Research ArticleExperimental Studies
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Eribulin Induction of Immunogenic Cell Death (ICD): Comparison With Other Cytotoxic Agents and Temporal Relationship of ICD Biomarkers

MINGFA ZANG, JIA ZHENG, XIAOYU AN, BO LI, HUAJUN YANG, BRIAN ERICKSON, RYAN KUNZ and BRUCE A. LITTLEFIELD
Anticancer Research January 2025, 45 (1) 39-53; DOI: https://doi.org/10.21873/anticanres.17391

Abstract

Background/Aim: Preclinical studies were undertaken to investigate whether eribulin’s known cytotoxic antimitotic effects are characterized by immunogenic cell death (ICD) as assessed by three established ICD biomarkers: extracellular released ATP, released HMGB1 and cell surface calreticulin. Materials and Methods: Using BT-549, Hs578T and MCF-7 breast cancer cell lines, antiproliferative IC50’s of eribulin, five other microtubule targeting agents (MTAs; ER-076349, vinblastine, vinorelbine, paclitaxel, docetaxel) and three DNA damaging agents (DDAs; doxorubicin, cisplatin, oxaliplatin) were determined. Results: Treatment of cells with 10×IC50 concentrations of all drugs in serum-free media resulted in time-dependent induction of cytotoxicity over DMSO controls. Measurement of ATP and HMGB1 released into conditioned media and appearance of cell surface calreticulin support eribulin’s ability to induce ICD. Compared to the other agents tested, eribulin’s potency as an ICD inducer was mid-range and shared with vinblastine, paclitaxel, doxorubicin and oxaliplatin. Interestingly, MTAs as a group appeared to be more potent inducers of ATP release compared to DDAs, whereas DDAs appeared to be more potent inducers of cell surface calreticulin compared to MTAs. Overall, drug effects on ATP release and cell surface calreticulin showed early peaking followed by rapid decline, while effects on HMGB1 release were generally slower and more prolonged. Conclusion: Our results support the concept that eribulin’s cytotoxic effects are associated with ICD. These findings provide impetus for investigating how eribulin-induced ICD may contribute to the larger spectrum of phenotypic and immunological effects by which eribulin exerts antitumor therapeutic benefits.

Key Words:

Eribulin, a macrocyclic ketone analog of the marine sponge natural product halichondrin B (1), is approved for clinical use in the USA, Japan, the European Union and many other global jurisdictions for certain patients with advanced breast cancer, liposarcoma or soft tissue sarcoma (2, 3). Originally defined mechanistically as a cytotoxic microtubule-targeting agent (MTA) that inhibits microtubule dynamics and irreversibly blocks cell cycle progression in mitosis resulting in apoptosis (1, 4-7), results from phase III clinical trials unexpectedly showed prolonged overall survival (OS) in breast cancer and liposarcoma patients without corresponding prolongation of progression-free survival (PFS; 2, 3). These clinical observations prompted further exploration of potential mechanisms that might affect the biology of residual tumors not killed outright by eribulin’s cytotoxic effects. The results of these translational studies showed that in addition to its known cytotoxic antimitotic effects, eribulin also triggers alterations in tumor phenotype and changes in the tumor microenvironment, including increased vascular perfusion, reduced tumor hypoxia, reversal of epithelial mesenchymal transition (EMT) and increased cellular differentiation, reduced experimental metastasis, and other phenotypic changes impinging on the host antitumor immune responses (8-20).

First reported in 2005, immunogenic cell death (ICD) involves release of so-called danger-associated molecular patterns (DAMPs), cellular components expressed on or released from dying cells that activate innate immune signaling pathways resulting in enhanced antitumor immune responses (21-26). Several molecular signatures of ICD are now well established, including extracellular release of both ATP and HMGB1 and translocation of calreticulin from the endoplasmic reticulum to the cell surface (22-26). Considering eribulin’s well documented cytotoxic effects (1, 4-7, 19, 20), the goal of the current study was to ask if eribulin’s cytotoxicity is characterized by ICD that might contribute to its reported effects on tumor immunology (12, 13, 15-18). For context, we asked this question in comparison with several other MTAs and DNA-damaging agents (DDAs). Our results add to the increasingly complex spectrum of mechanisms by which eribulin elicits therapeutically beneficial anticancer effects.

Materials and Methods

Test compounds. Eribulin mesylate (salt form used in Halaven® clinical formulation, Eisai Co. Ltd, Tokyo, Japan; hereinafter referred to as eribulin) and its biologically active C34,C35-diol synthetic precursor ER-076349 (1, 7) were synthesized by Eisai Co. Ltd. at its Kashima, Japan manufacturing facility. Vinblastine, paclitaxel, docetaxel, doxorubicin and oxaliplatin were obtained from Selleckchem (Shanghai, PR China). Vinorelbine was obtained from Tianjin Meilun Medical Products (Tianjin, PR China). Cisplatin was obtained from Qilu Pharmaceutical (Jinan, PR China). All compounds were prepared as 10 mM stocks in 100% (v/v) DMSO and stored frozen at −20°C in small aliquots until use.

