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Abstract
Background Emerging evidence suggests that the mechanism of chemotherapy-induced cell death may influence the antitumor immune response in patients with cancer. Unlike immunologically silent apoptosis, pyroptosis is a lytic and inflammatory form of programmed cell death characterized by pore formation in the cell membrane and release of proinflammatory factors. Gasdermin E (GSDME) has recently gained attention after cleavage of GSDME by certain chemotherapeutics has been shown to elicit pyroptosis. This study investigated the immunomodulatory effects of a mesothelin-targeting antibody drug conjugate (ADC) in mouse models of breast and colon cancer.
Methods The antitumor effects of the ADC were studied in EMT6 breast cancer and CT26 colon cancer syngeneic mouse models. The immunomodulatory effects of the ADC were assessed by analysis of tumor-infiltrating immune cells using flow cytometry. ADC mechanism of action was evaluated by morphology, biological assays, ADC-mediated cleavage of key effector proteins, and CRISPR/Cas9-mediated knockout (KO). Finally, the antitumor effect of ADC and Fms-like tyrosine kinase-3 ligand (Flt3L) combination therapy was evaluated in tumors expressing GSDME as well as in GSDME-silenced tumors.
Results The data demonstrated that the ADC controlled tumor growth and stimulated anticancer immune responses. Investigation of the mechanism of action revealed that tubulysin, the cytotoxic payload of the ADC, induced cleavage of GSDME and elicited pyroptotic cell death in GSDME-expressing cells. Using GSDME KO, we showed that GSDME expression is critical for the effectiveness of the ADC as a monotherapy. Combining the ADC with Flt3L, a cytokine that expands dendritic cells in both lymphoid and non-lymphoid tissues, restored control of GSDME KO tumors.
Conclusions Together, these results show for the first time that tubulysin and a tubulysin containing ADC can elicit pyroptosis, and that this fiery cell death is critical for antitumor immunity and therapeutic response.
- immunity
- dendritic cells
- drug therapy, combination
- tumor microenvironment
- therapies, investigational
Data availability statement
Data are available upon reasonable request. The data generated in this study are available within the article and its supplementary data files. Data are available from the authors upon reasonable request, with the permission of Bristol-Myers Squibb.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Antibody drug conjugates (ADCs) have been proven to be a successful targeted anticancer platform with accelerated development in recent years.
Pyroptosis plays an important role in antitumor immunity, and the clinical potential of inducing pyroptotic cell death in tumor cells is an emerging area of interest.
WHAT THIS STUDY ADDS
This is the first study to provide evidence that a microtubule-targeting payload can induce pyroptotic cell death and has identified a new mechanism of action for ADCs.
ADC treatment of gasdermin E (GSDME)-expressing tumors induces direct pyroptotic cell death in cancer cells and stimulates antitumor immunity.
The combination of ADC with dendritic cell-modulating Fms-like tyrosine kinase-3 ligand (Flt3L) restores tumor control in GSDME knockout tumors, potentially extending clinical benefit to patients with GSDME-silenced cancers.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This work has identified a new mechanism of action for ADCs, which suggests that patients with GSDME-silenced tumors may benefit from combined treatment with an ADC and Flt3L.
Background
In many cancers, tumor cells reduce or intrinsically resist apoptosis, which has led to the investigation of anticancer agents that induce alternate forms of cell death as a therapeutic strategy.1 Unlike apoptosis, which is considered to be immunologically silent,2 pyroptosis is an inflammatory form of programmed cell death characterized by membrane ballooning, cell membrane lysis and release of proinflammatory factors.3 Pyroptosis is mediated via cleavage of the gasdermin family of proteins. The best characterized gasdermins are gasdermin D, which is cleaved by caspase-1 or caspase-4 and caspase-5 (caspase-11 in mice), and gasdermin E (GSDME), which is cleaved by caspase-3.4 5 These activated caspases cleave the hinge region between the N-terminal and C-terminal domains of gasdermins, releasing the N-terminal domain that oligomerizes to form pores in the cell membrane, leading to cell swelling and ballooning,3 and the extracellular release of proinflammatory cytokines.2 The final common pathway of these forms of programmed cell death is an active process requiring oligomerization of the ninjurin-1 protein in the plasma cell membrane. The resulting large membrane pores mediate osmotic rupture of the plasma membrane, releasing the contents of the cytoplasm including damage-associated molecular pattern molecules.6 Pyroptosis has been identified as a defensive mechanism against intracellular pathogens by triggering inflammatory antimicrobial responses7; however, there is recent evidence suggesting that some chemotherapeutic drugs can evoke a switch from caspase-3-dependent apoptosis to pyroptosis in GSDME-expressing tumors.4 5
GSDME-mediated pyroptosis has been reported to be elicited by chemotherapeutics with varied mechanisms of action including DNA damage and microtubule stabilization, as well as through activation of both intrinsic and extrinsic programmed cell death pathways.8–10 Ultimately, these pathways induce caspase-3-dependent cell death, and as a result, GSDME has been described as the key protein that can convert caspase-3-mediated cell death from apoptosis to pyroptosis. Moreover, GSDME-mediated pyroptosis has been shown following chemotherapy to lead to immunogenic cell death (ICD) and enhanced antitumor immunity.11
Antibody drug conjugate (ADC) therapy has a growing place in the armamentarium of anticancer therapies. ADCs couple the exquisite specificity and tumor-targeting capability of monoclonal antibodies with the extreme potency of cytotoxins that are otherwise too toxic to deliver as systemic agents.12 As well as direct cell killing, ADCs that contain tubulin-targeting payloads such as maytansines and aurostatins can exert immunomodulatory activity via induction of dendritic cell (DC) maturation, proinflammatory cytokine production and effective T-cell priming resulting in antitumor activity.13 14 Tubulysins are antimitotic agents which, like the auristatins, contain a linear peptidic structure and vinca binding site.15 Tubulysins exhibit highly potent cytotoxicity by inhibiting tubulin polymerization and degrading microtubules during cell division. It had earlier been observed that tubulysin induced both apoptosis and caspase-3 activation in a human carcinoma cell line.15
Although systemic toxicity has prevented the development of tubulysins as stand-alone agents, tubulysin has been incorporated as a cytotoxic payload in ADCs.16 17 For example, BMS-986148 is a fully human antimesothelin IgG1 monoclonal antibody conjugated to tubulysin via a cleavable valine–citrulline linker and has been evaluated in a phase I/II a study in patients with advanced solid tumors, predominantly ovarian cancer and mesothelioma. BMS-986148 exhibited a low therapeutic index, with a small subset of patients having tumor shrinkage and a similarly small subset of patients experiencing dose-limiting toxicities including ill-understood off-target hepatotoxicity and presumed on-target pleuropericardial symptoms.18 Like the maytansines and auristatins, tubulysins have immunomodulatory effects via the induction of ICD in mouse models.19
DCs, as the key antigen-presenting cell of the immune system, play a critical role in the initiation of effective antitumor immunity20 with intratumoral DCs driving recruitment of antitumor effector cells.21 However, immunosuppressive factors within the tumor microenvironment (TME) often hamper antigen presentation by intratumoral DCs, inducing T-cell tolerance rather than immunity.22 Fms-like tyrosine kinase-3 ligand (Flt3L) is a cytokine that stimulates the proliferation and differentiation of bone marrow precursors to generate a range of mature haematopoietic cells including DCs.23 Administration of recombinant human Flt3L in both mice and humans has demonstrated marked expansion of DCs in lymphoid and non-lymphoid tissues,24–27 including in tumor tissues, and Flt3L has been evaluated as an immunotherapy agent in both preclinical28–30 and clinical trials for cancer treatment.31 32 DCs are essential to the immune response triggered by ICD, and we hypothesize that increasing DCs within the TME increases the effectiveness of anticancer agents.
