TT rapidly ablates melanoma xenograft tumors in a concentration dependent manner

We first performed a dose-response study in a xenograft model of human melanoma (MM649 human melanoma cells) to understand what concentrations of TT were required for effective tumor ablation. We assumed that TT was diluted twofold when injected into tumors (50 µL drug into a ~100 mm³ tumor) and that the drug would be homogenously distributed through the entire tumor volume. We subsequently found that I.T. doses of TT lower than 15 µg (266 µM or 27 nmole) were less efficacious at promoting tumor ablation (figure 1A). While partial ablation of the tumor site was observed with 7.5 µg (133 µM or 13.5 nmole) of the drug, tumors soon recurred at the edge of the primary lesion, suggesting that I.T. concentrations >133 µM/13.5 nmole were required for full efficacy in this model. To understand how TT acts at the cellular level in this context we modeled both tumor (MM649) and its associated vasculature (2H-11—SV40 immortalized murine endothelial cells) in vitro and determined the effects of therapeutically relevant doses of drug on cell morphology/survival over 12 hours. At these concentrations, TT induced a rapid necrosis in both cell lines as indicated by propidium iodide (PI) staining (figure 1B, C and online supplemental movie S1, S2). Morphologically, cells appeared to detach from the well surface and swell, forming a “halo”—like structure during the course of PI uptake. As expected, the kinetics of this cell death process were dose-dependent, with death occurring more rapidly at higher concentrations of TT (figure 1B, C). At subefficacious in vivo doses i.e. 100 µM, cell survival was minimally affected over the time course. PKC inhibition with BIS-1 had minimal effects on cell survival in MM649 and 2H-11 cells treated with either 500 or 300 µM TT, suggesting that cell death was largely PKC-independent in this context (figure 1D, E). Identical morphological changes to those seen in MM649 and 2H-11 cells were also observed in a larger panel of squamous cell carcinoma/melanoma cell lines cultured in two or three-dimensions and treated with both 300 and 500 µM TT, that is, cell rounding and cytoplasmic swelling (online supplemental figure 1).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Dose-response experiments in vivo indicate that high concentrations of tigilanol tiglate (TT) are required for therapeutic efficacy. (A) High concentrations of TT are required for efficacy in a human melanoma xenograft model. Established MM649 tumors in BALB/c Foxn1^nu nude mice were injected intratumorally with various concentrations of TT (50 µL) and tumor volume measured versus time. Tumor volume graphs, together with Kaplan-Meier analysis (% tumors <100 mm³) are depicted. n=6–10 tumors per concentration tested. Overall mouse survival is also shown. Both vehicle and 15 µg TT (27 nmole/266 µM) data sets were previously published in (19) and are reproduced here. (B, C) TT induces a dose-dependent disruption of plasma membrane integrity in tumor/endothelial cell lines at therapeutically relevant concentrations. MM649 (human melanoma) (B) and 2H-11 (immortalized mouse endothelial) (C) cells were incubated with various concentrations of TT or vehicle (Vehc.) in media containing 2 µg/mL PI. Images were acquired over time using an Incucyte. The percentage of PI+ve cells was determined per field of view over time ± SD. Boxes show cells undergoing cell swelling and necrosis. n=3. Scale bars: 100 µm. (D, E) Inhibition of PKC has minimal effects on MM649 (D) and 2H-11 (E) cell survival in response to TT treatment at therapeutically relevant doses. Cells in 96-well plate format were pre-incubated ± 5 µM BIS-1, after which they were treated with either 500 or 300 µM TT for the indicated times. BIS-1 remained on all wells for 24 hours. Cell survival was determined via MTS assay after 24 hours and compared with untreated controls. +: both compound and inhibitor remained on cells for 24 hours. Mean cell survival ± SD. is shown. n=3. Statistical analysis by two-way ANOVA with Sidak’s correction. Comparisons made between TT and TT + BIS-1 samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; PI, propidium iodide; PKC, protein kinase C; TT, tigilanol tiglate.

Although the cell death induced in vitro by TT appeared to be PKC-independent, our previous studies have suggested that TT-directed PKC/C1 domain activation plays a role in treatment efficacy in vivo. To investigate this further we repeated experiments with an inactive analog of TT (EBC-158) that is unable to activate PKC.17 We found that although EBC-158 could induce cell swelling and necrosis at therapeutically relevant concentrations in tumor and endothelial cells in vitro, the kinetics of this process were significantly delayed compared with TT (online supplemental figure 2). Consistent with previous studies we found that unlike TT, EBC-158 was not efficacious at inducing tumor ablation in vivo at 15 µg (258 µM or 26 nmole), suggesting that PKC/C1 domain activation plays a role in drug response in vivo (online supplemental figure 2A). Interestingly, we saw that tumors still underwent partial hemorrhagic necrosis after EBC-158 administration (online supplemental figure 2B), consistent with our in vitro data. Together, these results suggest that PKC/C1 domain activity, although not strictly necessary for inducing cell swelling and necrosis, may modulate the kinetics of this process, and may thus play a role in TT-directed tumor ablation.

