Background
Traditional treatments for lymphoma, such as chemotherapy and radiotherapy, have notable drawbacks, and passive immunotherapy using a monoclonal antibody is restricted to CD20-positive B cell lymphoma. In addition, resistance to the treatment is also observed [
1]. Therefore, new treatment types are urgently required. One such novel therapy is treatment with active immunotherapeutic agents, such as agonists of the TOLL-like receptors (TLRs), which facilitate the induction of prolonged antitumour immune responses.
TLRs are expressed on a series of immune cells, such as dendritic cells, macrophages, B cells, T cells and natural killer cells, and they recognise specific pathogen-associated molecular patterns (PAMPs). With the exception of TLR3, most TLR signalling depends on myeloid differentiation primary-response protein 88 (MYD88). The activation of MYD88-dependent signalling results in the activation of NF-κB, IFN regulatory factors (IRFs) and activator protein 1 (AP-1), leading to the release of proinflammatory cytokines and stimulatory molecules for the activation of immune cells [
2]. As the bridge between innate immunity and adaptive immunity, TLR signalling regulates cytokine production, T helper and effector cells as well as suppressor cells, such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) [
3-
6].
Recent studies have focused on TLR7, 8 and 9, which are localised intracellularly to the endosomal membranes. TLR7 activation by ligands, such as viral RNA or synthetic agonists, induces strong T
H-1-biassed immune responses and thus leads to a durable tumouricidal effect by supporting the activation of CD8+ T cells [
7]. In addition to the eradication of large primary tumours, the combined application targeting TLR7, 8 and 9 also established long-term antitumour immunity [
6]. For the treatment of lymphoma, Jonathan and colleagues established a phase II clinical study in which a TLR9 agonist (1018 ISS) was used with rituximab for follicular lymphoma [
8]. Enhanced antitumour efficacy was also found when radiotherapy was combined with the TLR9 agonist CpG DNA 1826 or the TLR7 agonist R848 [
9,
10].
In this study, we introduced a novel TLR7 agonist called SZU-101 and sought to investigate its immunogenicity and antitumour effects. We hypothesised that locally or systemically administered SZU-101 in combination with conventional chemotherapeutic agents would induce systemic antitumour immune responses. In addition, as one of the conventionally used chemotherapeutic agents for lymphoma, doxorubicin was selected for the combined treatment. Here, we demonstrated that intratumourally administered SZU-101 enhanced the therapeutic effectiveness of doxorubicin (DOX) through the generation of a systemic immune response. The combined application induced the release of proinflammatory cytokines, facilitated the maturation of dendritic cells and activated B cells and T cells, leading to the eradication of both primary and distant tumours in a murine lymphoma model. This study provides evidence for the translation of the combined active immunotherapy to early phase clinical trials for the treatment of lymphoma.
Discussion
Lymphoma is a type of malignant cancer in the lymphohematopoietic system, presenting as a solid tumour in lymph nodes, which is called nodal lymphoma, or as extranodal lymphoma in the lymphoid tissues of many organs, such as the spleen, bone marrow, skin, brain, liver and bowels. Recent studies have revealed that combined immunotherapy for lymphoma may help overcome the suppressive micro-environment, induce tumour-specific immune response and improve tumour clearance.
It was reported that the administration of a conventional chemotherapeutic agent, such as cyclophosphamide or DOX, in combination with TNF-α, enhanced the antitumour effects against large established tumours in a murine lymphoma model [
11,
12]. The standard cyclophosphamide, DOX, vincristine and prednisone (CHOP) therapy was also used in combination with a monoclonal antibody, rituximab (R-CHOP), for CD20-positive B cell lymphoma, or with bryostatin 1 for human diffuse large cell lymphoma [
13,
14]. On the other hand, for immunotherapy, conventional antitumour therapy, such as monoclonal antibodies, chemotherapeutic agents and radiotherapy, was applied in combination with TLR agonists, such as TLR9 agonist 1018 ISS, TLR9 agonist CpG DNA 1826 and TLR7 agonist R848 [
8-
10]. These findings suggested that combined therapy with conventional drugs at a carefully selected dose and course schedule could lead to advanced antitumour effectiveness in lymphoma treatment. In this study, we focused on establishing a therapeutic schedule with the proper dose for the treatment of lymphoma using a TLR7 agonist synthesised in our lab combined with chemotherapeutic drugs in a murine T cell lymphoma model.