Human breast cancer cell lines and in vitro antiproliferative assays. Triple negative breast cancer (TNBC) cell lines BT-549 and Hs578T, and hormone receptor positive (HR+) breast cancer cell line MCF-7 were maintained as part of Crown Bioscience’s OmniPanel™ in vitro cell line screening service in their Taicang City (PR China) laboratories. All OmniPanel™ cell lines are routinely assessed for mycoplasma and authenticated using short tandem repeat DNA profiling. Effects of test compounds on proliferation of BT-549, Hs578T and MCF-7 cell lines were assayed by Crown Biosciences under contract from Eisai Inc. using 3-day (BT-549, Hs578T) or 6-day (MCF-7) 96-well plate cell proliferation assays with ATP-based readouts (Cell Titer-Glo® Luminescent Cell Viability kit, Promega, Shanghai, PR China). Based on preliminary seeding density and cell growth rate determination studies, MCF-7 cells used 6-day assays due to much slower growth rates compared to BT-549 and Hs578T cells, for which 3-day assays were optimal. Vehicle controls consisted of 0.1% DMSO which represented the highest concentration used for any test agent. IC50 values for test agents were calculated by 4-parameter, variable-slope nonlinear regression analyses using GraphPad Prism (Boston, MA, USA, version 10.2.0).

Treatment of cells with test agents, preparation of serum-free conditioned media and cell harvesting for flow cytometry. Treatment of cells with 10×IC50 concentrations of test agents or vehicle control for analysis of induction of ICD was performed by Crown Bioscience under contract from Eisai Inc., with work performed in Crown Bioscience’s Taicang City, PR China laboratories. BT-549, Hs578T and MCF-7 cells were grown under standard conditions in serum-containing media and harvested while still in logarithmic growth phase. Cells were then plated into T-175 flasks in serum-containing media at sufficient density to achieve approximately 60% confluence after 24 h, at which time monolayers were washed twice for 10 min each at 37°C with serum-free medium containing 0.1% BSA. Fresh serum-free medium containing 0.1% BSA plus 10×IC50 concentrations of test agents or 0.1% DMSO as vehicle control was then added to the monolayers. Three T-175 flasks for each test agent at each time point were used to provide sufficient conditioned media for ATP and HMGB1 measurements and harvested monolayer cells for flow cytometric analysis of cell surface calreticulin.

After addition of test agents to the monolayers as above, conditioned media were removed at 12, 24 and 48 h (BT-549, Hs578T) or 24, 48, 96 and 144 h (MCF-7) and centrifuged at 300×g for 10 min at 4°C. Pellets from this first centrifugation, containing unattached living cells, dead cells and cellular debris, were briefly set aside to be combined with the attached monolayer cells harvested as described below. Supernatants from this first centrifugation were then recentrifuged at 10,000×g for 10 min at 4°C. Pellets from this second centrifugation were discarded and supernatants containing the final conditioned media were immediately frozen in aliquots at −80°C for later ATP and HMGB1 measurements. Following removal of conditioned media as above, cell monolayers were harvested with Accutase® (Sigma, Shanghai, PR China) per the manufacturer’s protocol. In preliminary studies (data not shown), Accutase® harvesting was selected over trypsin- and Versene™-based protocols based on lack of interference with flow cytometric detection of cell surface calreticulin. After cell detachment, the Accutase® solution containing harvested cells was combined with cell pellets from the first centrifugation step above, followed by resuspension, cell counting, and processing for flow cytometric measurement of the cell surface calreticulin, as described below.

Measurement of ATP released into serum-free conditioned media. Previously frozen aliquots of conditioned media prepared as above were thawed overnight at 4°C. ATP was measured using a recombinant luciferase-based ATP luminescence assay (ENLITEN® ATP Assay System; Promega, Shanghai, PR China) according to the manufacturer’s instructions.

Development of targeted mass spectroscopy (TMS)-based quantitative HMGB1 assay. A TMS-based quantitative assay for HMGB1 protein released by cells into serum-free conditioned media was developed by IQ Proteomics (Framingham, MA, USA) under contract from Eisai Inc. (Nutley, NJ, USA), as follows. Recombinant human HMGB1 (Bio-Techne, Devens, MA, USA) was digested with LysC (Fujifilm Wako Chemicals, Richmond, VA, USA). Resulting peptides were desalted on StageTips packed with Empore C18 material (3M, Maplewood, MN, USA) and vacuum dried. Four HMGB1 peptides ending in lysine were selected, avoiding methionine-containing peptides and those with fewer than seven amino acids. The selected peptides included HPDASVNFSEFSK, RPPSAFFLFC(CAM)SEYRPK, GEHPGLSI GDVAK and DIAAYRAK and were synthesized as stable heavy isotope peptides (SIL-peptides) for assay development and execution. In initial TMS work-up runs, all four peptides were detected down to pg/ml levels in serum-free DMEM media containing 0.1% BSA, the media used for collecting conditioned media after treating cells with the test agents (see above). Out of the four peptides, DIAAYRAK showed the lowest threshold of detectability in the above media, at 100 pg/ml.

