Introduction
The antitumor effects of checkpoint inhibitors have revealed the importance of immune-mediated antitumor defenses [
1]. Macrophages are crucial contributors to innate immunity and also function as antigen-presenting cells (APCs). However, macrophages are heterogeneous and can exert opposing functions that promote tumor development and progression [
2]. In particular, tumor associated macrophages (TAMs) displaying the M2 phenotype promote tumor growth and metastasis. In contrast, the M1 counterpart possesses proinflammatory and tumor suppressive properties [
2,
3]. Macrophages display plasticity and change their functional profiles in response to environmental stimuli. Thus, the reprogramming of macrophages toward M1 phenotype is believed to be a key target of antitumor immunotherapy [
4].
Cryptotanshinone (CT) is a well characterized compound derived from traditional Chinese medical (TCM). CT, initially extracted from the root of
Salvia miltiorrhiza. Bunge, is one of several tanshinone derivatives, including tanshinone I, IIA, and IIB and dihydrotanshinone [
5]. More recently, CT has been purified, synthesized, and biochemically characterized. Many researchers are currently investigating CT and have reported that CT exhibits direct cytotoxic effects on multiple types of cancer cells [
6‐
11]. We have recently demonstrated that CT exhibits dual antiproliferative effects on mouse Lewis lung carcinoma (LLC) cells as well as a dendritic cell (DC)-maturing effect (see accompanying paper by Liu et al., Cancer Immunol Immunother 2019), [
https://doi.org/10.1007/s00262-019-02326-8]. CT inhibits LLC proliferation by activating p53, downregulating cyclin B1 and Cdc2, and consequently resulting in G2/M cell-cycle arrest. In addition, CT promoted DC maturation, as evidenced by upregulation of costimulatory and MHC molecules, and elevated production of proinflammatory cytokines (e.g., TNFα, IL-1β, and IL-12p70), using a signaling pathway that relies on the presence of MyD88.
Immunotherapy of cancers with checkpoint inhibitor blocking antibodies, such as anti-PD-L1 or anti-CTLA4, is only effective for about ¼ of patients with preexisting tumor-infiltrating effector T cells [
12,
13]. The unresponsive cancer patients may need a greater boost of their tumor-specific T cells to achieve more successful immunotherapy with checkpoint inhibitor-blocking antibodies. Based on its dual antiproliferative effect on LLC and DC-maturing effect, we hypothesized that CT could perhaps be a good candidate to induce antitumor immunity in LLC-bearing immunocompetent mice. Indeed, CT together with anti-PD-L1 cured LLC-bearing mice with the induction of subsequent LLC-specific immunity as described in the accompanying paper by Liu et al. [
https://doi.org/10.1007/s00262-019-02326-8]. However, it remains to be determined [
1] whether CT can inhibit the proliferation of other cancer cells such as hepatocellular carcinoma (HCC) cells; [
2] whether CT can activate APCs other than DCs, such as macrophages; [
3] whether CT can induce tumor-specific immunity in mouse models other than LLC; and [
4] to determine the receptor and pathway used by CT to induce adaptive immunity.
In the current study, we investigated the potential antiproliferative effect of CT on Hepa1-6 cells and found that CT inhibited the growth of Hepa1-6 cells by inducing apoptosis through blockade of the JAK2/STAT3 signaling pathway. We also discovered that CT activates macrophages in an M1 polarized direction using the TLR7/MyD88/NF-κB signaling pathway. Furthermore, when treated with a combination of CT and anti-PD-L1, mice with established Hepa1-6 tumors were completely cured, with the generation of Hepa1-6-specific immunity. Thus, CT possesses the dual capacities to inhibit the growth of multiple tumors and promote antitumor immune responses.
Materials and methods
Mice and cell lines
C57BL/6, TLR7−/−, MyD88−/−, and immunodeficient nude mice (8–12 weeks old, female) were kept under specific pathogen-free conditions with water and food given ad libitum.