As the TLR7 was localised intracellularly, we introduced a fluorescence imaging technique and found that the synthesised compound can be transported to the cytoplasm (Additional file
2: Figure S2). We also demonstrated that SZU-101 can activate TLR7-NF-kB signalling in a TLR7-specific system at a low concentration of 1 μM after 6 h of stimulation or at a higher concentration of 5 μM after 4 h of stimulation. These findings suggested that SZU-101 is a TLR7 agonist and can induce downstream signalling through TLR7 activation.
It was reported that TLR7 activation could lead to strong T
H-1-biassed immune responses and induce the release of proinflammatory cytokines [
7,
15,
16]. In our study, we observed a high level of proinflammatory cytokines, such as TNF-α, IFN-γ and IL-12 in sera from SZU-101-treated tumour-bearing mice, while no induction of T
H-2 cytokines IL-4, IL-5 or IL-10 was detected (Figure
1D). We also observed that one T
H-2-biassed cytokine, IL-6 was significantly induced after agonism, which was in line with others’ reports that the activation of TLR7 by R848, 852A and imiquimod led to the release of the cytokines observed above through the TLR7-MYD88 pathway [
17,
10,
18]. The T
H-1 cytokines IFN-γ and IL-12 were significantly induced after 4-h treatment. However, more importantly, all induced cytokine productions were reduced to the basal level after 24-h treatment, providing a critical clue for planning the treatment schedule. Furthermore, TLR7 agonists could also significantly up-regulate the activation marker CD69 on B/T cells and activate dendritic cells [
16,
10]. In the study, we also found that the application of SZU-101 significantly induced the expression of the activation marker CD69 on B/T cells and stimulated the activation of dendritic cells (Figure
1C). These findings indicated that SZU-101 is a functional TLR7 agonist that can induce a strong immune response in tumour-bearing mice with such high effectiveness that the effect of SZU-101 on cytokine release and activation of immune cells was observed as early as 2 h after treatment.
In human lymphoma, the expression of TLR7 has been demonstrated in several studies. TLR7 was widely expressed in human follicular lymphoma (FL), diffuse large B cell lymphoma (DBCL) and peripheral T cell lymphoma (PTCL) as well as in other lymphocytic diseases, such as chronic lymphocytic leukaemia (CLL) [
19-
21]. In this study, as most of the murine lymphoma cells expressed endogenous TLR7 (Figure
2A), we sought to investigate whether SZU-101 had a direct effect on the tumour cells. The MTT assay results indicated that SZU-101 had no direct effect on EL4 tumour cells (Figure
2B), which was in line with others’ findings of another TLR7 agonist, imiquimod, on acute myeloid leukaemia (AML) cells, whereas the TLR7/8 agonist R848 may lead to terminal differentiation and suppressed cell proliferation in AML [
22].
For lymphoma treatment, the low immunogenicity and immunosuppression have been the most problematic issues in the development of anticancer therapies. As described above, the combined application of conventional antitumour therapies with immune stimuli may provide potential strategies for lymphoma treatment. For instance, for B cell lymphoma, the TLR1/2 agonist (Pam
3CSK
4) was applied with the conventional chemotherapeutic agent Ara-C, resulting in a synergistic anticancer effect through the up-regulation of immunomodulatory molecules [
23].
In our murine model of T cell lymphoma, the application of a conventional chemotherapeutic agent, DOX, which is a component of CHOP therapy, in combination with TLR7 agonist SZU-101, significantly improved survival compared with PBS treatment or DOX treatment alone. However, based on the survival data, no significance difference was observed between SZU-101-treated groups within 60 days post treatment (log-rank test, Figure
3C).
As the anticancer immunotherapy of TLR agonists could lead to the quick release and systemic dispersion of proinflammatory cytokines, the dose and administration method must be carefully selected [
24]. To determine the treatment schedule and administration method, we chose a relatively low dose of DOX (3 mg/kg) and applied DOX every 2 days, such that the total usage of DOX was approximately 50% less than a previous study on CHOP therapy in a murine model of lymphoma [
14]. Regarding the administration method, it was reported that the local administration of the TLR7 agonist imiquimod with anti-CD40 immunotherapy exhibited a strong antitumour effect in a murine model of malignant mesothelioma [
25]. However, others have reported that the systemic administration of R848 with radiotherapy primed durable antitumour immune responses in a murine model of lymphoma [
10]. In this study, we sought to include both local and systemic administration of SZU-101 to investigate the proper administration method for the combined therapy.