In this study, we examined the immunomodulatory effects of an antimouse mesothelin antibody conjugated to tubulysin via a cleavable valine–citrulline linker (mesoADC), the murine equivalent of BMS-986148,33 in syngeneic models of breast and colon cancers. This study revealed that, in addition to controlling tumor growth, mesoADC demonstrates immunomodulatory activity. Investigation into the mechanism of cell death revealed for the first time that a microtubule depolymerizing agent can induce GSDME-mediated pyroptotic cell death in GSDME-expressing cells and that suppression of GSDME expression alters the response of tumors to ADC therapy.
Materials and methods
Compounds and cell lines
EMT6 and CT26 cell lines were purchased from American Type Culture Collection and cultured in RPMI-1640 (Sigma) containing penicillin and streptomycin (Sigma) and 10% fetal bovine serum (FBS) (Bovogen Biologicals). Cells were negative for mycoplasma contamination tested using MycoAlert Mycoplasma Detection Kit (Lonza). Bone marrow-derived dendritic cells (BMDCs) were generated from BALB/c mice as previously described.34 MesoADC and the isotype control ADC were generated and provided by Bristol-Myers Squibb.33 Tubulysin M was purchased from Levena Biopharma. Recombinant human Flt3L was purchased from Sino Biological.
Flow cytometry analysis of cell lines
EMT6 and CT26 cell lines were stained for surface ADC binding using 1 µg/mL of mesoADC or isotype ADC or surface mesothelin expression with 10 µg/mL antimesothelin antibody (AM26533AF-N, Origene) or rat IgGa isotype control (559073, BD Pharmingen) followed by 2 µg/mL goat antimouse IgG Alexa Fluor 647 (421236, Life Technologies) or 4 µg/mL goat antirat IgG Alexa Fluor 488 (ab150165, Abcam). To detect intracellular GSDME expression, cells were fixed in 10% neutral buffered formalin (Sigma) then permeabilized with ice-cold methanol. Cells were stained with 1 µg/mL anti-GSDME antibody (ab215191, Abcam) or rabbit IgG isotype control (02–6102, Invitrogen) followed by 2 µg/mL goat antirabbit Alexa Fluor 647 (A32733, Life Technologies). Data acquisition was performed using a BD Accuri C6 Plus flow cytometer (BD Biosciences), and data analysis was performed using FCS Express software (De Novo Software).
Cell viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay
MTT viability assays were performed with EMT6 and CT26 cell lines as previously described.35 Briefly, EMT6 and CT26 cells were treated with increasing doses of Tubulysin M, mesoADC or isotype ADC in medium. The cells were cultured for 48–72 hours prior to the addition of 0.5 mg/mL MTT (Sigma-Aldrich), incubated for 3 hours at 37°C, MTT solution removed, and the resulting crystals dissolved in isopropanol. Optical absorbance was measured at 570 nm using a FLUOstar OMEGA microplate reader (BMG Labtech).
MesoADC internalization assay
EMT6 and CT26 cells were grown on 18 mm round sterile glass coverslips. The cells were washed with cold phosphate buffered saline (PBS), pulsed with 1 µg/mL mesoADC or isotype ADC for 30 min at 4°C followed by washing with cold PBS to remove excess antibody. Cells at time 0 were fixed immediately with 10% neutral buffered formalin for 10 min. Prewarmed media was added to the remaining cells and placed at 37°C for 30 and 60 min followed by washing and fixing as described previously. Cells were blocked 5% BSA/0.1% Triton-X 100 in PBS for 30 min, followed by 2 µg/mL goat antimouse IgG Alexa Fluor 488 (Molecular Probes, cat# A-11001). Cells were washed and counterstained with 0.5 µg/mL 4′,6-diamidino-2-phenylindole (ThermoFisher). Images were captured using the LSM700 confocal microscope (Zeiss).
Animal experiments
Female BALB/c mice (6–8 weeks old) were inoculated subcutaneously in the right flank with 106 EMT6, CT26, EMT6 CRISPR control, or EMT6 GSDME CRISPR KO cells. Tumor size was measured using electronic calipers and tumor volume was determined using the calculation (a2×b)/2, where a is the shortest diameter and b is the longest diameter of the tumor. Mice were randomly allocated to treatment groups when tumor volume reached approximately 40–60 mm3, ensuring that each treatment group had the equivalent numbers of small and larger tumors and a similar mean tumor volume. Unless otherwise indicated, mice were treated with a single intravenous injection of vehicle control (PBS), isotype ADC or mesoADC at 4.4 mg/kg, which had previously been determined as a tolerable dose. For experiments where mice were coadministered Flt3L, mice were given 10 µg recombinant human Flt3L or PBS control by subcutaneous injection every day for 10 days. Mice were monitored daily using a clinical record sheet, and body weight and tumor volume were measured at least three times per week. Mice were humanely euthanized when tumor volume reached 1000 mm3 or earlier if a predefined clinical score was exceeded.
