IL-20 antagonist suppresses PD-L1 expression and prolongs survival in pancreatic cancer models

Lu, Shao-Wei; Pan, Hong-Chin; Hsu, Yu-Hsiang; Chang, Kung-Chao; Wu, Li-Wha; Chen, Wei-Yu; Chang, Ming-Shi

doi:10.1038/s41467-020-18244-8

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Published: 14 September 2020

IL-20 antagonist suppresses PD-L1 expression and prolongs survival in pancreatic cancer models

Nature Communications volume 11, Article number: 4611 (2020) Cite this article

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) and cancer-associated cachexia (CAC) are multifactorial and characterized by dysregulated inflammatory networks. Whether the proinflammatory cytokine IL-20 is involved in the complex networks of PDAC and CAC remains unclear. Here, we report that elevated IL-20 levels in tumor tissue correlate with poor overall survival in 72 patients with PDAC. In vivo, we establish a transgenic mouse model (KPC) and an orthotopic PDAC model and examine the therapeutic efficacy of an anti-IL-20 monoclonal antibody (7E). Targeting IL-20 not only prolongs survival and attenuates PD-L1 expression in both murine models but also inhibits tumor growth and mitigates M2-like polarization in the orthotopic PDAC model. Combination treatment with 7E and an anti-PD-1 antibody shows better efficacy in inhibiting tumor growth than either treatment alone in the orthotopic PDAC model. Finally, 7E mitigates cachexic symptoms in CAC models. Together, we conclude IL-20 is a critical mediator in PDAC progression.

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Introduction

Pancreatic ductal adenocarcinoma (PDAC) often has a poor prognosis, even if it is diagnosed early. Oncogenic Kras mutation in mouse PDAC is the signature event and critical for tumor initiation¹. Mutations in the proto-oncogene KRAS and tumor-suppressor gene TP53 indicate a poor prognosis in PDAC¹. The LSL-Kras^G12D; Trp53^flox/flox; Pdx-1-Cre (KPC) mouse model has been established as a clinically relevant PDAC model that develops many key features of human PDAC and a robust inflammatory response². In the PDAC microenvironment, proinflammatory cytokines secreted by tumor cells and infiltrating immune cells, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, have been shown to modulate PDAC progression and immune evasion³.

Tumor-associated macrophages (TAMs) are mainly differentiated from monocytes and recruited into tumors by cytokines⁴. In general, macrophages can be differentially polarized into a classical M1-like phenotype for inhibiting tumors or an alternative M2-like phenotype for promoting tumorigenesis and immunosuppression. Distinct from classical macrophages, TAMs are often functionally transformed by the tumor microenvironment and have an M2-like phenotype that promotes PDAC progression⁵. In addition, M2-like TAMs have been shown to contribute to immunosuppressive microenvironments in PDAC⁶. Inhibitors of immune checkpoint pathways have been linked to the promotion of tumor immune surveillance⁷. Programmed death-ligand 1 (PD-L1) is one of the ligands that binds to programmed death-1 (PD-1) on T cells and attenuates the immune response by downregulating the activity of antitumor T cells⁸. In the PDAC microenvironment, PD-L1 is highly expressed in cancer cells, facilitating immune escape and cancer progression⁷. Blockade of PD-L1 has been shown to inhibit tumor growth in a mouse model⁹. However, a previous study indicated that targeting PD-L1 for PDAC therapy was unsuccessful, as the response rate was <3.1% and there was no response to monotherapy¹⁰. In addition to its immunosuppressive microenvironment, PDAC is accompanied by tissue fibrosis, which facilitates cancer progression¹¹. It has been shown that pancreatic tumors activate fibroblasts, leading to enhanced pancreatic tissue fibrosis¹². In addition, the chronic activation of cancer-associated fibroblasts driven by the inflamed tissue microenvironment promotes pancreatic stromal remodeling that boosts tumor growth and progression¹³. Therefore, combined therapies that target tissue fibrosis pathways and enhance tumor immune surveillance may be important for treating PDAC.

During the progression of pancreatic cancer, cancer-associated cachexia (CAC), which involves progressive loss of body weight, wasting of the skeletal muscle, and atrophy of adipose tissues, often occurs in patients^14,15. PDAC is often accompanied by CAC in later stages, which is the major cause of poor survival in these patients, and the 5-year survival rate is <5%^16,17. CAC-induced fat loss is primarily due to increased lipolysis and the browning of white adipose tissue (WAT)¹⁸. The increased lipolysis is associated with the activation of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) in adipose tissue¹⁹. In patients, the anti-lipolytic effect of insulin on adipocytes is decreased, which stimulates lipolysis in CAC²⁰. The browning of WAT is associated with increased expression of uncoupling protein 1 and increased lipid mobilization and energy expenditure during cachexia²¹. Inhibition of lipolysis and WAT browning has been shown to protect against CAC²¹. Proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, and interferon (IFN)-γ, are crucial mediators of CAC, and targeting these cytokines has been suggested as a potential intervention for cachexia and prevention of metabolic imbalance²². However, the mechanisms underlying the crosstalk between cancer cells and tumor-associated immune cells during PDAC progression and CAC development remain elusive.