TT induces a dose-dependent pyroptosis involving activation of the intrinsic/extrinsic pathways of apoptosis and GSDME cleavage

We next investigated the effects of TT on mitochondrial function in MM649 and A-431 (human SCC) cells in vitro. Here, we found that TT induced loss of mitochondrial membrane potential (ΔΨ_m) around 10–30 min post administration, dependent on dose (figure 2A, online supplemental movie S3, S4). We subsequently found that drug administration led to the release of mitochondrial cytochrome c either concomitantly with (500 µM TT) or soon after (300 µM TT) loss of TMRM staining in HeLa cells expressing cytochrome c-EGFP, demonstrating that drug-induced loss of ΔΨ_m culminated in mitochondrial outer membrane permeabilization (MOMP; online supplemental figure 3A,B and online supplemental movie S5). Further experimentation in MM649 cells expressing Tom20-mEmerald revealed that mitochondrial swelling also occurred after TT-induced loss of TMRM staining at both 500 and 300 µM (approximately 30–40 min post initial administration of compound; online supplemental figure 3C,D). This progressed to an increase in Tom20-mEmerald signal in large vacuolar type structures that also stained positive for LysoTracker Deep Red (online supplemental figure 3E, F and online supplemental movie S6), indicative of an increase in mitophagy. As expected, mitochondrial swelling and induction of mitophagy occurred at later time points in cells treated with 300 µM TT and was not as extensive as observed with 500 µM of the compound. Together, these data demonstrated that TT promotes loss of ΔΨ_m, followed by mitochondrial swelling, MOMP and the upregulation of mitochondrial degradation prior to cell death, the kinetics of which ultimately depended on TT dose.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Therapeutically relevant concentrations of TT induce mitochondrial dysfunction, pyroptosis and terminal necrosis in vitro and in vivo. (A) Loss of mitochondrial membrane potential (ΔΨ_m) and cytoplasmic vacuolization occur prior to cell swelling and plasma membrane rupture in TT-treated cells. MM649 and A-431 cells were incubated with media containing 20 nM TMRM (red) and 50 nM SYTOX^TM Green (green). Treated cells were subsequently imaged at different time points using confocal spinning disk fluorescence microscopy. White boxes: cells showing cytoplasmic vacuolization. Red boxes: cells showing plasma membrane swelling prior to lysis. White arrow heads: membrane blebbing. 60×mag. Scale bars: 20 µm. Note the presence of DNA fragmentation in 300 µM treated MM649 and A-431. (B) TT induces the formation of intracellular vacuoles. MM649 cells were treated with TT at the indicated concentrations for 1 hour and stained with toluidine blue prior to fixation and sectioning. 100×mag. (C, D) Reduction in intracellular ATP levels occurs prior to lysis (LDH release) in TT treated cells. A-431 and MM649 cells plated in 96-well plate format were treated with 300 or 500 µM TT. At distinct time points, intracellular ATP levels (C) or extracellular LDH release (D) were determined. Mean values ± SD are plotted in each graph. n=3. Statistical analysis by two-way ANOVA with Dunnett’s correction. Comparisons to vehicle treated controls. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) Analysis of cell death pathway induced in response to 500 and 300 µM TT. WCE obtained at the indicated time points from MM649 and A-431 cells were separated by SDS-PAGE and transferred to nitrocellulose in preparation for immunoblotting. Immunoblotting was performed with the indicated antibodies. Arrows indicate full-length protein or processed fragments. tBid=truncated and active Bid fragment. GSDMD: p43 is inactive while p30 is active. GSDME: p35 is active. (F) Knockout of GSDME, CASP3 and CASP7 protects cells from both 300 and 500 µM TT. Knockout (KO) cells plated in 96-well plate format were treated with either 500 or 300 µM TT for the indicated times. % cell survival was determined via MTS assay after 24 hours and compared with untreated controls. +: TT remained on cells for 24 hours. Mean % cell survival ± SD is shown. n=4. The lower panel shows immunoblotting analysis of KO cell lines with the indicated antibodies. Statistical analysis by two-way ANOVA with Dunnet’s correction. Comparisons made to wt treated controls. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (G) Intratumoral injection of TT gives rise to similar morphological changes, including the formation of intracellular vacuoles, in MM649 tumor cells in vivo. MM649 xenograft tumors established in BALB/c Foxn1^nu nude mice were treated with TT and then fixed in preparation for TEM analysis. ANOVA, analysis of variance; ATP, adenosine triphosphate; LDH, lactate dehydrogenase; TEM, transmission electron microscopy; TT, tigilanol tiglate; WCE, whole cell extract; wt, wild-type.