Tumor analysis
Tumors were harvested from mice and prepared as single-cell suspensions using the mouse tumor dissociation kit and GentleMACS Octo Dissociator (Miltenyi Biotec) following the manufacturer’s instructions. Red blood cell (RBC) lysis was performed using RBC lysis buffer (eBioscience) as per the manufacturer’s protocol. The cells were stained with antibodies against mouse CD45 BUV395, CD3e BUV395, CD49b Pan-NK Cells BV421, CD8A PE-Cy7, CD4 BV786, MHC II (I-A/I-E) BV480, CD11b BB515, CD11c BV711, CD103 APC, F4/80 PE, CD86 FITC, CD80 PE, CD40 PE, CD25 PE, Foxp3 Alexa Fluor 647 and interferon gamma (IFN-γ) Alexa Fluor 488 (BD Biosciences). Fixed Viability Stain 700 (BD Biosciences) was used to exclude dead cells. Data acquisition was performed on the BD LSR Fortessa cell analyzer (BD Bioscience), and data analyzed using FCS Express software.
FoxP3 expression was detected using the Transcription Factor Buffer Set (BD Biosciences) according to the manufacturer’s instructions. For detection of IFN-γ production in T cells, tumor suspensions were stimulated with 5 ng/mL phorbol 12-myristate-13-acetate (Sigma) and 500 ng/mL ionomycin (Sigma) in the presence of GolgiStop (BD Pharmingen) for 4 hours at 37°C. Staining was carried out using the Cytofix/perm with GolgiStop kit (BD Biosciences) according to the manufacturer’s instructions.
Intereleukin (IL)-1β, IL-6, and IL-12 ELISA
BMDCs were cultured with 10 ng/mL, 100 ng/mL or 1000 ng/mL tubulysin M in media for 24 hours. Supernatant was collected and analyzed using IL-1β ELISA (elisakit.com), IL-6 ELISA (elisakit.com), and mouse IL-12 p40/70 ELISA (RayBiotech) following manufacturer’s instructions.
In vitro maturation of BMDCs using mesoADC
EMT6 and CT26 cells were cultured with mesoADC, isotype ADC or vehicle control at the doses indicated. After 48 hours, BMDCs were added to each well and co-cultured for a further 24 hours. All cells were harvested and stained with antibodies against mouse CD45 BUV395, CD11c BV711 and CD86 FITC. Data acquisition was performed on the BD LSR Fortessa cell analyzer and data analysis performed using FCS Express software.
Live cell confocal microscopy
EMT6 cells were seeded in a µ-Slide 8 Well ibiTreat chamber slide (Ibidi). Cells were cultured with PBS vehicle control, 1 µg/mL isotype ADC, 1 µg/mL mesoADC or 0.01 µg/mL tubulysin M for 24 hours for still images or 30 hours for time lapse imaging. 1 µg/mL propidium iodide (PI) (Sigma) was added to the culture medium before imaging. Still images were captured using the LSM700 confocal microscope. Time lapse images were captured on the CellVoyager CV1000 spinning disk confocal microscope (Olympus) with images captured every 217 s.
Lactate dehydrogenase (LDH) release assay
EMT6 cells were treated with increasing doses of mesoADC or tubulysin M in medium. After 24 hours’ incubation with tubulysin M or 40 hours with mesoADC, supernatant was harvested and LDH was measured using the CytoTox96 LDH release kit (Promega) according to the manufacturer’s instructions.
Immunoblotting
Cells were lysed in buffer containing 50 mM Tris, pH 7.4, 150 mM sodium chloride, 1% Triton-X 100, cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Sigma) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were separated by SDS–PAGE and transferred to a PVDF membrane. Blots were probed with 0.5 µg/mL anti-GSDME antibody or 4 µg/mL antiactivated caspase-3 antibody (AB3623, Millipore) and visualized by chemiluminescence using a ChemiDocMP imager (Bio-Rad).
GSDME CRISPR/Cas9-mediated knockout (KO) in EMT6 cells
Mouse DNF5A CRISPR/Cas9 KO plasmid (sc-424934), mouse DFNA5 HDR Plasmid (sc-424934-HDR) and Control CRISPR/Cas9 plasmid (sc-418922) were purchased from Santa Cruz. CRISPR plasmid and HDR plasmid were cotransfected into EMT6 cells using Lipofectamine 2000. After 72 hours, the cells were selected with 1 µg/mL puromycin. Selected cells were expanded, and single-cell sorting of RFP-positive cells was performed using a MoFlo Astrios cell sorter (Beckman Coulter). Clones were screened using intracellular flow cytometry and western blotting for GSDME expression. The selection cassette was removed using a Cre Vector (sc-418923, Santa Cruz) and a single-cell sort was performed on RFP negative cells.
Annexin and PI assay
EMT6 cells were treated with 10 µg/mL mesoADC or 0.1 µg/mL tubulysin M for 40 and 48 hours, collected, washed twice with PBS, and stained using the annexin V-iFluor 647 Apoptosis Detection Kit (Abcam). Cells were stained with 1 µg/mL PI prior to performing data acquisition on a BD Accuri C6 Plus cell analyzer. Analysis of flow cytometry data were performed using FCS Express software.
Statistical analysis
Statistical analyses were performed using GraphPad Prism V.9.0 software. Comparison of two groups was performed by unpaired two-tailed t-test. Data of three or more groups were analyzed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. IC50 was calculated using linear regression and dose–response curve fitting. Longitudinal analysis of tumor growth was carried out by linear mixed effect modeling using the TumGrowth web tool (https://kroemerlab.shinyapps.io/TumGrowth/). P values were calculated by testing whether tumor growth slopes were dissimilar between treatment groups using type 2 ANOVA and pairwise comparisons across groups. Tumor growth rates were calculated using a method adapted from Hather and colleagues.36 The tumor volumes were log-transformed and linear regression was applied to determine the slope for each tumor. Rate-based growth was then calculated for each treatment group relative to the growth rate of the PBS-treated tumor and time. Kaplan-Meier median survival curves were compared using log-rank (Mantel-Cox) test. Statistical significance was reached when the p value was <0.05.