Hepa1-6 hepatoma cell line (CRL-1830) and EG7 thymoma cell line (CRL-2113) used in the present study were maintained in DMEM (Meditech) supplemented with 10% FBS (Hyclone) and 2 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol at 37 °C in a humidified incubator with 5% CO2.
Cell proliferation assay
Hepa1-6 cells (5 × 103/well) were seeded in triplicate in round-bottomed 96-well plates in complete DMEM (0.2 ml/well) and incubated in the presence or absence of the indicated concentration of CT at 37 °C in a CO2 incubator for 48 h. Tritiated thymidine (3H-TdR, New England Nuclear, North Billerica, MA) was added at 0.5 μCi/well for the last 4 h of culture. The cultures were harvested on a membrane using a 96-well automatic harvester (INOTECHAG IH-280, Dottikon, Switzerland). The filter mat and scintillation fluid were placed into a bag, which was sealed and assessed for 3H-TdR incorporation (CPM) using an automatic MicroBeta counter (Wallac).
A CCK8 (Sigma-Aldrich, St. Louis, MO, USA) assay was performed to assess cell viability in Hepa1-6 cells treated with different concentrations of cucurbitacin I, according to the manufacturer’s instructions.
Cell cycle and apoptotic assays by flow cytometry (FACS)
The cycle distribution was analyzed by FACS analysis after staining with propidium iodide (PI) solution. Briefly, Hepa1-6 cells were treated with CT for 48 h and fixed with 75% ethanol. Next, the cells were incubated with 500 µL of a solution containing 50 µg/mL PI and 0.1% Triton X-100 in the dark and analyzed by FACS. To further analyze the apoptosis induction effects of CT, the cell apoptosis was detected as described previously [
8]. After treatment with CT for 48 h as described above, both attached cells and floating cells were harvested, stained with PI and Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s instructions, and analyzed by FACS.
Generation and treatment of bone marrow-derived macrophages (BMM)
Mouse BMM were generated as described previously [
14]. To measure surface marker of mouse BMM, adherent cells were grown to confluence in 24-well plates (at about 5 × 10
5/well) in a CO
2 incubator in the presence or absence of various reagents at concentrations specified for 48 h before immunostaining.
Immunostaining and FACS
Cultured BMMs were detached as described by Han et al. [
14]. The BMMs were resuspended at 1.0 × 10
6 cells/1 ml in PBS and then incubated with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Alexa Fluor
® 647-anti-mouse CD206 (clone MR5D3, BD Pharmingen). For immunoprofiling of tumor-bearing mice, single cell suspensions of Hepa1-6 tumors or the draining lymph nodes (dLN) at 1 × 10
6 cells/sample were immunostained with a combination of some of the following antibodies, such as FITC-anti-mouse CD4 (clone GK1.5, Tonbo), PE-anti-mouse CD11b (clone M1/70, BD), PerCP-Cy5-anti-mouse-B220 (clone RA3-6B2, Tonbo), APC-anti-mouse-CD11c (clone HL3, BD), eFluor450-anti-mouse CD45 (clone 30-F11, eBioscience), APC-Cy7-anti-mouse-CD8 (clone 53-6.7, Tonbo), eFluor450-anti-mouse CD44 (clone IM7, eBioscience), APC-anti-mouse CD62L (clone MEL-14, eBioscience), eFluor450-anti-mouse CD8 (clone 53-6.7, eBioscience), and eFluor660-anti-mouse CD107a (clone 1D4B, eBioscience). Data of the stained samples were acquired using an LSR II flow cytometer (BD) and analyzed using the software FlowJo (Tree Star Inc., Ashland, OR).
Total RNA isolation and cDNA synthesis
Total RNA from mouse BMM was isolated according to the protocol of RNeasy Micro Kit (Qiagen, Hilden, Germany, Cat: 74,004). Total RNA from Hepat1-6 tumors was extracted using TRIzol (Invitrogen, Cat: 1,559,026) and purified using the RNeasy Micro Kit RNA. The purity and concentration of isolated RNA samples were determined by measuring absorption at 260 nm wavelength using an NanoDrop ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE). cDNA was then synthesized from the RNA using the RT2 First Strand Kit (Qiagen, Cat: 330,401).