Consistent with the mitochondrial swelling, further interrogation of our live cell imaging showed that loss of ΔΨ_m was followed by vacuolization of the cytosol at both concentrations of the drug tested (figure 2A, white dashed boxes). This rapidly progressed to the cytoplasmic swelling and necrosis (figure 2A, red dashed boxes) we had previously observed in our Incucyte-based assays. Similar swollen, vacuolar structures were also seen in toluidine blue stained sections of MM649 cells treated with TT at both 300 µM and 500 µM (figure 2B). Luminescence-based assays also revealed that TT induced a rapid and significant reduction in intracellular ATP levels in both A-431 and MM649 cells, the kinetics of which were dependent on treatment dose (figure 2C). Furthermore, lactate dehydrogenase (LDH) release assays confirmed concomitant plasma membrane rupture in both cell lines (figure 2D). TT-induced cell death could also be significantly delayed at both concentrations through the use of a chloride channel blocker and TRPM4 inhibitor, NPPB and 9-phenathrol, suggesting that loss of cell viability in response to drug may also involve an ion channel/TRPM4-based mechanism (online supplemental figure 4 A,B).

While cytoplasmic swelling/necrosis predominated in drug treated cells, at 300 µM TT membrane blebbing was especially apparent in both cell types prior to plasma membrane rupture (figure 2A, white arrows). Interestingly, the large majority of nuclei treated with 500 µM TT remained intact, while nuclear fragmentation or shrinking was extensively observed with 300 µM TT (figure 2A, red dashed boxes). This suggested that apoptotic-based pathways were active or had time to proceed at lower doses of drug. Consistent with this, we observed caspase-8, caspase-9, caspase-3, caspase-7, Bid and poly (ADP-ribose) polymerase (PARP) cleavage in response to TT at 300 and 500 µM treatment in both cell types with slightly different kinetics (figure 2E and online supplemental figure 4C). While differences were found between cell types in terms of caspase/Bid/PARP cleavage, drug treatment consistently led to Mcl-1 downregulation in both MM649 and A-431 cells at 500 µM and 300 µM (online supplemental figure 4C). Consistent with the induction of cytoplasmic swelling and necrosis, we also found that the pore forming protein GSDME was cleaved and activated (N-terminal 45 KDa fragment) during TT-mediated cell death. Neither MLKL phosphorylation or activating cleavage of GSDMD were observed after TT administration, suggesting that TT does not promote necroptosis or gasdermin D-dependent pyroptosis (figure 2E). Use of a pan-caspase inhibitor (Q-VD-OPh) prior to the administration of TT also had some inhibitory activity in MM649 cells at 300 µM (online supplemental figure 4D). Manipulating components of the intrinsic apoptotic machinery that act at or adjacent to mitochondria also protected cells to some extent from TT treatment. Indeed, overexpression of Bcl-2 in HeLa cells or deletion of Apaf1 in MEFs protected cells from both 300 µM and 500 µM TT (online supplemental figure 4 E,F). In contrast, deletion of Bax and/or Bak in MEFs appeared to protect cells to a significant extent from 300 µM but not 500 µM TT (online supplemental figure 4F). Consistent with a role for caspase activation and GSDME cleavage in TT-mediated pyroptosis, CRISPR mediated knockout of GSDME, caspase 3/caspase 7 and caspase 3/caspase 7/GSDME protected cells from both 300 µM and 500 µM TT (figure 2F), although this was transient at 500 µM TT. Together, these data demonstrate that therapeutically relevant concentrations of TT induce a caspase-dependent pyroptosis involving GSDME pore formation in cells.

To understand whether a similar morphological response occurred in vivo after TT administration, that is, the appearance of swollen vacuolar structures followed by cell necrosis, we also treated MM649 tumors in nude mice with TT and prepared samples for electron microscopy. Indeed, we observed similar morphological characteristics in vivo after injection of TT (30 µg or 533 µM) (figure 2G), indicating that our in vitro studies held relevance for drug response in vivo.

TT binds to endoplasmic reticulum membranes and promotes the activation of mitochondrial/endoplasmic reticulum stress responses

As TT likely acts as a DAG mimetic, we hypothesized that it might induce pyroptosis and terminal necrosis in a manner independent of PKC/C1 domains by binding to and perturbing the function of organellular membranes. To investigate this, we took advantage of a fluorescent analog with physiochemical and biological properties similar to those of TT (known as TT-A). Due to the known interplay between mitochondria and the endoplasmic reticulum (ER) in cell physiology and the fact that mitochondrial function was affected, we decided to overexpress fluorescently tagged mitochondrial and ER resident proteins (Tom20-mEmerald and Sec61-RFP) to identify colocalization with TT-A by fluorescence microscopy. As expected, TT-A (300 µM) rapidly accumulated into membrane structures including both mitochondria and ER, 10 min after addition (figure 3A,B and online supplemental movie S7). At later time points (40–70 min), we observed the formation of globular structures containing TT-A which did not co-localize with either Tom20-GFP or Sec61-RFP but appeared to co-localize with lysosomal structures as indicated by LysoTracker Deep Red staining (figure 3 C,D and online supplemental movie S8), consistent with induction of a lysosome mediated degradation pathway. TT-A induced cell death also occurred approximately 1–2 hours after treatment with this concentration of drug. In contrast to TT-A, TT (300 µM) appeared to accumulate predominantly in ER membranes after 15 min rather than mitochondria (figure 3E). Strikingly, we also observed a large amount of TT in the “cytosolic” fraction. Immunoblotting suggested that this fraction was heavily contaminated with ER membranes. However, given we had previously seen TT-A form droplet-like structures after administration, we reasoned that TT may also be processed into lipid droplets. Indeed, it is well known that ER components (including protein disulphide isomerase - PDI) are frequent contaminants in these preparations.23 24 Furthermore, due to the buoyancy of lipid droplets, they are not removed from crude cytoplasmic extracts during the high-speed centrifugation step required for ER isolation. Consistent with this hypothesis, we found that TT induced the formation of lipid droplet structures in MM649 cells as indicated by Nile red staining (online supplemental figure 5A). Thus, TT accumulates in ER membranes and is possibly processed into lipid droplet-like structures that undergo attempted breakdown via a lysosomal-based pathway.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