Results
MesoADC potency is dependent on antigen expression
The binding of mesoADC to the murine breast cancer cell line EMT6 and murine colorectal cancer cell line CT26 was analyzed by flow cytometry. MesoADC showed higher binding to EMT6 cells compared with CT26 cells (figure 1A), and this was confirmed to be due to higher mesothelin expression using a mesothelin antibody (online supplemental figure 1A). To determine whether internalization of the antibody occurred, cells were incubated with mesoADC and analyzed 30 and 60 min later by fluorescence microscopy, which confirmed trafficking of the ADC from the cell surface into the cell (figure 1B). Importantly, no cell surface binding was observed using the isotype control ADC, demonstrating the specific binding of mesoADC (online supplemental figure 1B). Next, the cells were treated with mesoADC and cell viability was examined after 72 hours. MesoADC was most potent against EMT6 cells with an IC50 of 0.25 µg/mL compared with 9.5 µg/mL for CT26 cells (figure 1C). Analysis of viability following treatment of the cells with tubulysin alone (the ADC payload) demonstrated equivalent IC50 for both cell lines (0.001 µg/mL). This suggests that the different IC50 values for ADC treatment between the cell lines is a result of differential antigen expression and ADC binding, rather than a difference in response to the free drug itself.
Supplemental material
In vitro characterization of mesoADC. (A) Flow cytometry analysis of mesoADC binding to EMT6 and CT26 cells. Data are presented as representative histograms (left) and MFI (right). Data are presented as MFI normalized to isotype ADC±SEM, n=3. (B) EMT6 (top) and CT26 (bottom) cells were pulsed with 1 µg/mL mesoADC for 30 min then chased for 30 and 60 min at 37°C. Representative images, n=3 independent experiments. (C) EMT6 and CT26 cells were exposed to increasing doses of mesoADC (left and middle) or tubulysin (right). Viability was assessed using MTT assay after 72 hours for mesoADC and 48 hours for tubulysin. Data are presented as a percentage of vehicle control, mean±SEM (n=3). ADC, antibody drug conjugate; DAPI, 4′,6-diamidino-2-phenylindole; MFI, mean fluorescence intensity.
Antitumor activity of mesoADC in a murine model of breast cancer
Having demonstrated a more potent effect of mesoADC on EMT6 cells in vitro, we next wanted to determine if mesoADC could be used to treat mice bearing EMT6 tumors. Tumor-bearing mice were given a single intravenous dose of PBS vehicle or escalating doses of mesoADC (figure 2A). A dose-dependent antitumor effect was observed, with growth inhibition correlating with increasing mesoADC dose. Comparison of the maximum tested dose of mesoADC (4.4 mg/kg) to PBS vehicle or isotype control ADC confirmed the antitumor effectiveness of the ADC up to day 18 (figure 2B), and these results were also reflected in a significant decrease in tumor growth rate up to day 18 (online supplemental figure 2A,B). Interestingly, despite showing limited efficiency in vitro, mesoADC treatment also exhibited tumor control up to day 18 in the CT26 model (online supplemental figure 2C,D).
MesoADC reduces tumor growth and stimulates immunological memory in the syngeneic EMT6 tumor model. (A) EMT6 tumor–bearing mice were treated with escalating doses of mesoADC or (B) maximum dose (4.4 mg/kg) of mesoADC or isotype ADC, or PBS control. Initiation of treatment is indicated by arrows. Data are presented as mean tumor volume±SEM (dotted lines), n=4–5 per group. Longitudinal analysis of tumor growth was carried out by linear mixed effect modeling. (C) Individual growth curves and (D) survival of mice treated with PBS vehicle or 4.4 mg/kg isotype ADC or mesoADC. (E) Mice that achieved CR were rechallenged with EMT6 cells, with naïve mice used as controls (n=5). *P<0.05, **P<0.01. ADC, antibody drug conjugate; CR, complete response.
We next wanted to determine whether mesoADC treatment could induce immunological memory. Mice treated with a single intravenous dose of vehicle, 4.4 mg/kg of mesoADC or isotype ADC were monitored for 45 days or until the tumors reached endpoint size (figure 2C). ADC treatment increased median survival compared with the PBS vehicle control, but this did not reach statistical significance (figure 2D). A complete response was seen in 40% (2/5) of mice treated with mesoADC and 20% (1/5) of mice treated with isotype ADC. In mesoADC-treated mice that were not cured, there was an initial period of tumor control followed by rapid tumor outgrowth. We also observed slowed tumor growth rate in mice treated with our non-targeting isotype ADC, a phenomenon that has been observed by others,37 38 and which is likely caused by premature payload release and/or processing by tumor-associated macrophages (TAMs). Cured mice were rechallenged with a subcutaneous inoculum of EMT6 cells, resulting in rejection of tumors in all challenged mice (figure 2E), suggesting that mice may have survived tumor rechallenge because of immunological memory. This result was obtained irrespective of whether the ADC was antigen-targeting or non-targeting and suggests that the tubulysin payload contributed to an immunologically based antitumor effect mediated by tubulysin-containing ADCs.
MesoADC treatment alters tumor-infiltrating lymphocyte populations
To investigate the effects of mesoADC on immune cell populations within the tumor, we performed immune profiling on single-cell suspensions from the EMT6 tumors of mice treated with escalating doses of mesoADC. MesoADC treatment induced a dose-dependent increase in the percentage of intratumoral immune (CD45+) cells (figure 3A). Analysis of T-cell populations revealed no change in overall T-cell numbers following mesoADC treatment, but analysis of CD4+ and CD8+ T-cell subpopulations revealed a significant decrease in CD4+ T cells and increase in CD8+ T cells with increasing mesoADC dose (figure 3A,B). These effects were specific to mesoADC, as these changes were not observed with isotype ADC treatment (online supplemental figure 3). No significant changes in the percentage of intratumoral natural killer (NK) cells, NKT cells, macrophages, or DCs were observed after mesoADC treatment (figure 3A).