Assaying released HMGB1 in serum-free conditioned media using TMS-based quantitative HMGB1 assay. Conditioned media samples prepared as above were thawed and denatured in 4 M urea, 1% (w/v) sodium dodecyl sulfate (SDS), HALT protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA), 100 mM 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS) buffer, pH 8.0, followed by dithiothreitol (DTT) reduction and iodoacetamide alkylation of free cysteines. The ‘single-pot, solid-phase-enhanced, sample preparation’ (SP3) technology protocol (27, 28) was applied to purify proteins across all samples in these studies. Briefly, magnetic beads were prepared by combining E3 and E7 Sera-Mag Carboxylate-Modified Magnetic SpeedBeads (1:1 v/v) (Cytiva, Marlborough, MA, USA). Beads were washed three times with high pressure liquid chromatography (HPLC)-grade water prior to resuspension in lysis buffer (4 M urea, 1% SDS, 100 mM EPPS, pH 8.0) for a final bead slurry concentration of 50 μg/μl. Approximately 20 μl of the pre-washed bead slurry and 140 μl 100% ethanol were added to each 50 μl sample containing 50 μg of protein (4 M urea, 1% SDS, 100 mM EPPS, pH 8.0). Proteins were allowed to bind and were incubated with the bead slurry for 15 min at 25°C with gentle end-over-end rotation. Following protein binding, supernatants were discarded, and the bead-captured proteins were washed three times with 80% ethanol followed by air drying. The purified protein-capture beads were resuspended in 100 mM EPPS, pH 8.0 and digested with LysC protease at a protease:protein ratio of 1:10. Following protein digestion, 1 pmol of each of the four SIL-peptides was added to each test sample to serve as quantitative internal markers. The digested peptides (supernatant+SIL peptides) were purified and separated from the magnetic beads. Three post-digestion washes were performed to wash and ensure elution of all peptides from the beads, using the following buffers sequentially: 100 mM EPPS, pH 8.0, 0.2% formic acid and 80% acetonitrile/0.1% formic acid. With every wash and elution, the supernatant was transferred and combined with the initial supernatant and dried to completion by vacuum centrifugation. Peptides were desalted on StageTips and vacuum dried.

TMS data were acquired using an Orbitrap Lumos mass spectrometer operating in nano-flow mode using a nLC-1200 UHPLC system (ThermoFisher Scientific). Separation was achieved using an in-house packed C18 column (Sepax GP-C18 resin, 1.9 μm) packed to a length of 35 cm with a 75 μm inner diameter. Peptides were separated over a 35 min linear gradient, 0 to 65% Buffer B (80% acetonitrile, 0.1% formic acid) balanced with Buffer A (0.1% formic acid). Data were acquired via custom targeted assay with the instrument operating in tMS2 mode. Precursors were isolated using the quadrupole and fragmented via HCD fragmentation. MS2 spectra were collected in the Orbitrap at 60K resolution. The number of targets was small so retention times were not used; instead, the mass list was scanned for the entire method. The peptide mass list and charge states used in the TMS method are as follows (listed in sequence of “PEPTIDESEQ”, charge state, light m/z, heavy m/z): HPDASVNF SEFSK, 3, 488.8966, 491.5680; RPPSAFFLFC(CAM)SEYRPK, 3, 668.0050, 670.6764; GEHPGLSIGDVAK, 3, 427.2262, 429.8976; and DIAAYRAK, 2, 454.2534, 458.2605.

TMS data analysis. LC-MS data were analyzed with the open-source Skyline software package (www.skyline.ms). After TMS analysis, concentrations of HMGB1 protein in conditioned media (‘endogenous HMGB1’) were determined by comparing areas under the curve (AUC) for endogenous peptides (light isotopes) with SIL-peptides (heavy isotopes), the two being clearly distinguishable for all four peptides. Since each peptide occurs only once in the HMGB1 molecule, concentrations of endogenous peptides are equivalent to concentrations of HMGB1 protein molecules in the original conditioned media. Final HMGB1 protein concentrations in conditioned media were thus scored as averages of determinations made with each of the four individual peptides.

Flow cytometric measurement of cell surface calreticulin. Cells were harvested after treatment with eribulin and comparators in serum-free media as described above and analyzed for expression of cell surface calreticulin using standard flow cytometry procedures. Briefly, cells were incubated with rabbit anti-human calreticulin monoclonal antibody [Calreticulin (D3E6) XP® Rabbit mAb #12238; Cell Signaling Technology, Shanghai, PR China] or isotype control [Rabbit (DA1E) mAb IgG XP® Isotype Control #3900; Cell Signaling Technology, Shanghai, PR China], followed by washing and incubation with fluorochrome-conjugated secondary antibody [goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody conjugated with Alexa Fluor™ 647; Invitrogen™/Thermo Fisher Scientific, Beijing, PR China]. Flow cytometry was performed on a BD C6 flow cytometer (BD Biosciences, San Jose, CA, USA), using initial forward scatter area (FSC-A)/side scatter area (SSC-A) gating to gate out cellular debris then FSC-A/forward scatter height (FSC-H) gating to select single cells, followed by analysis of cell surface calreticulin using the antibody strategies detailed above.

Statistics. Statistical analyses were performed using GraphPad Prism (Boston, MA, USA, version 10.2.0) with p<0.05 considered statistically significant. When used for multiple comparison strategies, drug-treated versus DMSO control group values were compared using two-way ANOVA with Geisser-Greenhouse correction employing Dunnett’s multiple comparison techniques with individual variances computed for each comparison.

Results

Antiproliferative effects of eribulin and comparators against human breast cancer cell lines in vitro. Antiproliferative effects of eribulin and comparators against two TNBC (BT-549, Hs578T) and one HR+ breast cancer (MCF-7) cell line were assessed and quantified (Figure 1, Table I). Test agents were from two broad categories: six MTAs including eribulin, its biologically active C34,C35-diol synthetic precursor ER-076349 (1, 7), vinca alkaloids vinblastine and vinorelbine and taxanes paclitaxel and docetaxel, and three DDAs including DNA intercalator doxorubicin and platinum compounds cisplatin and oxaliplatin. As a group, MTAs were two to three log orders more potent than DDAs against the three cell lines, with eribulin and ER-076349 consistently showing the highest activities (lowest IC50s), followed by the taxanes and vinca alkaloids. Within DDAs, doxorubicin consistently showed one to two log order higher activity compared to the platinum compounds.