TT binds to ER membranes and induces activation of mitochondrial/ER stress signaling and the integrated stress response. (A, B, C, D) A fluorescent TT analog, TT-A, initially binds to mitochondrial/ER membranes (t=10 min) but accumulates in lysosomes at later time points (t=40+ min). MM649 cells overexpressing mEmerald-Tom20 and mCherry-Sec61 beta were incubated with 300 µM TT-A, then visualized via spinning disk confocal microscopy. Mitochondrial (A) and ER (B) based colocalization with TT-A can be observed after 10 min (white arrow heads). At later time points, TT-A accumulates in structures that do not colocalize with mitochondrial/ER markers, but localize in lysosomes (C and D). In (D) MM649 cells were stained with 100 nM LysoTracker Deep Red and 20 nM TMRM prior to incubation with TT-A. Images were acquired using a spinning disk confocal microscope. 60×mag. In (C) white arrows show TT-A with no colocalization with mitochondrial/ER markers. In (D) white arrows show colocalization between TT-A and LysoTracker Deep Red. Scale bars in (A–D) 20 µm. (E) TT accumulates in ER. MM649 cells treated with vehicle/300 µM TT and isolated after 30 min were subjected to subcellular fractionation and HPLC-MS analysis. The median concentration values of TT acquired in each compartment are shown in box plot format. n=4. Mitochondrial (Tom20) and ER (PDI) markers were used to assess the purity of subcellular fractions via immunoblotting (lower panel). (F) TT induces ER stress and activation of the integrated stress response. MM649 cells were treated with the indicated concentrations of TT and WCE generated at distinct time points. Protein (30 µg) was separated via SDS-PAGE and immunoblotting performed with the indicated antibodies. Arrows indicate differentially migrating species. (G, H) Inhibition of Ire1α, but not PERK, protects cells from lower doses of TT. Cells in 96-well plate format were incubated ± 5 µM Kira6 (G) or 5 µM GSK2606414 (GSK: H) for 1 hour prior to incubation with 500/300 µM TT for the indicated times. Cell survival was determined via MTS assay after 24 hours and compared with untreated controls. +: compound and inhibitor remained on cells for 24 hours. Mean cell survival ± SD is plotted in each graph. n=3. Statistical analysis by two-way ANOVA with Sidak’s correction. Comparisons were made between TT and TT + Kira6/GSK samples. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; PDI, protein disulphide isomerase; ER, endoplasmic reticulum; TT, tigilanol tiglate; WCE, whole cell extract.

Given that TT bound to ER membranes, we investigated whether stress signaling pathways associated with this organelle were activated. Treatment of MM649 cells with 500 µM TT led to the rapid phosphorylation (t=10 min) of Ire1α, PERK, PKR, p38 and JNK1/2, indicative of activation of the ER/mitochondrial unfolded protein response (UPR^ER/mt) and stimulation of stress kinase signaling (figure 3F). Consistent with this, we also found that eiF2α was rapidly phosphorylated in response to TT, demonstrating rapid activation of the integrated stress response (ISR) (figure 3F). A similar outcome was observed in cells treated with 300 µM TT, although activation of p38 and PKR occurred at later time points and was maintained for at least 120 min. The change in PERK migration due to phosphorylation was confirmed through the use of PhosTag and phosphatase-based experiments (figure 3F, online supplemental figure 5 B,C). Inhibition of Ire1α (kinase and RNase inhibition with Kira6) but not PERK, significantly protected cells from lower doses of TT (figure 3 G,H). Bcl-2, in addition to inhibiting Bax/Bak-dependent MOMP, has also recently been shown to inhibit Ire1 and as a result facilitate tissue homeostasis.25 This might also help explain why Bcl-2 overexpression protects cells against TT-induced pyroptosis (online supplemental figure 4E). Thus, TT binding to ER membranes induces upregulation of UPR^mt/ER signaling, ISR induction and promotion of a cell death pathway that may involve Ire1α activation.