Effects of mesoADC on lymphocyte subsets in the EMT6 model. EMT6 tumors were harvested after ADC administration, and (A) the percentage of live CD45+ leukocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD49b+ CD3− NK cells, CD49b+ CD3+ NKT cells, F4/80+ CD11b+ macrophages and CD11c+ MHCIIhigh dendritic cells was determined. The data are presented as mean±SEM, with n=5–13. (B) Representative density plots depicting changes in the CD4+ and CD8+ T-cell populations following exposure to escalating doses of mesoADC. Percentages of live CD3+ cells are shown. (C) Dissociated tumors were analyzed for CD4+CD25+FoxP3+ Tregs or IFN-γ-producing CD3+ and CD8+ T-effector cells. For IFN-γ analysis, whole tumor digests were incubated for 4 hours with PMA/ionomycin and monensin. The data are presented as mean±SEM, with n=4–5 per group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ADC, antibody drug conjugate; IFN-γ, interferon gamma; NK, natural killer; PMA, phorbol 12-myristate 13-acetate.
To better understand the changes observed in tumor-infiltrating lymphocytes in response to treatment, single-cell suspensions were analyzed for regulatory T (Treg) cells and IFN-γ-producing effector T cells (Teff). A lower frequency of intratumoral Tregs in mesoADC treated tumors was observed as well as a significantly increased proportion of IFN-γ-producing CD3+ and CD8+ T cells, and a significant increase in the CD8+IFN-γ+ Teff-to-Treg ratio with high-dose ADC treatment (figure 3C). Taken together, these results suggest a shift toward antitumor T-cell immunity following mesoADC treatment.
MesoADC triggers maturation of DCs
Microtubule-depolymerizing payloads have been shown to induce DC maturation leading to antitumor activity.13 14 We therefore investigated whether the tubulysin payload or mesoADC could mature DCs in vitro using BMDCs. Following 24 hours’ culture of BMDCs in the presence of tubulysin, we analyzed the surface expression of DC maturation markers MHC-II, CD40, CD80, and CD86 and observed a significant dose-dependent upregulation of all markers (online supplemental figure 4A). Supernatant from DC cultures was analyzed for the release of proinflammatory cytokines IL-1β, IL-6 and IL-12 following exposure to tubulysin. These cytokines have been shown to play critical roles in the regulation of T-cell function and are important for antitumor immune responses.39 A dose-dependent upregulation of all three proinflammatory cytokines was detected in response to tubulysin (online supplemental figure 4B).
Since tubulysin can mature DCs, we next wanted to determine whether mesoADC had the same effect. To do this, we co-cultured mesoADC-treated EMT6 and CT26 cells with BMDCs for 24 hours then measured the expression of costimulatory molecules on the DCs. MesoADC treatment of EMT6 cells was able to induce maturation of co-cultured DCs in vitro, as demonstrated by a dose-dependent upregulation of CD86 expression (figure 4A). This was specific to mesoADC as this response was not seen with the isotype ADC. In contrast, mesoADC treatment of CT26 cells did not induce DC maturation (figure 4A), most likely because the lower mesothelin expression in CT26 cells resulted in extracellular release of free drug in concentrations too low to induce detectable DC maturation. Having demonstrated that mesoADC can mature DCs in vitro, we analyzed intratumoral DCs from mice treated with mesoADC. Single-cell suspensions of EMT6 tumors from mice treated with increasing doses of mesoADC were analyzed by flow cytometry. This revealed a significant increase in the maturation marker CD86 on both CD45+ immune cells as well as on intratumoral DCs (figure 4B). Overall, both mesoADC and tubulysin can induce phenotypical maturation of DCs both in vitro and in vivo.
MesoADC induces maturation of DCs in vitro and in EMT6 tumors. (A) MesoADC-treated EMT6 or CT26 cells were cocultured with BMDCs for 24 hours. CD86 expression was measured on DCs using flow cytometry. Data are presented as representative histograms (left) and MFI (right). Data are presented as mean fold change from BMDCs+cells±SEM, n=3. (B) EMT6 tumor suspensions from mice treated with mesoADC were analyzed by flow cytometry for the expression of CD86 on CD45+ cells (left) and intratumoral CD11c+ DCs (middle and right). Normalized CD86 MFI and percentage of CD86+ cells are represented. The data are presented as mean±SEM, with n=5–9. **P<0.01, ****P<0.0001. BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; MFI, mean fluorescence intensity; ns, not significant.
MesoADC and tubulysin induce pyroptosis
Recent studies have shown that certain chemotherapeutics can cleave GSDME to drive pyroptosis.5 8 9 As both the EMT6 and CT26 cells used in this study express GSDME,5 11 we examined whether tubulysin could induce pyroptotic cell death in these lines. We performed confocal live cell imaging of EMT6 cells following exposure to both mesoADC and tubulysin. The cells demonstrated evident cytoplasmic swelling, and the addition of PI into the culture media confirmed loss of membrane integrity (figure 5A). Similarly, time lapse confocal microscopy allowed visualization of the cytoplasmic swelling and membrane ballooning over time (figure 5B and online supplemental videos 1 and 2). Pyroptosis is commonly associated with LDH release from the dying cell as the membrane ruptures.40 To assess this, EMT6 cells were exposed to increasing doses of tubulysin and mesoADC for 24 and 48 hours, respectively. This showed an increasing release of LDH with increasing dose of tubulysin or mesoADC (figure 5C).
Supplementary video
Supplementary video
MesoADC and tubulysin induce pyroptosis and cleavage of GSDME in EMT6 cells. (A) Confocal live cell imaging of EMT6 cells treated with vehicle, 1 µg/mL isotype ADC, 1 µg/mL mesoADC or 0.01 µg/mL tubulysin for 24 hours with addition of PI (red). (B) Time-lapse live cell confocal imaging of EMT6 cells exposed to 1 µg/mL mesoADC or 0.01 µg/mL tubulysin for 30 hours with addition of PI (red). Time points indicate time elapsed from the first image (hours:min:s). (C) EMT6 cells were exposed to increasing doses of tubulysin and mesoADC for 24 and 40 hours, respectively, and LDH release into culture media was measured. Data are presented as a percentage of maximum LDH release, mean±SEM, n=3. (D) Immunoblotting of GSDME FL, GSDME N and cleaved caspase-3 in EMT6 cells treated for 40 hours with vehicle, 1 µg/mL isotype ADC or 1 µg/mL mesoADC or treated for 24 hours with 2 µg/mL actinomycin D as a positive control.5 Arrows indicate cells of interest. *P<0.05, **P<0.01. ADC, antibody drug conjugate; GSDME, gasdermin E; GSDME FL, full-length gasdermin E; GSDME N, N-terminal gasdermin E; LDH, lactate dehydrogenase; PI, propidium iodide.