We generated a murine tumour model, in which two tumours were implanted on the same mouse to assess the local and systemic antitumour effect of the treatments. On the local tumour, which was directly treated with the drugs, the combined therapy led to significant improvement of the tumouricidal effect, especially 1 week post treatment, whereas the tumours in other groups began to re-grow (Figure
4A). However, in the distant tumour, which was not directly treated, DOX showed relatively low effectiveness compared with other treatments. We also observed that only DOX in combination with intratumourally administered SZU-101 (as local administration) exhibited a durable antitumour effect on the distant tumours, while tumour re-growth was noted in other groups of treatments, including the treatment of DOX in combination with intraperitoneally administered SZU-101 (as systemic administration; Figure
4B). These findings suggested that combined therapy with DOX and SZU-101 by local administration exhibited the best therapeutic performance in the murine model of EL4 T cell lymphoma by generating a strong local antitumour effect and inducing a systemic immune response against a distant tumour.
As the major effect of the activation of TLR7 is the induction of IFN-γ [
18], we observed that when EL4 tumour cells were co-cultured with lymphocytes from the surviving mice treated by SZU-101, especially with the lymphocytes from the CoAd i.t. group, IFN-γ release was significantly increased (Figure
5A). We also found increased IL-6 induction when EL4 cells were co-cultured with lymphocytes from intratumourally administered SZU-101-treated mice (Figure
5A), suggesting that the intratumourally administered SZU-101 may have triggered an IL-6-based tumour-specific memory immune response. Furthermore, it was reported that the activation of TLR1/TLR2 signalling by a synthetic bacterial lipoprotein exhibits potential antitumour capabilities of up-regulating CTL function [
26]. In this study, we found a similar result for TLR7, which suggested that the CTL function of the lymphocytes from SZU-101-treated surviving mice was significantly increased. In fact, lymphocytes from the CoAd i.t. group exhibited a 200% enhancement in CTL function (Figure
5B). Although the underlying mechanisms of the enhanced antitumour immune response by this combined therapy are unclear, based on the current data, we speculate that intratumourally administered SZU-101 enhances the process of antigen presentation on the tumour site where the tumour-specific antigens were concentrated by DOX-induced tumour cell death, while simultaneously activating immune cells to generate a durable systemic antitumour effect.
Methods
Mice, cell lines and animal model
The C57BL/6 mice used in this study were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China). The EL4 T cell lymphoma cells were maintained in DMEM (Gibco) with 10% FBS and 100 U/ml penicillin-streptomycin.
The C57BL/6 mouse lymphoma model was developed through the subcutaneous implantation of EL4 cells. Briefly, 1 × 10
6 EL4 cells were inoculated subcutaneously on day 0 of each experiment. The mice were ready for experiments on day 7, when the tumours were approximately 500–1,000 mm
3; the tumour volume was calculated using the following equation: [tumour volume = short axis
2 × long axis/2] [
27]. A visible tumour size of 20 mm in diameter was defined as the endpoint criterion, and those mice that met the criterion were sacrificed according to the AVMA guidelines on euthanasia.
All animal experiments were performed with the approval of Laboratory Animal Welfare and Ethics Committee, School of Medicine, Shenzhen University.
Western blotting
The anti-TLR7 antibody was purchased from Santa Cruz Biotechnology and was applied to confirm TLR7 expression in the mouse lymphoma cell lines EL4, TK-1 and A20. Cell lysates were subjected to electrophoresis using 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary and secondary antibodies with proper blocking procedures and finally exposed to X-ray film (Fuji).
HEK-BLUE assay
The TLR7 agonist SZU-101 was synthesised as described in Additional file
2: Figure S2. HEK-BLUE hTLR7 cells were purchased from InvivoGen. The cells stably expressed human TLR7 and a SEAP reporter, which can be used to detect TLR7 agonism through the activation of NF-kB signalling. The cells were maintained in selective DMEM growth medium with an additional 10 μg/ml blasticidin and 100 μg/ml Zeocin™. After incubation with different doses of SZU-101, the cells were tested using the HEK-BLUE detection kit according to the manufacturer’s instructions. The TLR7 agonist imiquimod and purified mouse TNF-α were used as positive controls. The induction of TLR7 activation can be visualised and assessed by reading the OD at 620–655 nm.