TT promotes the release/externalization of damage associated molecular patterns from cancer cells

As we had observed both caspase-8 activation and ISR induction following TT treatment, we reasoned that TT may promote immunogenic cell death (ICD) during the transition to pyroptosis/necrosis. We therefore analyzed additional markers of ICD in TT-treated cells, including the generation of reactive oxygen species (ROS), ATP release, calreticulin externalization, HMGB1 release and type I interferon (IFN) production.26 In addition, we also investigated whether TT could induce activation of nuclear factor kappa B (NF-κB) dependent responses given that these have also been associated with the immunogenicity of cell death. We found that TT stimulated a significant increase in ROS production over time in response to 300 µM of drug in both MM649 and A-431 cells (figure 4A). Drug exposure also induced extracellular ATP release in a dose-dependent manner from both MM649 and A-431 cells (t_max=10–30 min (500 µM) and 60 min (300 µM); figure 4B), which appeared to occur in concert with plasma membrane disruption (figure 4C) suggesting that it occurred postmortem. ELISA assays demonstrated that HMGB1 release, while low, was significant in both A-431 and MM649 cells at later time points (figure 4D). Flow cytometry also revealed that drug treatment induced significant calreticulin externalization in both cell types (figure 4E). Use of an NF-κB luciferase reporter system17 demonstrated that while TT could induce upregulation of NF-κB mediated transcription in HeLa cells at lower concentrations (50–100 µM), this did not occur in MM649 and A-431 cells (online supplemental figure 6A,B,C). Furthermore, although we observed TT-induced secretion of interleukin (IL)-6, IL-8, CXCL9 and CXCL10 in some cell lines at lower concentrations of drug (50–100 µM), we failed to see evidence of bona fide STING activation in response to TT as indicated by a lack of type I IFN secretion (online supplemental figure 6D,E,F). Consistent with the induction of ICD in vitro, we also observed eiF2α phosphorylation and the formation of calreticulin puncta in MM649 human melanoma xenografts treated with TT, suggesting that ICD also occurred in vivo (figure 4F, G, H). Similar or improved responses were observed in the mouse melanoma line, B16-F10-OVA (online supplemental figure 7).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4

TT induces biomarkers of immunogenic cell death in vitro and in vivo. (A) TT promotes ROS production in MM649 and A-431 cells. Cells stained with 5 ng/mL DHE were incubated with 300 µM TT for the indicated times and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values are shown ± SD. n=4. (B) TT promotes extracellular ATP release. Cells were treated with TT (300/500 µM) for the indicated time periods and cell culture supernatants analyzed for ATP content. Mean relative luminescence units (RLU) are shown ± SD. n=4. (C) TT-induced ATP release occurs concomitantly with plasma membrane disruption. Again, MM649 and A-431 cells were treated with TT and the levels of intracellular (intATP) and extracellular (extATP) determined over time, together with plasma membrane breakage via SYTOX^TM Green uptake (SYTOX). n=4. (D) TT promotes HMGB1 release. Cells were treated with TT (300/500 µM) for the indicated time periods and cell culture supernatants analyzed for HMGB1 content via ELISA. Mean fold change values (compared with vehicle treated controls) are shown ± SD. n=4. (E) TT promotes calreticulin externalization prior to membrane rupture. Cells were treated with TT (300/500 µM) for the indicated time periods and subsequently incubated with LIVE/DEAD far red prior to staining for cell surface calreticulin. Cells were analyzed via flow cytometry. MFI values in box plot format are shown for each graph. n=4. (F, G) Treatment of tumors with TT results in eiF2α phosphorylation in vivo. Tumors (75–100 mm³; three tumors per condition) treated with vehicle or 30 µg (533 µM/54 nmole) TT I.T. and isolated after 1 hour for immunohistochemistry (F) or over a 1–12 hours time course for immunoblotting (G) (30 µg protein) with anti-phospho-eiF2α and total eiF2α. Scale bar: 200 µm. (H) TT promotes calreticulin externalization in vivo. MM649 tumors (75–100 mm³) were treated with vehicle or 30 µg (54 nmole) TT I.T. and isolated after 4 hours. Representative image from 4 hours treated sample. n=2. Scale bar: 50 µm. Statistical analysis by two-way ANOVA with Sidak’s correction in (A) and Dunnett’s correction in (B, E). A mixed effects analysis with Dunnett’s correction was performed for data in (D). Comparisons were made to vehicle-treated controls in all cases. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; DHE, dihydroethidium; I.T., intratumoral; ROS, reactive oxygen species; TT, tigilanol tiglate.

TT promotes the development of T-cell directed antitumor responses in vitro and in vivo

To determine whether the death induced by TT was immunogenic, we first performed co-culture experiments in which drug-treated cancer cells (B16-F10-OVA) pre-stained with 5-chloromethylfluorescein diacetate (CMFDA) were incubated with immature bone marrow derived cells (BMDCs) isolated from C57BL/6 J mice. Flow cytometry indicated that drug-treated cells were phagocytosed effectively by CD11c⁺ cells, the extent of which was dependent on drug concentration and treatment time (figure 5A and B). Imaging of several CD11c⁺CMFDA^mid cells from the 500 µM TT treatment sample confirmed antigenic uptake, as indicated by the presence of intracellular green puncta (figure 5C; white arrowheads).