Quantitative real-time polymerase chain reaction (qPCR)
qPCR was performed using a LightCycler 480 II (Roche Life Sciences, Branford, CT, USA), the RT2 SYBR Green/ROX qPCR Master Mix (Qiagen, Cat: 330,523), and the specific primer pairs (sTable 1). The cycling conditions for the qPCR amplification were: hot start for 10 min at 95 °C; amplification for 40 cycles at 95 °C for 15 s, 55 °C for 35 s, and 72 °C for 30 s. The transcript levels were then normalized to that of a house-keeping gene (i.e., β actin or GAPDH) and then the data were analyzed using the ΔΔCT method through Qiagen’s GeneGlobe Data Analysis Center.
Western blot analysis
Western blotting analysis was performed as described previously [
15]. Briefly, cells treated with CT for 48 h were lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentrations were quantitated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Frederick, MD, USA). Total protein 30 µg/lane were loaded onto SDS–polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). The membranes were blocked and incubated with (1:1000) rabbit anti-p-STAT3 (Cell Signaling, Cat: 9145L, Tyr 705), anti-STAT3 (Cell Signaling, Cat: 4904S), anti-p-JAK2 (Cell Signaling, Cat: 8082), anti-JAK2 (Cell Signaling, Cat: 3230), anti-I-kBα (Cell Signaling, Cat: 9242), anti-GAPDH (Cell Signaling, Cat: 2118) overnight at 4 °C and (1:2000) horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Cat: 70,741) for 1 h at room temperature. The protein bands were visualized using the G-BOX Chemi system (Syngene, Frederick, MD).
Cytokine quantitation
TNFα and IL-12p70 in the culture supernatants were quantitated by human and mouse Customary Cytokine Arrays following the manufacturer’s protocol (MesoScale Diagonostics, Rockville, MD).
Mouse tumor model and treatment
Female mice (C57BL/6,
n = 5–10, 8–12-weeks old) were injected subcutaneously with 0.1 ml PBS containing Hepa1-6 (2 × 10
7/ml) or EG7 (2 × 10
5/ml) or into left or right flank regions as previously reported [
16,
17]. The appearance and size of tumors, as well as the mouse body weight, were monitored twice weekly. The length (
L) and width (
W) of tumors were measured with a caliper. Tumor size was calculated by the formula: (
L ×
W2)/2. Hepa1-6-bearing mice were treated with intratumoral (i.t.) injection of CT alone or in combination with anti-PD-L1 as indicated. In some experiments, tumor-bearing mice were simultaneously treated with intraperitoneal (i.p.) administration of 200 μg of either control rat IgG (clone 2A3, BioXcell, West Lebanon, NH), anti-mouse CD4 (clone GK1.5, BioXcell), anti-mouse CD8α (clone 53-6.72), or anti-mouse NK1.1 (clone PK136, BioXcell). For the analysis of leukocyte infiltration in Hepa1-6 tumor tissue, the tumors were removed and dissociated into single cell suspensions using an enzymatic cocktail consisting of collagenase I, II, and VI, deoxyribonuclease I, and elastase as previously reported [
18].
Statistical analysis
Student’s t tests were performed for parametric comparisons between two groups. A two-way analysis of variance (ANOVA) was used to analyze tumor volume difference between groups. Differences in survival curves were considered statistically significant by the log-rank survival analysis. All experiments were performed at least three times, and the results of one representative experiment or the mean of multiple experiments are shown. All statistical analyses were conducted using GraphPad Prism software (version 7, GraphPad Software, San Diego, CA).
Discussion
In this study, we developed a successful immunotherapeutic vaccination regimen of Hepa1-6 tumors based on cytotoxic effect and the immune-activating effect of CT with potentiation of the antitumor therapeutic effects by addition of the checkpoint inhibitor (anti-PD-L1).