Figure 1.
Figure 1.

Antiproliferative effects of eribulin and comparator cytotoxic agents on (A) BT-549, (B) Hs578T and (C) MCF-7 human breast cancer cells. Inhibition of cell proliferation by the indicated concentrations of eribulin, ER-076349, vinblastine, vinorelbine, paclitaxel, docetaxel, doxorubicin, cisplatin and oxaliplatin was measured after 3-day (BT-549, Hs578T) or 6-day (MCF-7) drug exposures in 96-well plate ATP-based cell proliferation assays. Data shown are means of triplicate determinations at each point and represent percents of cell growth observed in control wells treated with 0.1% DMSO. See Materials and Methods. ATP, Adenosine triphosphate; DMSO, dimethyl sulfoxide.

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Table I.

Antiproliferative activities of eribulin and comparator cytotoxic agents against three established lines of human breast cancer cells.

Interestingly, at the highest concentrations of MTAs for all cell lines, dose/response curves tended to drop to horizontal plateau tails in the 10-30% range (Figure 1), suggesting MTAs may induce senescence for the fraction of cells that is not killed outright by cytotoxicity resulting from disrupted MT processes. In contrast, such horizontal plateau tails were not seen at the highest concentrations of DDAs, with dose/response curves consistently nearing or reaching 0%. These observations suggest that while cells may escape cytotoxicity to senescence for the MT-based mechanisms of MTAs, a ‘senescence safe harbor’ against cytotoxicity is unavailable to cells challenged by DDAs with DNA damaging mechanisms, resulting in more complete cytotoxicity at the top concentrations of DDAs.

Induction of cytotoxicity by 10×IC50 concentrations of eribulin and comparators in serum-free media. Using IC50 values determined in Table I, 10×IC50 values were calculated and used as surrogates for maximally effective doses capable of inducing cytotoxicity. Since measurement of ICD markers ATP and HMGB1 would require extended incubations in serum-free media, it was important to establish that DMSO control cells remained sufficiently viable during serum-free incubations to see additional cytotoxic effects of the test agents. As shown in Figure 2, all three cell lines treated with 0.1% DMSO maintained good viability in serum-free media containing 0.1% BSA for up to 48 h (BT-549, Hs578T) or 144 h (MCF-7) such that additional cytotoxic effects of the drugs can be seen. Treatment with 10×IC50 concentrations of drugs led to numerical decreases in viable cell numbers relative to DMSO controls, with the exception of doxorubicin and cisplatin at 12 h in Hs578T cells and eribulin and ER-076349 at 24 h in MCF-7 cells. The observed decreases in viable cell numbers over DMSO controls are as expected from treatment with 10×IC50 drug concentrations and are consistent with drug induced cytotoxicity.

Figure 2.
Figure 2.

Cytotoxic effects of 10×IC50 concentrations of eribulin and comparators. Effects of 10×IC50 concentrations of the indicated agents on total viable cell numbers of (A) BT-549, (B) Hs578T and (C) MCF-7 cells recovered following incubation for the indicated times in serum-free media containing 0.1% BSA. Cell numbers represent single flow cytometric determinations of total viable cells recovered from three T-175 flasks for each cell line/drug/time combination, with flow cytometric determinations using sequential FSC-A/SSC-A gating to disregard small cellular debris, FSC-A/FSC-H gating to capture only singlet cell events, and annexin V/propidium iodide gating to count only viable cells. See Materials and Methods. Harvested conditioned media and cell preparations here also supported the ATP, HMGB1 and calreticulin evaluations presented in Figures 3-7 below. Horizontal dashed lines at each time indicate viable cell numbers obtained with 0.1% DMSO vehicle controls. ATP, Adenosine triphosphate; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; FSC-A, forward scatter area; FSC-H, forward scatter height, SSC-A, side scatter area.

Interestingly, MTAs eribulin and paclitaxel triggered cytotoxicity faster in BT-549 and Hs578T TNBC cells at the early 12 h time point compared to cisplatin, whereas at the later 24 and 48 h time points, cisplatin and oxaliplatin led to more complete cytotoxicity compared to the MTAs. Within DDAs, cisplatin and oxaliplatin were consistently more potent cytotoxicity inducers compared to doxorubicin in BT-549 and Hs578T TNBC cells. In HR+ MCF-7 cells, cytotoxicity from eribulin and ER-076349 lagged behind almost all other MTAs and DDAs at 24 and 48 h, yet by 96 and 144 h, eribulin, ER-076349 and the two vinca alkaloids overtook DDAs to end with more complete final cytotoxicity. Interestingly, doxorubicin, which had shown unremarkable cytotoxicity against BT-549 and Hs578T cells, showed more robust cytotoxicity against MCF-7 cells at all time points. Combined with observations of MTA top dose plateau tails seen in Figure 1, such patterns suggest temporal, mechanistic and cell type-related differences in cytotoxic responses between MTAs and DDAs.

