, 21 hours or 18 hours, respectively, to give a total culture time. (B) Schematic representation of the measurement of ATP and HMGB1 release in the coculture of BMDCs with ferroptotic MCA205 cells shown in figure 6C, D. Viable or dying cancer MCA205 cells (induced with either RSL3 or MTX as positive controls) were harvested and cocultured with BMDCs for 3 hours (for ATP) or 24 hours (for HMGB1). Next, the supernatants were collected and analyzed for ATP using CellTiter-Glo Luminescent Cell Viability Assay (Promega) and for HMGB1 with ELISA kit (IBL-Hamburg). (C) The concentration of ATP in the coculture of BMDCs with ferroptotic MCA205 cells after 3 hours of coincubation. The values are the means±SEM of four independent measurements performed in triplicates. Two-way ANOVA was used to calculate the statistical significance (*p<0.05, **p<0.01). (D) The concentration of HMGB1 in the coculture of BMDCs with ferroptotic MCA205 cells after 24 hours. The values are the means±SEM of three independent measurements performed in duplicates. One-way ANOVA with Tukey’s multiple comparison test was used to calculate the statistical significance: ****p<0.0001. For positive control, BMDCs were cocultured with MCA205 cells killed by MTX (2 µM, 24 hours). Activation and maturation profiles of BMDCs from these experiments are shown in figure 3A–C. BMDCs, bone-marrow derived dendritic cells; HMGB1, high-mobility group box 1; MTX, mitoxantrone; PBS, phosphate-buffered saline; RSL3, RAS-selectivelethal 3.

Figure 7

Graphical abstract—timespan of immunogenicity of ferroptotic cancer cell death. After receiving ferroptotic stimuli, cancer cells start to undergo cell death (Phase 0) and release DAMPs (eg, ATP and HMGB1). After 3 hours of ferroptosis induction, cancer cells are in phase I, which is the early ferroptotic cell death stage, where the highest levels of ATP and HMGB1 are reached. At this time point, early ferroptotic cancer cells are engulfed by BMDCs and promote their activation and maturation, as evidenced by analyses of their phenotypic markers (MHCII, CD80, and CD86) and the pro-inflammatory cytokine IL-6. In addition, if early ferroptotic cells in Phase I are injected into mice, they lead to protective immunity in the mouse tumor prophylactic vaccination model if the adaptive immune system is intact. In the late ferroptotic phase II, cancer cells are no longer immunogenic. Because all the supernatants (SN) from the late ferroptotic cancer cells (treated with RSL3 for 24 hours) are removed and no DAMPs (ie, HMGB1, ATP) are present anymore, which are required for their immunogenicity. Notably, although late ferroptotic cells were engulfed by BMDCs, they failed to induce BMDC phenotypic activation and maturation in vitro or to induce an effective antitumor immune response. It is crucial that phase I ferroptotic cell death occurs in the presence of antigen-presenting cells and should preferably proceed in vivo. During that stage (ie, phase I), most of ATP and HMGB1 and possibly other DAMPs are released or reaching the maximal levels, thereby creating the best adjuvanticity effect of early ferroptotic cancer cells. These data show that the stage of cell death is a key aspect of the immunogenicity of ferroptotic cancer cells and demonstrate that ATP and HMGB1 released after ferroptosis induction may act as immunogenic signals. BMDCs, bone-marrow derived dendritic cells; DAMPs, damage-associated molecularpatterns; HMGB1, high-mobility group box 1.