MTS assay
A CellTiter® 96 MTS assay (Promega) was performed according to the manufacturer’s instructions. Briefly, 1 × 104 EL4 cells were seeded in each well with PBS, SZU-101 (3 μg/μl), DOX (2 μg/μl) or SZU-101 combined with DOX. Cell viability was evaluated after a 24-h incubation.
Flow cytometry
On day 7, the tumour-bearing mice were treated with 3 mg/kg SZU-101 or 4 mg/kg DOX by intraperitoneal administration. Mouse spleens were collected at 2, 4 or 24 h post treatment on day 8, and the splenocytes were prepared by removing the red blood cells with RBC lysis buffer (BioLegend) after separating the cells through a 70-μm cell strainer. Approximately 1 × 106 cells were stained with corresponding florescence antibodies and analysed by FACSCalibur flow cytometry (BD Biosciences). The antibodies for flow cytometry were purchased from BioLegend and included the following: anti-CD11c-Alexa488, anti-CD86-PerCP/Cy5.5, anti-CD80-Alexa647, anti-CD3-Alexa488, anti-CD19-Alexa647 and anti-CD69-PerCP/Cy5.5.
ELISA
The tumour-bearing mice were intraperitoneally administered SZU-101 (3 mg/kg) or DOX (4 mg/kg) on day 7. Sera samples were collected at 2, 4 or 24 h post treatment on day 8 and stored at −20°C. An ELISA for multiple cytokines in peripheral blood was performed using a Ready-SET-Go!® ELISA kit (eBioscience) according to the manufacturer’s instructions.
Drug administration and treatment schedule
EL4 lymphoma cells were inoculated subcutaneously at two sites on the back of each mouse (approximately 20 g in weight) on the left and right side. The SZU-101 stock was prepared in DMSO at a concentration of 60 μg/μl, and DOX was prepared in PBS at a concentration of 2 μg/μl. The drugs for the mice in each group were prepared as described in Table
1.
Table 1
Dose of SZU-101 and DOX in drug treatments
Ctrl | - | - | -/50 μl | -/100 μl |
DOX | - | 1.6 μg/μl; 4 mg/kg | 50 μl/- | -/100 μl |
SZU-101 i.t. | 1.2 μg/μl; 3 mg/kg | - | 50 μl/- | -/100 μl |
SZU-101 i.p. | 0.6 μg/μl; 3 mg/kg | - | -/50 μl | 100 μl/- |
CoAd i.t. | 1.2 μg/μl; 3 mg/kg | 1.6 μg/μl; 4 mg/kg | 50 μl/- | -/100 μl |
CoAd i.p. | 0.6 μg/μl; 3 mg/kg | 1.6 μg/μl; 4 mg/kg | 50 μl/- | 100 μl/- |
DOX was applied on days 1, 4 and 7 of each course of treatment, and SZU-101 was applied every 24 h. A total of seven applications of SZU-101 and three applications of DOX were applied for one course. Tumour growth was measured daily, and long-term survival was evaluated.
Cytotoxicity assay
Tumour-bearing mice treated with different drugs or combinations were sacrificed, and lymphocytes from each mouse were collected. The effector cells were incubated with EL4 mouse T cell lymphoma cells at an effector:target cell ratio of 10:1. The cytotoxicity experiment was performed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit according to the manufacturer’s instructions.
Statistical analysis
Each group of assay included at least five samples for in vitro assays or ten mice for in vivo assays (except one mouse died in SZU-101 i.t. group). Statistical analysis was performed using SPSS 16 (IBM) and data was shown as mean ± standard error of the mean (SEM). Data was analysed by one-way ANOVA and a value of p < 0.05 was considered statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JZ, WJ, GJ and DZ designed the study. JZ, SH, JD, ZW and XC performed the experiments. WL and GJ provided the SZU-101 agonist and other drugs. JZ, SH, GJ and DZ analysed the data and wrote the manuscript. JZ and SH are considered as co-first authors. All authors read and approved the final manuscript.