Effects of cytotoxic concentrations of eribulin and comparators on cellular release of ATP. ATP is a known biomarker for ICD (22-26). Concentrations of ATP released by cells into serum-free conditioned media were assessed using a recombinant luciferase-based ATP luminescence assay to determine if cytotoxicity induced by eribulin and comparators at 10×IC50 concentrations (Figure 2) showed characteristics of ICD (Figure 3). In BT-549 cells, treatment with 10×IC50 concentrations of eribulin led to rapid appearance of extracellular ATP at 12 h, with levels increasing further at 24 and 48 h (Figure 3A). ATP induction by the three other MT-depolymerizing MTAs (ER-076349, vinblastine, vinorelbine) was greater than eribulin at all 3 time points, while MT stabilizers (paclitaxel, docetaxel) showed only transient and small numerical increases over control at 12 h, effects which disappeared by 24 h. None of the three DDAs (doxorubicin, cisplatin, oxaliplatin) showed ATP increases relative to DMSO at any time point. These results thus indicate that, in BT-549 cells, MT depolymerizing MTAs, including eribulin, robustly trigger ATP release, while MT stabilizing MTAs as well as DDAs are ineffective ATP inducers.

Figure 3.
Figure 3.

Effects of cytotoxic concentrations of eribulin and comparators on cellular release of ATP. Effects of 10×IC50 concentrations of eribulin and comparators on release of ATP into extracellular serum-free conditioned medium in (A) BT-549, (B) Hs578T and (C) MCF-7 cells were assessed as described in the Materials and Methods. Horizontal dashed lines represent ATP levels seen with 0.1% DMSO controls at each time point. Results represent mean±range of duplicate determinations. Statistical analyses for drug-treated versus DMSO control groups were performed separately at each time point using two-way ANOVA with Geisser-Greenhouse correction employing Dunnett’s multiple comparison techniques with individual variances computed for each comparison. Statistical significance indicated as *p<0.05; **p<0.01. ANOVA, Analysis of variance; ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide.

ATP release from Hs578T cells was evaluated using eribulin and a smaller set of comparators (Figure 3B). In these cells, eribulin triggered large ATP increases at 12 h, with the effect diminishing at 24 h and virtually gone by 48 h. Unlike BT-549 cells, paclitaxel triggered ATP release at 12 and 24 h, although its effect at both time points was smaller than those seen with eribulin. Except for a modest numerical increase in ATP release by oxaliplatin at 12 h, the three DDAs were ineffective inducers of ATP release in Hs578T cells, although statistically significant decreases in released ATP were seen for these three drugs at 48 h.

Interestingly, compared to the two TNBC cell lines, MCF-7 cells showed very low overall levels of ATP release under any condition, with extracellular ATP concentrations only in the 0.01-0.17 nM range, compared to 0.1-5.0 nM levels for BT-549 and Hs578T cells (Figure 3C). Treatment with eribulin and all other agents numerically increased ATP release at 48 h, with MT depolymerizing MTAs as a group showing larger ATP increases compared to DDAs. However, none of the increases seen at 48 h achieved statistical significance, and such effects had disappeared entirely by 96 and 144 h (Figure 3C), possibly reflecting the low number of viable cells seen at these late time points (Figure 2C).

Overall, the results of ATP release studies in BT-549, Hs578T and MCF-7 cells suggest that MTAs including eribulin are generally more robust early inducers of ATP release compared to DDAs, with few if any effects of DDAs in the two TNBC lines, and numerical effects only comparable to MTAs in the HR+ MCF-7 line.

Effects of cytotoxic concentrations of eribulin and comparators on cellular release of HMGB1. Concentrations of extracellular HMGB1, another known biomarker for ICD (22-26), were assessed following treatment with cytotoxic concentrations of eribulin and comparators using a TMS-based quantitative HMGB1 assay (Figure 4). In BT-549 cells, only slight numerical increases were seen for most drugs including eribulin at 12 h (Figure 4A). However, at 24 and 48 h, HMGB1 release by most MT depolymerizing MTAs increased numerically, with the largest increases seen with vinblastine and vinorelbine. At these time points, increases by ER-076349 surpassed those of eribulin, with both of these surpassing the increases seen with the MT stabilizer paclitaxel. Although the three DDAs (doxorubicin, cisplatin, oxaliplatin) showed no effects at 12 and 24 h, roughly equivalent numerical increases by the three compounds were seen at 48 h, in contrast to the previously seen total lack of DDA effects on ATP at all timepoints in BT-549 cells (Figure 3A).

Figure 4.
Figure 4.

Effects of cytotoxic concentrations of eribulin and comparators on cellular release of HMGB1. Effects of 10×IC50 concentrations of eribulin and comparators on the release of HMGB1 into extracellular serum-free conditioned medium in (A) BT-549, (B) Hs578T and (C) MCF-7 cells were assessed. Horizontal dashed lines represent HMGB1 levels seen with 0.1% DMSO controls at each time point. Results represent mean±standard error of the mean (SEM) of quadruplicate mass spectroscopy determinations made from four different HMGB1-specific peptides; see Materials and Methods. Statistical analyses for drug-treated versus DMSO control groups were performed separately at each time point using two-way ANOVA with Geisser-Greenhouse correction employing Dunnett’s multiple comparison techniques with individual variances computed for each comparison. Statistical significance indicated as *p<0.05. ANOVA, Analysis of variance; DMSO, dimethyl sulfoxide.