In vitro and in vivo studies have demonstrated that CT inhibits cell proliferation in a variety of cancer cell lines [
6,
10,
25‐
29]. More recent reports have shown that CT inhibits the proliferation of lung cancer cells and leukemia cells by affecting insulin growth factor-1 receptor signaling and protein synthesis, respectively [
30,
31]. In our study, CT inhibited the growth of Hepa1-6 both in vitro and in vivo (Figs.
1,
4 and
5). It has been reported that CT induced a G
1/G
0 cell-cycle arrest in rhabdomyosarcoma (Rh30) and prostate cancer (DU145) cells by downregulating expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb) [
25], but that CT induced a G
2/M cell-cycle arrest in lung (A549) cells via upregulating expression of cyclin-dependent kinases (CDK) [
6]. The present study showed that CT did not affect the cell cycle of Hepa1-6 cells, suggesting that CT inhibited Hepa1-6 cells’ proliferation by another pathway.
CT has been reported to induce cell death in tumor cells [
6]. In the previous study we showed that CT was antiproliferative for mouse LLC cells by upregulating p53 and downregulating cyclin B1 and Cdc2 and consequently inducing G2/M cell-cycle arrest [
https://doi.org/10.1007/s00262-019-02326-8]. In the present study, CT inhibited proliferation by the induction of apoptosis in the Hepa1-6 cells (sFig. 2). Previous studies have shown that CT suppresses the proliferation of ovarian cancer cells and induced apoptosis of pancreatic as well as prostate cancer cells via the STAT3 signaling pathway [
8,
10,
32‐
34]. STAT3, regulated by Janus kinases (JAKs), is constitutively activated in most human malignant tumors, and is involved in the proliferation, angiogenesis, immune evasion and has anti-apoptotic effects in cancer cells [
35], including HCC [
36]. In our study, CT decreased STAT3 phosphorylation in a dose- and time-dependent manner. CT can affect the STAT3 signaling pathway either directly or by alterations of certain upstream regulators [
14,
22]. Our study demonstrated that CT also markedly inhibited the activities of JAK2 when the duration of CT exposure was increased to 2 h. Thus, the inhibition of JAK2/STAT3 signaling pathway may provide significant therapeutic benefits to HCC patients. On the other hand, the tumor was only minimally inhibited in nude mice, but more suppressed in immunocompetent mice by CT. This indicates that the antiproliferative effect is less contributory to inhibiting the tumor than the immune effects of CT. Nevertheless, these two effects may cooperate in suppressing tumors in immunocompetent mice.
TAMs are derived from circulating monocyte precursors [
37] and are important regulators of tumorigenesis [
38]. It has been well documented that TAMs promote the development of tumor, and their infiltration is highly correlated with poor prognosis [
39‐
42]. Furthermore, Fan QM et al. report that TAM represents a dominant myeloid population infiltrating Hepa1-6 tumors [
43]. However, macrophages with an M1 phenotype exhibit phagocytic and antigen-presenting activity, produce Th-1-activating cytokines, and mediate cytotoxic functions, including anticancer activity. Based on our observations of greater antitumor responses by immunocompetent than immunodeficient mice to CT in vivo (Fig.
4), we hypothesized that CT, in addition to activating DCs [
https://doi.org/10.1007/s00262-019-02326-8] might also influence macrophage polarization. Our data show that CT was capable of promoting BMM polarization toward M1 phenotype in vitro with upregulation of CD80 and CD86, and the production of TNFα and IL-12p40 proinflammatory cytokines (Fig.
2). In addition, Hepa1-6 tumors of mice treated with CT elevated the levels of expression of iNOS, IFNα, IFNβ, IL-12p40, and TNFα, but not IL-10 or TGFβ1 (Fig.