Cleavage of GSDME at the hinge region is critical for the generation of the pore-forming N-terminal fragment that drives membrane pore formation in pyroptosis.4 5 We evaluated the ability of mesoADC to cleave GSDME using western blot. Exposure of EMT6 cells to mesoADC for 40 hours resulted in cleaved GSDME and caspase-3 (figure 5D). Taken together, these results showed that both tubulysin and mesoADC can induce pyroptotic cell death.
KO of GSDME in EMT6 cells converts pyroptosis to apoptosis and alters immune cell infiltrates in EMT6 tumors
GSDME expression is suppressed in many cancers and has been suggested to be a tumor suppressor gene.5 We therefore wanted to determine if the mechanism of mesoADC-induced cell death would be altered if GSDME expression were silenced. We used CRISPR/Cas9 to KO GSDME expression in EMT6 cells (figure 6A). Live-cell imaging of GSDME KO cells demonstrated cell rounding, blebbing, and shrinkage associated with apoptosis after exposure to both mesoADC and tubulysin in contrast to the control cells, which demonstrated the characteristic cytoplasmic swelling associated with pyroptosis (figure 6B). Addition of PI to the culture media demonstrated delayed uptake of the dye by the GSDME KO cells, suggesting that the cell membrane was still intact, as seen in apoptosis. Annexin V and PI uptake of GSDME KO cells by flow cytometry showed an increase in early apoptotic (annexin V+/PI−) cells following exposure to mesoADC and tubulysin compared with control cells (figure 6C), indicating a switch toward apoptotic cell death with GSDME KO. Taken together, these results suggest that the mechanism of mesoADC-induced cell death is altered in GSDME KO cells.
KO of GSDME in EMT6 cells converts pyroptosis to apoptosis and alters immune cell infiltration and DC phenotype in EMT6 tumors. (A) Immunoblotting analysis of GSDME expression in CRISPR control and GSDME KO EMT6 cells. (B) Live cell imaging of EMT6 CRISPR control or GSDME KO cells treated with vehicle, 10 µg/mL mesoADC for 48 hours or 0.1 µg/mL tubulysin for 30 hours with addition of PI (red). Arrows indicate cells of interest. (C) EMT6 CRISPR control or GSDME KO cells were exposed to 10 µg/mL mesoADC and 0.1 µg/mL tubulysin for 40 and 48 hours, stained with annexin V and PI and analyzed by flow cytometry. Data are presented as representative flow plots (i) and as mean percentage of early apoptotic cells±SEM, n=3 (ii). (D) Tumor growth curves from mice bearing EMT6 parental, CRISPR control or GSDME CRISPR KO tumors. Data represented as mean tumor volume±SEM (dotted lines). (E) Percentages of live intratumoral CD45+ leukocytes, CD3+ T cells, ratio of CD4+:CD8+ T cells, NKT cells (CD49b+ and CD3+), NK cells (CD49b+ and CD3−), F4/80+CD11b+ macrophage, CD11c+ MHCIIhigh DCs, CD103+ DCs, CD11b+ DCs and CD103+ DC:CD11b+ DC ratio are shown. The data are presented as mean±SEM for all panels, n=13, except for parental, where n=3. *P<0.05, **P<0.01. DC, dendritic cell; GSDME, gasdermin E; KO, knockout; NK, natural killer.
We next wanted to determine if GSDME KO would affect tumor growth. EMT6 parental cells, CRISPR control cells and GSDME CRISPR KO cells were injected subcutaneously in mice and tumor growth was monitored for 16 days. Equivalent tumor volumes and tumor growth rates among CRISPR control and GSDME CRISPR KO groups of mice were observed (figure 6D). Immune profiling of tumors revealed a significant decrease in CD45+ immune cells as well as a significant decrease in DCs in the GSDME KO tumors (figure 6E). Further investigations of the DC phenotype revealed a significant increase in CD103+ CD11b− cross-presenting DCs in GSDME KO tumors, as well as a significant decrease in CD11b+CD103− DCs (figure 6E). In contrast, analysis of DC maturation markers revealed a significant reduction in major histocompatibility complex II (MHCII) and CD40 expression on DCs from GSDME CRISPR KO tumors compared with control tumors (online supplemental figure 5), a phenotype known to be associated with tolerance and suppression of CD8+ T-cell immunity.41
Combination of mesoADC with Flt3L restores tumor control in GSDME KO tumors
We next wanted to determine if the switch from pyroptosis to apoptosis in GSDME KO cells affected the response to ADC treatment in vivo. We demonstrated that GSDME KO had no significant effect on tumor growth or survival in the absence of treatment when compared with control tumors (figure 7A and online supplemental figure 6). After a single dose of 4.4 mg/kg mesoADC, a significant reduction in median survival for mice bearing GSDME KO tumors (23 days) compared with CRISPR control mice (29.5 days) was observed (figure 7B).
MesoADC shows reduced effectiveness in GSDME KO tumors, which is restored when combined with Flt3L. Mice bearing EMT6 CRISPR control or GSDME CRISPR KO tumors were treated with vehicle control or mesoADC at 4.4 mg/kg alone or with 10 µg hFlt3L/day for 10 days. Mice were monitored for 45 days. Data are presented as Kaplan-Meier survival curves, n=6–8. (A) Comparison of survival for mice bearing CRISPR control and GSDME CRISPR KO tumors. (B) Comparison of CRISPR control+ADC and GSDME CRISPR KO+ADC. (C) Comparison of GSDME CRISPR KO with ADC and Flt3L alone, and in combination. Pairwise comparison was performed for each treatment compared with GSDME CRISPR KO. (D) Summary of median survival and complete response for each treatment group. *P<0.05, **P<0.01, ***P<0.01. ADC, antibody drug conjugate; Flt3L, Fms-like tyrosine kinase-3 ligand; GSDME, gasdermin E; KO, knockout.