• Download figure
• Open in new tab
• Download powerpoint

Figure 5

TT-directed pyroptosis is immunogenic in vitro. (A, B, C) TT-treated B16-F10-OVA cells are effectively phagocytosed by immature BMDCs in vitro. TT-treated B16-F10-OVA cells (pre-stained with CMFDA) were incubated with immature BMDCs for 4 hours, stained with anti-CD11c-APC and then subsequently analyzed via flow cytometry (A). Blue boxes: CD11c+ve cells with/without CMFDA uptake. Red boxes: CD11c+CMFDA_mid cells, that is, cells that have phagocytosed dying cancer cell fragments. Median % CD11c+CMFDA_mid cells observed for each treatment were plotted via box plot format (B). n=4. CD11c+CMFDA_mid cells were also imaged to confirm antigenic uptake (C). White arrows indicate intracellular CMFDA-stained fragments. Statistical analysis by two-way ANOVA with Dunnett’s correction. Comparisons were made to vehicle treated controls. (D) TT-treated B16-F10-OVA cells can promote the activation/proliferation of OVA-directed CD4 and CD8 T-cell responses in vitro. BMDCs from PTPRCA mice (CD45.1⁺CD45.2⁻) pre-incubated with TT treated cells (16 hours) were co-incubated with CFSE stained T cells from OT-I (CD8⁺Va2⁺Vb5⁺ T cells from CD45.1⁻CD45.2⁺ mice) and OT-II (CD4⁺Va2⁺Vb5⁺ T cells from CD45.1⁻CD45.2⁺ mice) mice. Early activation (anti-CD69) and proliferation (CFSE dilution) markers were assessed in live cells at t=2 (OT-I) or 4 (OT-II) days via flow cytometry. The % of non-activated (CD69⁻ Prolif_neg), early activated (CD69⁺ Prolif_neg), activated (CD69⁺ Prolif_pos) and late activated (CD69⁻ Prolif_pos) T cells±SD are depicted in each graph. n=3. Statistical analysis by two-way ANOVA with Tukey’s correction. See comparisons below main graphs for statistical data. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; BMDC, bone marrow derived cells; CFSE, carboxyfluorescein succinimidyl ester; CMDFA, 5-chloromethylfluorescein diacetate; TT, tigilanol tiglate.

Given the uptake by immature BMDCs, we next investigated whether drug could help promote the activation of antigen-specific T-cell responses in vitro (see online supplemental figure 8 for gating strategy). Consistent with our previous results, we found that prior treatment of B16-F10-OVA cells with TT led to significant activation and proliferation of both OVA-directed CD4 and CD8 T cells when combined with BMDCs in mixed lymphocyte reaction assays, as indicated by carboxyfluorescein succinimidyl ester (CFSE) dilution and CD69 upregulation (increase in CD69⁺ prolif_pos and CD69^– prolif_pos cells; figure 5D). Incubation of BMDCs with B16-F10-OVA cells that had undergone freeze thaw cycles failed to activate CD4/CD8 T-cell responses, and the removal of BMDCs from TT-treated samples also led to a similar result (figure 5D). Thus, TT-mediated cell death in cancer cells promotes the development of antigen-specific T-cell responses in vitro.