In Hs578T cells, eribulin showed minor trends of increased HMGB1 release at 12 and 24 h, which increased substantially at 48 h (Figure 4B). MTA paclitaxel showed a similar trend, lagging only slightly behind eribulin. Doxorubicin, which had been completely ineffective in stimulating ATP release in Hs578T cells, showed a slowly rising trend of increased HMGB1 release starting at 12 h and ending at 48 h, with values only slightly below paclitaxel. Similarly, cisplatin, which had not stimulated ATP release in these cells, showed an increased trend of HMGB1 release at 24 and 48 h.

Finally, in MCF-7 cells, only small numerical increases in HMGB1 release were seen with most drugs at 24 and 48 h (Figure 4C), although notably, the actual HMGB1 concentrations at these time points (5-10 nM) were roughly equivalent to the higher stimulated levels seen with BT-549 and Hs578T cells. HMGB1 release in DMSO controls continued to increase numerically at 96 and 144 h, despite the fact that overall cytotoxicity by DMSO alone at these time points remained relatively low and flat (Figure 2C). However, bearing in mind the HMGB1 increases seen with DMSO alone, at 96 h HMGB1 release seemed to peak for eribulin, the two vinca alkaloids, docetaxel and doxorubicin. By 144 h, values for all drugs were approximately equal to DMSO alone, except the two platinum compounds, which dropped dramatically well below DMSO levels at this latest time point.

Overall, the results of the HMGB1 release studies in BT-549, Hs578T and MCF-7 cells suggest that the onset of HMGB1 effects are generally slower than those seen with ATP, and that MTAs are generally more robust early inducers compared to DDAs, similar to patterns seen with ATP release. On the other hand, the rather slow onset of HMGB1 effects tended to climb to maxima at late time points, not diminishing at later times as seen with ATP. This observation might reasonably be explained by the relatively longer stability of HGMB1 in extracellular medium compared to ATP, which might be subject to ATP hydrolysis with increasing time in conditioned media.

Effects of cytotoxic concentrations of eribulin and comparators on expression of cell surface calreticulin. Cell surface expression of calreticulin, another known biomarker for ICD (22-26), was assessed by flow cytometry following exposure to 10×IC50 concentrations of eribulin and comparators. As shown in Figure 5, numerical trends of eribulin’s calreticulin effects in all 3 cells lines were generally lower and slower to manifest than several comparators, particularly the three DDAs including cisplatin, oxaliplatin and doxorubicin. In BT-549 cells, eribulin had little or no effect at 12 and 24 h, times at which numerical increases of calreticulin positivity were already evident by cisplatin (Figure 5A). At 24 to 48 h, eribulin’s effect increased but remained smaller than that of vinblastine. In Hs578T cells, eribulin had no effect until 48 h, whereas doxorubicin and cisplatin showed major numerical increases at both 24 and 48 h (Figure 5B). In the slower growing MCF-7 cells, eribulin had little effect at 24 and 48 h, but showed moderate numerical stimulation of calreticulin at 96 h, roughly equivalent to that seen with the three DDAs, doxorubicin, cisplatin and oxaliplatin (Figure 5C). Overall, the results of the cell surface calreticulin analysis suggest that eribulin has only low to modest ability to stimulate this ICD biomarker, with smaller numerical effects and slower onsets compared to most other comparators.

Figure 5.
Figure 5.

Effects of cytotoxic concentrations of eribulin and comparators on cell surface calreticulin. Effects of 10×IC50 concentrations of eribulin and comparators on cell surface calreticulin in (A) BT-549, (B) Hs578T and (C) MCF-7 cells were assessed by flow cytometry. Horizontal dashed lines represent cell surface calreticulin levels seen with 0.1% DMSO controls at each time point. Data represent percentages of cell surface calreticulin positive cells determined by flow cytometry following sequential FSC-A/SSC-A gating to disregard small cellular debris, FSC-A/FSC-H gating to capture only singlet cell events, then staining for cell surface calreticulin. See Materials and Methods for details. DMSO, Dimethyl sulfoxide. FSC-A, forward scatter area; FSC-H, forward scatter height, SSC-A, side scatter area.

Temporal appearance of ATP, HMGB1 and cell surface calreticulin as markers of ICD. Patterns of drug effects on ATP, HMGB1 and cell surface calreticulin in Figure 3, Figure 4 and Figure 5 above suggested that the timing of appearance of the three ICD markers might differ and that MTA and DDA subclasses of drugs might differentially affect the individual markers. To evaluate these points in more detail, data were aggregated by normalizing individual ICD marker expression to DMSO controls at each time point, averaging across the three cell lines and plotting results from either all drugs together or from the MTA and DDA subclasses separately. Although MCF-7 cells used a longer time course relative to BT-549 and Hs578T cells, and Hs578T cells employed a smaller subset of drugs relative to BT-549 and MCF-7 cells, the data aggregation approach appears to support the concept of different time courses and subclass sensitivities of the three ICD markers (Figure 6). Thus, ATP release in response to drug treatment as a whole occurred rapidly by 12 h, with 6-fold increases over DMSO controls that rapidly declined at 24 h, 48 h and later time points (Figure 6A). As a subclass, MTA drugs appeared to be more effective ATP inducers than DDAs, with the latter showing almost no effect over DMSO controls at any time point. In contrast to ATP, HMGB1 response trends were both smaller and slower, numerically reaching only 2- to 3-fold over DMSO controls at 48 h, with slow declines thereafter (Figure 6B). As subclasses, MTAs and DDAs appeared to show roughly equivalent potencies as HMGB1 inducers. Finally, the appearance of cell surface calreticulin seemed to be an early and robust response to drug treatment at 12 to 24 h, where a 10- to 20-fold numerical calreticulin increase was seen for all drugs together (Figure 6C). Interestingly, while calreticulin’s early response (Figure 6C) was shared with ATP (Figure 6A), MTA and DDA subclass drugs behaved in an opposite manner concerning the two ICD markers: DDAs appeared to be much more effective at inducing calreticulin compared to MTAs (Figure 6C), while MTAs appeared to be much more effective at inducing ATP compared to DDAs (Figure 6A). Finally, comparison of the three ICD markers together supports the rapid early appearance of both ATP and cell surface calreticulin and a much smaller and slower appearance of HMGB1 (Figure 6D).