Having shown that GSDME KO alters DC numbers and phenotype within the tumors, we hypothesized that the addition of Flt3L may restore DC numbers and improve effectiveness of the ADC in mice bearing GSDME KO tumors. To test this, we treated mice bearing GSDME CRISPR KO tumors with mesoADC and Flt3L alone or in combination. Flt3L treatment significantly increased median survival (12.0 days vehicle vs 20.5 days Flt3L) as did mesoADC treatment alone (12 days vehicle vs 23 days mesoADC) (figure 7C). The combination of mesoADC and Flt3L not only significantly increased survival (12 days vehicle vs 29 days mesoADC and Flt3L) but also resulted in a median survival time that was equivalent to mesoADC treatment in CRISPR control tumors (29.0 days of GSDME CRISPR KO with ADC and Flt3L vs 29.5 days of CRISPR control with ADC). Moreover, mesoADC and Flt3L treatment led to complete resolution of one of the tumors. In CRISPR control tumors, the addition of Flt3L to mesoADC treatment provided no advantage with equivalent median survival times and complete resolution of the tumors compared with mesoADC alone (figure 7D and online supplemental figure 6). Interestingly, Flt3L treatment alone in mice with GSDME-expressing tumors did not result in changes in tumor growth or median survival (online supplemental figure 7), suggesting that Flt3L treatment may only be effective in tumors lacking GSDME expression.
Discussion
In this study, we describe the antitumor and immunomodulatory activity of a mesothelin-targeting ADC in mouse models of breast and colon cancer. Investigations into the mechanism of cell death demonstrated that tubulysin, either alone or as part of an ADC, cleaves GSDME leading to pyroptosis in GSDME-expressing cells. Although the immunomodulatory effects of tubulysin have previously been described,19 this is the first study to show that a microtubule depolymerizing agent and indeed an ADC can elicit pyroptotic cell death. With the recent resurgence of ADC therapies stemming from technology improvements and marked by a growing number of Food and Frug Administration (FDA) approvals, this preclinical study identifies a new mechanism of action for ADCs which may have ramifications for how ADC therapies are applied in patients with cancer.
Using syngeneic immunocompetent tumor models, we demonstrated that mesoADC can control tumor growth and elicit immunological memory. To investigate the effect of antigen density on ADC response, we used two different murine models, EMT6 and CT26 cells, which have high-level and low-level expressions of mesothelin, respectively. As expected, the CT26 cell line had reduced cytotoxicity in vitro after ADC treatment, with a 40-fold higher IC50. Nevertheless, ADC therapy was effective in the CT26 syngraft model. Hence, even tumors with reduced expression of the target antigen may be susceptible to treatment with mesoADC. This could be in part due to mesoADC-induced pyroptotic cell death, with a study by Wang and colleagues demonstrating that pyroptosis in fewer than 15% of tumor cells was sufficient to clear an entire tumor in animals.42
Microtubule depolymerizing agents, such as the maytansines and aurostatins, which have FDA approval for clinical use in ADCs, demonstrate a potent ability to mature DC so that they become immunogenic rather than tolerogenic.13 14 Likewise, data from this current study have shown that tubulysin stimulates DC maturation as measured by upregulation of costimulatory molecules as well as the release of proinflammatory cytokines. Moreover, upregulation of CD86 on intratumoral myeloid cells and DCs was observed following mesoADC treatment in mice bearing EMT6 tumors. These results are consistent with those of a previous study demonstrating similar immunomodulatory properties of a HER2-targeting tubulysin-based ADC.19 In addition, we also observed changes in post-treatment tumorous T-cell populations, with significantly reduced CD4+ T-cell infiltration, mirrored by an increase in CD8+ T cells. Further analysis of the T-cell phenotype revealed a decrease in Treg cells and an increase in IFN-γ-secreting T cells, resulting in an increased intratumoral T-effector to Treg ratio, indicative of an antitumor immune response.43 Similarly, other studies examining the immunomodulatory effects of ADC treatments also demonstrated an altered antitumor T-cell phenotype following treatment.14 19 44
Interestingly, in the EMT6 tumor model, we observed evidence of an antitumor response with immune characteristics after administration of the isotype ADC. Initially, we observed rapid tumor growth, then slower tumor shrinkage followed by tumor regrowth except for one mouse that was cured. The valine-citrulline linker in the isotype ADC is stable in human plasma but unstable in mouse plasma,45 46 thus prematurely releasing the payload into circulation. In addition, TAMs have been shown to break down ADCs, even non-targeting ADCs, resulting in bystander killing of tumor cells.37 47 48 Consequently, TAM-mediated generation and release of free tubulysin may kill tumor cells and stimulate an antitumor immune response.
Recent studies have shown that certain chemotherapeutics can elicit caspase-3-mediated pyroptosis in GSDME-expressing tumors, leading to cell death and antitumor immune responses.5 8 9 In this study, we have provided the first evidence showing that a microtubule-targeting payload and tubulysin-containing ADC can induce pyroptotic cell death. Treatment of GSDME-expressing cells with mesoADC or tubulysin demonstrated morphological hallmarks of pyroptosis, release of LDH and cleavage of GSDME. CRISPR-mediated KO of GSDME altered cell death morphology and increased the proportion of apoptotic cells after ADC treatment, suggesting that the GSDME KO converted programmed cell death from pyroptosis to apoptosis. Although we have identified ADC-mediated pyroptosis in a breast cancer model, these findings could be applied to a broad range of GSDME-expressing cancers including melanoma, renal carcinoma, and lung, breast, and esophageal cancers.49 Likewise, we speculate that any tubulysin-containing ADC could mediate pyroptosis in GSDME-expressing tumors and future studies will evaluate other microtubule inhibitors and ADC payloads for their ability to elicit pyroptosis.
In addition to tumor cells, chemotherapy can induce pyroptosis in GSDME-expressing normal tissues such as spleen, kidney, and intestine, thus contributing to chemotherapy-related toxicity.5 Given that advanced chemistries have increased the therapeutic index of recent ADCs, ADCs may represent a drug delivery method that can maximize tumor cell pyroptosis while minimizing pyroptosis of normal cells, which offsets the off-target toxicities expected from conventional cytotoxic chemotherapy.
Unlike Zhang et al, who found increased growth of GSDME KO EMT6 tumors,11 we did not observe any differences in the in vivo growth of GSDME KO and CRISPR control EMT6 tumors. In our GSDME KO tumors, we observed reduced proportions of CD45+ cells and DCs of altered phenotype but without evident changes in the proportions of T cells and macrophages as found in the other study. Differences in the GSDME KO approach and in the tumor site50 may account for these different findings. We used a non-integrating approach for GSDME KO because we and others found that lentiviral-mediated CRISPR KO induces constitutive Cas9 expression, which can be immunogenic and lead to tumor clearance.51 52 We also used a subcutaneous rather than an orthotopic location for tumor inoculation.