6e and sFig. 7), further indications that CT treatment activated macrophages toward M1 type in vivo. Given the APC function of macrophages and the contribution of M1 macrophages in potentiating antitumor immunity, it is presumed that activation of macrophages toward M1 type by CT plays a role in its immunotherapeutic effects on Hepa1-6 tumors.
To determine the signaling pathway utilized by CT, we have identified a receptor for which it acts as an agonist. Using TLR7
−/− and MyD88
−/− mouse BMM, we found that the absence of TLR7 and MyD88 adaptor molecule blocked induction of M1 polarization and the production of TNFα and IL-12 proinflammatory cytokines by CT (Figs.
3b–d). These findings indicate that signaling in response to CT is TLR7/MyD88-dependent. The activation of MyD88, a key adaptor protein that participates in propagation of TLR downstream signal transduction pathways in turn leads to subsequent activation of NF-κB. Our data show that CT treatment downregulated I-κBα both in mouse BMM and human HEK293 cell expressing TLR8 genes (data not shown), which enables p50/p65 complex to translocate from the cytosol to the nucleus, bind to promoters and activate NF-κB upregulation of the production of proinflammatory cytokines. TLR7
−/− and MyD88
−/− mouse BMM failed to show downregulation of I-κBα by CT (Fig.
3d). Thus, stimulation of mouse BMM by CT to produce TNF-α and IL-12p40 proinflammatory cytokines required TLR7/MyD88/NF-κB signaling pathway. However, our study does not rule out the possibility that CT may bind and form complexes with some available endogenous ligands for TLR7 and thus induce TLR7 activation.
Immunosuppression is a huge challenge for antitumor therapy. M2-polarized TAMs are well known to directly inhibit the immune response of CD8
+ T cells via the production of immunosuppressive factors such as IL-10 and TGF-β [
44]. Moreover, secreted factors from tumor cells also increase the expression of programmed cell death 1 ligand (PD-L1) in monocytes and macrophages and cytotoxic T lymphocyte antigen 4 (CTLA-4) ligands are constitutively expressed on regulatory T cells [
45]. Inhibitory signals from these immune checkpoints suppress the proliferation of CD8
+ T cells and weaken the tumoricidal activity of cytotoxic T cells. In our study, mice bearing established s.c. Hepa1-6 tumors were cured by a combination of CT and anti-PD-L1 and the resultant tumor-free mice exhibited Hepa1-6-specific antitumor immune responses and immunological memory (Fig.
5). Thus, a reasonable hypothesis would be that CT activates tumor-infiltrating APCs including macrophages and DCs that lead to the activation of T cells, whose adaptive antitumor activity could be further enhanced by M1 polarization of macrophages and reduction of immune suppression through treatment with anti-PD-L1. Indeed, CT treatment of Hepa1-6-bearing mice resulted in the selective upregulation of an array of genes indicative of DC maturation, activation of TAMs toward M1 type, Th1 polarization, and generation of antitumor immune defense, including CXCL9-11, IFNγ, perforin, granzyme B, iNOS, IFNα, IFNβ, IL-12p40, and TNFα in the tumor tissue (Fig.
6e and sFig. 7). Additionally, CT treatment promoted the generation of effector/memory T cells and Hepa1-6-specific functional CTLs in dLNs of Hepa1-6-bearing mice (Fig.
6c, d and sFigs. 5–6). The curative anti-Hepa1-6 therapeutic effect of treatment with CT plus anti-PD-L1 was dramatically reduced by depleting CD8 T or CD4 cells, further substantiating the notion that induction of adaptive antitumor T cell immunity is a major contributor of CT’s antitumor effect.
In conclusion, the present study suggests that CT is a potential therapeutic for the treatment of human hepatoma. However, because of its short half-life, CT has not been used yet in clinical trials as a cancer therapeutic. Despite short half-life, the in vivo administration of CT together with a checkpoint inhibitor successfully eradicated the tumor and induced a long-term immunity in mice. Further investigations on how to improve the stability and efficacy of CT are warranted.
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