Figure 6.
Figure 6.

Time course of ICD biomarker expression in breast cancer cell lines. (A-C): Time course of effects of all tested drugs, MTAs only or DDAs only on expression of (A) released ATP, (B) released HMGB1 and (C) cell surface calreticulin. (D): Time course comparison of the effects of all tested drugs on the three ICD markers as indicated. Values represent means of data from all three cell lines representing expression of the indicated ICD markers normalized to DMSO controls at each time point. ATP, Adenosine triphosphate; DDAs, DNA damaging agents; DMSO, dimethyl sulfoxide; ICD, immunogenic cell death; MTAs, microtubule targeting agents.

Comparison of ICD marker scores of individual drugs and MTA and DDA subclasses. Finally, the potencies of the individual drugs as ICD inducers were compared. ICD marker scores were generated to represent maximal DMSO-normalized effects of each drug on each marker for each cell line regardless of time point. These ICD marker scores were then averaged across the three cell lines either separately for each marker (Figure 7A) or aggregated together for an overall ICD marker score (Figure 7B). For ATP, most MTA subclass drugs including eribulin led to 5- to 8-fold maximal numerical increases in ATP, although docetaxel only showed a weak 2-fold change. In contrast, DDA subclass drugs were largely without effect on ATP, with only oxaliplatin leading to a small 2.5-fold numerical increase over DMSO (Figure 7A, left). For HMGB1, maximal changes were consistently low for both MTAs and DDAs, with eribulin, vinorelbine, doxorubicin and cisplatin all leading to only 3-fold numerical increases (Figure 7A, middle). Overall, cell surface calreticulin showed the greatest maximal effects of the three ICD markers: MTAs including eribulin led to 3- to 6-fold maximal calreticulin increases, with DDA drugs inducing even larger maximal effects with numerical increases of 11- to 32-fold (Figure 7A, right). Combining results from the three individual ICD markers into single overall ICD marker scores, results indicate that MTAs and DDAs lead to 4- to 6-fold and 5- to 12-fold maximal effects over DMSO, respectively (Figure 7B). Overall, among the drugs evaluated, eribulin appears to be centrally positioned in terms of ICD induction potency.

Figure 7.
Figure 7.

Individual and overall ICD marker scores. Maximum DMSO-normalized values for each ICD marker for each drug at any time point were calculated individually for each cell line, then averaged across all three cell lines. (A) Mean±standard error of the mean (SEM) of individual DMSO-normalized ICD marker scores across the three cell lines. (B) Overall ICD marker scores as mean±SEM of individual DMSO-normalized ICD marker scores from (A). Horizonal dashed lines represent DMSO controls normalized to themselves. Brackets indicate drugs categorized as MTAs or DDAs (see text). ATP, Adenosine triphosphate; DMSO, dimethyl sulfoxide; ICD, immunogenic cell death.

Discussion

The current work was undertaken to determine if eribulin’s known cytotoxic effects are characterized by ICD as assessed by three well-known ICD biomarkers: extracellular ATP, extracellular HMGB1 and cell surface calreticulin (22-26). Similarly to its marine natural product parent halichondrin B, eribulin was originally defined solely as an antimitotic MTA that irreversibly blocks cell cycle progression in mitosis, leading to cancer cell death by apoptosis (1, 4-7). Unexpectedly, results of phase III clinical trials in both breast cancer and soft tissue sarcoma (2, 3) showed prolonged OS without corresponding increases in PFS. Translational work undertaken to understand this discordance revealed eribulin’s hitherto unknown effects on residual tumors, tumor cells and the tumor microenvironment, including reversal of EMT, increased cellular differentiation, increased vascular perfusion resulting in reduced tumor hypoxia and reductions in experimental metastasis (8-11). Other studies then pointed to eribulin-induced alterations in tumor phenotype (14, 29, 30), immunological effects such as activation of cGAS-STING innate immune signaling and clinical observations of increased tumor-associated CD8+ T cells and decreased immuno-suppressive markers including PD-1, PD-L1/L2 and FOXP3 (12, 13, 15-18). While the phenotypic effects of eribulin on residual surviving cancer cells may be mediated by both cellular signaling effects and epigenetic regulation (14, 29-32), the question of whether eribulin can contribute to beneficial host immune responses stemming from its cell killing cytotoxic abilities currently remains unanswered. Considering that ICD is a mechanism by which dying cancer cells contribute to host antitumor immune responses (21-26), we investigated whether eribulin’s cytotoxic, as opposed to phenotypic, effects are characterized by ICD.