To test whether TT promoted ICD in vivo, we performed additional experiments using the immunogenic CT-26 colon carcinoma model. Two CT-26 tumors (75–100 mm³) were implanted via subcutaneous (s.c.) injection in the flanks of immunocompetent BALB/c mice and treated I.T. with either vehicle or 7.5, 15 or 30 µg TT. Tumors that recurred before day 10 in the TT-treated cohorts were reinjected with drug. 20 days after the initial treatment, cured mice were reinjected with CT-26 cells at a spatially distinct site to determine whether a bona fide antitumor immune response had been generated (figure 6A). In contrast to I.T. injection of vehicle, administration of TT ablated 73–83% of CT-26 tumors over a broad concentration range (figure 6B and C). While all tumors initially responded to treatment, a small fraction recurred at the edge of the lesion site (figure 6B). In the 15 µg and 7.5 µg TT treatment groups, 2 and 7 tumors were re-treated, respectively. Interestingly, drug efficacy was largely comparable among the concentrations of TT tested under these conditions, although less tumors ultimately recurred at 30 µg (five tumors) compared with 15 and 7.5 µg TT (eight tumors). Remarkably, I.T. treatment efficacy was significantly reduced when similar experiments were performed in nude mice (figure 6C, online supplemental figure 9A), demonstrating a requirement for T cells to prevent tumor recurrence. Consistent with the in vitro data, we also found that prior treatment with TT protected immunocompetent (figure 6D, E) but not nude (figure 6E, online supplemental figure 9B) mice from rechallenge with the same cell line. Experiments using AH1 directed tetramers, splenocytes and IFN-γ staining also confirmed that TT promoted the development of functional, AH-1 specific T cells in these mice (figure 6F, G). We also performed a gold standard vaccination assay to determine whether TT acted as a true ICD inducer. Here, we found that mock injection (phosphate-buffered saline (PBS)) or vaccination of mice with CT-26 cells that had undergone prior freeze-thaw (F/T) cycles failed to protect these mice against rechallenge with live cancer cells (online supplemental figure 10): 15/15 tumors growing in PBS-treated mice and 10/15 tumors growing in F/T treated mice). Furthermore, protection against rechallenge was not observed in mice vaccinated with CT-26 cells treated with 150 µM TT (13/15 tumors developed after rechallenge). Conversely, vaccination of mice with cells that had been treated with either doxorubicin or 300/500 µM TT protected mice against subsequent injection of live CT-26 cells (online supplemental figure 10): 0/15 and 4/15 tumors growing in 300 µM and 500 µM TT-treated mice, respectively). However, we found that several tumors formed at the vaccination site in mice administered 300/500 µM TT-treated cells, indicating that a population of cells could recover from TT treatment under these conditions. Although this was so, the fact that tumor growth was significantly inhibited on the rechallenge site by vaccination with TT-treated cells suggest that TT is a bona fide ICD-inducing agent.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6

TT promotes the development of T-cell dependent antitumor immunity in vivo. (A) Schematic diagram of experimental design. BALB/c mice with CT-26 tumors were treated with TT, allowed to resolve and cured mice rechallenged with CT-26 cells. (B) TT ablates CT-26 tumors in BALB/c immunocompetent mice. Mice with two distinct CT-26 tumors were injected with the indicated concentrations of TT. 15 mice per condition, n=30 tumors per group. *Indicates number of tumors reinjected with TT. (C) T cells are required to prevent the relapse of TT-injected tumors in immunocompetent mice. Kaplan-Meier analysis of individual CT-26 tumors (% tumors <100 mm³) in immunocompetent and immunodeficient (nude) mice injected with the indicated concentrations of TT. Data acquired from tumor growth curves detailed in (B) and in online supplemental figure 9A (immunodeficient (nude) mice). Statistical analysis by log-rank (Mantel-Cox) test. (D, E) TT promotes the development of antitumor immunity. Naïve and TT-cured mice were rechallenged with CT-26 cells. Tumor growth curves are depicted for each condition (D). Vehicle: n=15 mice. 30 µg TT: n=10 cured mice. 15 µg and 7.5 µg TT: n=9 cured mice. Kaplan-Meier analysis of data acquired in (D) is shown in (E) together with data acquired from experiments in nude mice (see online supplemental figure 9B). Statistical analysis by log-rank (Mantel-Cox) test. (F, G) TT treatment leads to the development of tumor-directed T cells in the periphery. Splenocytes isolated from naïve and rechallenged mice detailed in (D) were incubated with AH-1 peptide, stained with AH-1 directed tetramers (F) or anti-IFN-γ (G) together with LIVE/DEAD Aqua, anti-CD3, CD4, CD8 and analyzed via flow cytometry. Representative flow cytometry dot plots, together with box plots (median values) of replicate data (% AH-1 tetramer⁺ CD8⁺ or % IFN-γ⁺ CD8⁺ T cells) are shown. Vehicle: n=9; 30 µg TT: n=8; 15 µg and 7.5 µg TT: n=6. Statistical analysis by Student’s t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. IFN, interferon; TT, tigilanol tiglate.

TT promotes T-cell infiltration into tumors

We next sought to understand whether TT could promote damage associated molecular pattern (DAMP) release and T-cell recruitment to tumors in a “cold”, immune checkpoint inhibitor (ICI)-refractory tumor model of melanoma. To investigate this, B16-F10-OVA cells were implanted s.c. into C57BL/6 mice (two tumors per mouse) and treated with either vehicle or 15 µg TT once they reached 200 mm³. Remnant tumors were isolated on Days 1 and 4 post I.T. injection (online supplemental figure 11A). Immunohistochemistry and immunofluorescence revealed that TT induced significant loss of nuclear HMGB1 staining, together with punctate calreticulin staining indicative of membrane externalization within tumors 24 hours post-treatment that was not observed with vehicle treatment (online supplemental figure 11B,C). Treatment with TT also leads to significant recruitment of CD3⁺ cells into these tumors at day 4 (online supplemental figure 11D,E). Although there was a marked increase in CD8⁺ T cells in this scenario, the large majority of CD3⁺ cells at this time point appeared to be CD4⁺ cells (online supplemental figure 11F,G).