Although DCs were not analyzed by Zhang et al,11 we found reduced intratumoral DCs of altered phenotype in GSDME KO tumors. We showed an increased type 1 conventional DC (cDC1):type 2 conventional DC (cDC2) ratio in GSDME KO tumors, which may indicate a shift toward CD8+ T cell-mediated immunity. In GSDME KO tumors, we found an increased proportion of the uncommon and cross-presenting cDC1 (CD103+ CD11b−), which may favor CD8+ T cell-driven responses.21 53 We also found a decreased proportion of CD11b+ cDC2 (CD11b+ CD103−), which may either be immunosuppressive54 or promote CD4+ T-cell priming.55 In contrast, we found that DCs in GSDME KO tumors had reduced expression of the activation markers, MHCII and CD40, thus suggesting a switch toward tolerizing DCs in GSDME KO tumors.41 Although the immunophenotypical analysis may suggest a shift toward cDC1, the overall reduction in DC numbers, together with the altered maturation, suggests that the DC response in GSDME KO tumors is impaired and may contribute to the reduced effectiveness of mesoADC.
The DC-expanding cytokine, Flt3L,24 25 has been shown to reduce tumor growth in preclinical mouse models.28–30 In this study, Flt3L treatment alone did not alter growth of GSDME-expressing EMT6 syngraft tumors. In contrast, Flt3L treatment controlled GSDME KO tumor growth and significantly increased median survival compared with control. Interestingly, the Flt3L-responsive murine tumors, B16 melanoma and EL4 lymphoma, have silenced GSDME expression.5 11 These data suggest that the improved responsiveness to Flt3L treatment of GSDME-silenced tumors may result from Flt3L treatment, providing a quantitative and qualitative correction of the DC impairment we observed in GSDME KO tumors.
Furthermore, treating mice with GSDME-expressing tumors with both Flt3L and mesoADC did not significantly prolong survival than that afforded by mesoADC treatment alone. In contrast, use of this combination treatment in mice bearing GSDME KO tumors improved median survival compared with either treatment alone. Given that Flt3L-expanded DCs are immature or only partially activated,26 56 we propose that, by boosting intratumoral DC numbers, Flt3L treatment of mice bearing GSDME KO tumors results in partial shrinkage of tumors. Furthermore, combination treatment with Flt3L and mesoADC results in the intratumoral generation and release of tubulysin, which is sufficient to fully mature the Flt3L-expanded DCs. Consequently, the antitumor immune response toward GSDME KO tumors is restored, resulting in tumor shrinkage that is equivalent to that observed following mesoADC treatment of GSDME-expressing tumors.
In addition to caspase-3, granzyme B released from cytotoxic T cells and NK cells has been shown to cleave GSDME leading to pyroptosis.11 We found that mesoADC treatment increased the number of intratumoral cytotoxic T cells. Hence, in GSDME-expressing tumors, we hypothesize that, via granzyme B, the newly recruited CD8 and NK cells induce further pyroptosis of tumor cells, thereby establishing a positive feedback loop. GSDME has been proposed as a tumor suppressor gene, with expression suppressed in many cancers,5 thus potentially restricting the effectiveness of pyroptosis-inducing drugs to the smaller number of patients with GSDME-expressing tumors. However, we show here that combining a pyroptosis-inducing ADC with the DC-expanding cytokine, Flt3L, can permit control of GSDME KO tumors to be restored to the extent observed for mesoADC treatment of mice with GSDME-expressing tumors. This is a clinically meaningful preclinical result that suggests that patients with GSDME-suppressed tumors may benefit from combination therapy with an ADC and Flt3L.
It is increasingly acknowledged that the relationship between pyroptosis and apoptosis is complex, and that both pathway intermediates such as caspase-3 and effectors such as GSDME can variously be employed to determine the kind of programmed cell death.57 Here, however, it is important to recognize that treatments that induce tumor cell pyroptosis may sidestep the resistance of many cancers to treatment-induced apoptosis. Data presented herein demonstrate that mesoADC treatment of mice with GSDME-expressing tumors results in antitumor effects mediated both by tubulysin-induced pyroptotic tumor cell death and tubulysin-induced DC maturation together with the recruitment of pyroptosis-inducing cytotoxic T cells to the tumor. In GSDME KO tumors, the therapeutic combination of mesoADC and Flt3L compensates for the effects of mesoADC-induced apoptosis by boosting the number and antigen-presenting functions of intratumoral DCs, thus restoring antitumor immunity. Therefore, mesoADC and Flt3L combination therapy provides a novel approach that could extend the clinical benefit of ADC therapy to patients with GSDME-suppressed tumors.
Supplemental material
Data availability statement
Data are available upon reasonable request. The data generated in this study are available within the article and its supplementary data files. Data are available from the authors upon reasonable request, with the permission of Bristol-Myers Squibb.
Ethics statements
Patient consent for publication
Ethics approval
All animal experiments were approved by the University of South Australia Animal Ethics Committee, Adelaide, Australia (approval number 47-17) and conducted following institutional ethical guidelines.
Acknowledgments
We acknowledge funding support for this study from Bristol Myers Squibb (BMS). We are grateful to Dr Sanjeev Gangwar and his team at BMS for the provision of the mesoADC and isotype ADC. We thank the Health Services Charitable Gifts Board (Adelaide) for their ongoing financial support. The graphical abstract was created using www.biorender.com.
References
Supplementary materials
Supplementary Data
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Footnotes
Contributors NLW: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, validation, writing (original draft, review, and editing) and guarantor. AHS: data curation, formal analysis, investigation, methodology, resources, validation, and writing (original draft, review, and editing). VL: data curation, formal analysis, investigation, methodology, and writing (original draft, review, and editing). PC: methodology, resources, and writing (review and editing). HW: data curation, formal analysis, methodology, and writing (review and editing). MPB: conceptualization, formal analysis, funding acquisition, project administration, resources, validation, and writing (original draft, review, and editing).
Funding This work was supported by Bristol-Myers Squibb and the Health Services Charitable Gifts Board (Adelaide).
Competing interests There are no competing interests.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.