Initial studies determined the antiproliferative potencies (IC50s) of eribulin compared to several other MTAs (ER-076349, vinblastine, vinorelbine, paclitaxel, docetaxel) and DDAs (doxorubicin, cisplatin, oxaliplatin). As expected, MTAs were generally 2- to 3-log orders of magnitude more potent than DDAs in the three breast cancer cell lines tested, with MTA IC50s in the sub- to low-nM range and DDA IC50s in the sub- to low μM range (Figure 1, Table I). When used at higher 10×IC50 concentrations, time-dependent cytotoxic effects of all drugs tested were confirmed as shown by decreased viable cell numbers in comparison to DMSO controls (Figure 2).

We next asked if cytotoxicity by these drugs had characteristics of ICD, as assessed by established ICD biomarkers including extracellular ATP, extracellular HMGB1 and cell surface calreticulin. Despite drug-to-drug and cell line specific variabilities, results suggest that at least some degree of ICD was induced by all drugs tested including eribulin (Figure 3, Figure 4, Figure 5). Several aspects of the observed variabilities were of interest. For instance, MTAs as a group appeared to be more potent inducers of extracellular ATP release than DDAs, whereas DDAs as a group appeared to be more potent inducers of cell surface calreticulin than MTAs (Figure 6). Also, while MTAs and DDAs seemed roughly equipotent in inducing HMGB1 release, the overall magnitude of HMGB1 values over DMSO controls was considerably smaller than the ATP and calreticulin effects. Finally, ATP and cell surface calreticulin effects tended to peak quickly, while HMGB1 effects were smaller and slower. As such, our results agree with reports of drug-specific and temporal differences in the appearance of various ICD biomarkers following exposure of cells to cytotoxic challenges (22-26).

The large size of this study, which included up to nine test agents for each of three cell lines, three to four time points and four readouts (cell number, ATP, HMGB1, calreticulin), demanded both limitations on numbers of replicates as well as multiple comparison statistical approaches with high stringencies for significance. As a result, much of the data failed to achieve formal statistical significance and should be interpreted cautiously. However, the facts that (i) subclasses of drugs tended to track together as a group, (ii) ICD marker effects tended to follow logical time courses in parallel with loss of viable cells due to cytotoxicity, and (iii) many of the comparator drugs tested are already known to induce ICD, provides some measure of confidence that the numerical trends reported here reflect induction of IDC. Nevertheless, more detailed studies of individual drugs and ICD markers will be required to fully illuminate the ICD responses of eribulin and comparators inferred from our results.

Conclusion

A recent report showed that eribulin treatment of a P-glycoprotein knockout subline of 4T1 mouse mammary carcinoma cells led to release of the ICD markers ATP and HMGB1 (33). Our results extend these mouse findings to human breast cancer cells, with the additional inclusion of a third ICD marker, cell surface calreticulin. In our studies, treatment of BT-549, Hs578T and MCF-7 human breast cancer cells with cytotoxic concentrations of eribulin led to time-dependent increases in measured levels of extracellular ATP and HMGB1 and cell surface calreticulin, with some variability seen between the different cell lines. Among agents tested, eribulin was neither the most potent nor the weakest inducer of ICD markers, but rather occupied a mid-range position shared with vinblastine, paclitaxel, doxorubicin and oxaliplatin (Figure 7). Considering eribulin’s known ability to activate innate immune signaling and tumor-host immune responses (12, 13, 15-18), we speculate that a complex interplay of cytotoxicity-associated ICD from dead and dying cells together with phenotypic changes of surviving cells (8-20) contributes to therapeutically beneficial antitumor immune effects. Our results thus provide impetus for further preclinical and clinical investigation of eribulin’s multifaceted mechanisms to determine how best to leverage its potential as an antitumor immune response modifier.

Footnotes

  • Authors’ Contributions

    MZ, JZ, XA, BL and HY contributed to cell-based study designs, execution of cell proliferation, ATP and calreticulin assays, analyzing results and reviewing the manuscript. BE and RK designed and implemented the TMS-based quantitative HMGB1 assay, and contributed to writing and reviewing the manuscript. BAL conceptualized the study, set experimental strategy, contributed to experimental design, analyzed all data and primarily wrote the draft and final versions of the manuscript.

  • Conflicts of Interest

    MZ, JZ, XA, BL and HY are or were full-time employees of Crown Bioscience Inc., where some studies in this work were performed under contract from Eisai Inc. BE and RK are full-time employees of IQ Proteomics, which performed other studies in this work under contract from Eisai Inc. At the time of this work, BAL was a full-time employee of Eisai Inc., which discovered, developed and currently manufactures and markets eribulin mesylate (as Halaven®) according to FDA-approved indications in the USA and indications in other countries as approved by the relevant regulatory authorities. BAL now serves as a part-time contractor for Eisai Inc.

  • Received September 18, 2024.
  • Revision received November 13, 2024.
  • Accepted November 18, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Eribulin Induction of Immunogenic Cell Death (ICD): Comparison With Other Cytotoxic Agents and Temporal Relationship of ICD Biomarkers
MINGFA ZANG, JIA ZHENG, XIAOYU AN, BO LI, HUAJUN YANG, BRIAN ERICKSON, RYAN KUNZ, BRUCE A. LITTLEFIELD
Anticancer Research Jan 2025, 45 (1) 39-53; DOI: 10.21873/anticanres.17391
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