Combination of TT with immune checkpoint therapy improves therapeutic outcomes

We next sought to understand whether TT could improve the efficacy of checkpoint inhibition (anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) therapy) in the B16-F10-OVA model. A schematic of the treatment protocol can be seen in figure 7A. Again, B16-F10-OVA cells were implanted s.c. into C57BL/6 mice (two tumors per mouse) and IgG, anti-PD-1, anti-CTLA-4 or a combination of the two administered 2 days prior to I.T. injection of vehicle or TT into both target lesions (30 and 15 µg TT). Antibody was subsequently administered every 2 days for a further three cycles. B16-F10-OVA tumors were non-responsive to anti-PD-1, anti-CTLA-4 or anti-PD-1/CTLA-4 combination therapy (with I.T. injection of vehicle) as previously observed by others.27 Similar to our prior experiments, tumors were significantly responsive to I.T. injection of both 15 and 30 µg TT+IgG compared with both IgG+vehicle and anti-PD-1+vehicle, anti-CTLA-4+vehicle and anti-PD-1/anti-CTLA-4+vehicle treatments. However, ≥45% of treated lesions recurred in these mice around 10–30 days post-treatment (figure 7B, C; online supplemental figure 12). While combination treatment with 15 µg TT and anti-CTLA-4 had a substantial but ultimately non-significant effect on tumor recurrence and mouse survival, combination of TT at this dose with anti-PD-1 or anti-PD-1/anti-CTLA-4 had no additive effect compared with treatment with 15 µg of TT alone (figure 7B). However, the use of 30 µg TT in combination with anti-PD-1 led to less tumor recurrence and improved overall survival compared with 30 µg TT alone (figure 7C; online supplemental figure 12B). A similar but statistically significant outcome was observed in anti-PD-1/CTLA-4 combination experiments (figure 7C; online supplemental figure 12C). These data suggest that immune checkpoint therapy can combine effectively with TT to improve anticancer responses, prevent tumor recurrence and improve survival.

• Download figure
• Open in new tab
• Download powerpoint

Figure 7

TT combines with immune checkpoint inhibitor therapy to improve antitumor responses in an immune checkpoint inhibitor-refractory model of melanoma. (A) Schematic of the dual treatment regime. Mice with two distinct B16-F10-OVA tumors on their hindquarters were administered IgG/anti-PD-1/anti-CTLA-4 or a combination thereof via i.p. injection prior to I.T. injection of both tumors with vehicle or TT (15/30 µg). Mice received antibody every 2 days after I.T. injection for a further three cycles. (B, C) TT combines with checkpoint inhibitors to prevent tumor recurrence and improve overall survival. Kaplan-Meier analysis of individual tumors <100 mm³ (upper panels) and overall mouse survival (lower panels). n=20 tumors treated per condition. 10 mice per condition. (D) Schematic of the abscopal treatment regime. Mice with two distinct B16-F10-OVA tumors on their hindquarters were administered IgG/anti-PD-1/anti-CTLA-4 or a combination thereof via i.p. injection prior to I.T. injection of a single tumor with vehicle or TT (15/30 µg). Mice received antibody every 2 days after I.T. injection for a further three cycles. (E, F) TT combines with anti-CTLA-4 and anti-CTLA-4/PD-1 to inhibit the growth of non-injected B16-F10-OVA tumors and improve mouse survival. Kaplan-Meier analysis of individual tumors <100 mm³ and of mouse survival. n=10 mice per condition, except for 15 µg TT anti-IgG+vehicle (anti-CTLA-4 and anti-PD-1 cohorts) and 30 µg TT anti-IgG+vehicle (anti-CTLA-4 cohort) where n=5–6. Statistical analysis by log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.CTLA-4, cytotoxic T-lymphocyte-associated protein 4; i.p, intraperitoneal; I.T., intratumoral; PD-1, programmed cell death 1; TT, tigilanol tiglate.

To further investigate the potential of checkpoint inhibitor/TT combinations in this model, we performed an experiment to understand whether immune checkpoint therapy, together with I.T. administration of TT to a single target tumor could affect the growth of non-injected lesions (anenestic response). Tumors were generated as above and IgG, anti-PD-1, anti-CTLA-4 or a combination administered 2 days prior to I.T. injection of vehicle/TT into one tumor (30 and 15 µg TT). Again, checkpoint inhibitor/s were administered for a further three cycles every 2 days (figure 7D). As expected, TT-injected tumors (+ IgG only) were significantly ablated at both 15 and 30 µg TT, in contrast to vehicle treated lesions (figure 7E, F; online supplemental figure 13; “Injected” panels). A significant or almost significant change in the growth of non-injected B16-F10-OVA lesions and subsequent survival was observed in anti-CTLA-4+TT and anti-PD-1/CTLA-4+TT combinations only (at both 15 and 30 µg TT), when compared with either anti-CTLA-4+vehicle, anti-PD-1/CTLA-4+vehicle or IgG+TT-treated mice (figure 7E, F; online supplemental figure 13; “Non-injected” panels). Thus, anti-CTLA-4 or anti-PD-1/CTLA-4 combination immunotherapy improves the response of non-injected lesions to TT treatment and leads to increased survival.