The proinflammatory cytokine IL-20 is a member of the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26. IL-20 is expressed predominantly by monocytes, dendritic cells, epithelial cells, and endothelial cells²³. In addition, IL-20 receptor is constitutively expressed in epithelial cells and endothelial cells²⁴. IL-20 has been shown to affect multiple cell types by activating a heterodimeric receptor complex of either IL-20R1/IL-20R2 or IL-22R1/IL-20R2 (ref. ²⁵). Under physiological conditions, IL-20 is primarily involved in epidermal cell, keratinocyte, and monocyte differentiation. IL-20 induces signal transduction through the signal transducer and activator of transcription factor-3 pathway during keratinocyte proliferation and normal skin differentiation²⁴. In immune regulation, IL-20 induces the production of chemokine ligand 2 by monocytes and regulates immune defense mechanisms^24,26. Under pathological conditions, IL-20 expression is significantly upregulated in several inflammatory diseases, such as psoriasis, rheumatoid arthritis, atherosclerosis, renal failure, stroke, osteoporosis, and cancer^{27,28,29,30,31,32,33,34,35}.

Our previous studies have demonstrated that IL-20 promotes tumor growth and increases metastasis in hepatocellular carcinoma, breast cancer, prostate cancer, and oral cancer^27,28,29,30. We developed the anti-IL-20 monoclonal antibody (mAb) 7E, which was shown to neutralize the in vitro and in vivo activity of human and mouse IL-20, and showed that 7E treatment effectively alleviated inflammation and suppressed tumor growth in breast cancer and prostate cancer mouse models^28,29. However, it is not clear whether IL-20 correlates with the progression of PDAC or whether targeting IL-20 could be an effective treatment for PDAC.

In this study, we investigate the role of IL-20 in PDAC and evaluate the efficacy of IL-20 blockade by 7E in an orthotopic tumor model and a KPC mouse model. We find that IL-20 blockade by 7E prolongs survival and alleviates pancreatic fibrosis in both mouse models, reduces the expression of the immunosuppressive molecule PD-L1 on tumor cells, inhibits tumor growth, and mitigates TAM infiltration. In addition, 7E treatment mitigates CAC manifestations, including reducing tumor-associated weight loss, maintaining the food intake, and reducing lipolysis in adipose tissue, in CAC models. Together, our results indicate that targeting IL-20 with 7E represents a therapeutic strategy for treating PDAC and CAC.

Results

IL-20 was correlated with PD-L1 level and survival in patients

We performed immunohistochemical (IHC) staining to analyze the expression of IL-20 in tumor tissue samples from 72 patients with PDAC. IL-20 was strongly expressed in tumor cells and neoplastic ductal epithelial cells (Fig. 1a; left panel) but only lowly expressed by ductal epithelial cells in nontumorous pancreatic tissue (Fig. 1a; right panel). IL-20 expression was remarkably correlated with overall survival, and relatively high IL-20 expression predicted poor survival (Fig. 1b; left panel). There was a significantly negative correlation between high expression of IL-20 and survival in patients with advanced-stage PDAC (Fig. 1b; right panel). PD-L1 is highly expressed in PDAC and plays an important role in tumor progression. Thus we stained the 72 tumor tissue samples with an anti-PD-L1 antibody and compared the expression of IL-20 and PD-L1 in these patients. PD-L1 was expressed in the patients with PDAC (Fig. 1c), and IL-20 was significantly correlated with PD-L1 expression (r = 0.6203, p < 0.0001; Fig. 1d).

**Fig. 1: Poor clinical outcomes in pancreatic cancer were associated with high IL-20 expression in patients and targeting IL-20 prolonged survival and inhibited PD-L1 and αSMA expression in KPC mice.**

We determined the pathogenic role of IL-20 in vivo by using Pdx-1-Cre (KPC) mice, which spontaneously develop pancreatic tumors that mimic the progression of PDAC in humans^2,36. IHC staining confirmed the upregulation of IL-20 expression in pancreatic tumor tissue samples from KPC mice compared with pancreatic tissue samples from wild-type (WT) mice (Fig. 1e). Neutralization of IL-20 with 7E (6 mg/kg, twice a week; treatment begun at the age of 25 days and continued until death) significantly prolonged the survival of KPC mice compared with control phosphate-buffered saline (PBS) or mouse immunoglobulin G (mIgG) treatment (Fig. 1f). In addition, 7E treatment effectively reduced the number of Ki-67⁺ proliferating cells in comparison to PBS and mIgG treatment (Fig. 1g).

PD-L1 is a ligand of PD-1 and expressed on tumor cells. Previous studies have shown that PD-L1 plays a role in regulating tumor growth, and this molecule is a new therapeutic target^37,38. Pancreases from KPC mice treated with PBS or mIgG displayed prominent expression of PD-L1, and significant inhibition of PD-L1 expression was observed in 7E-treated mice, which had a longer life span than the mice in the two control groups (Fig. 1h).

Pancreatic fibrosis plays an important role in the pathogenesis of PDAC. We previously reported that IL-20 acts as a fibrogenic factor in liver fibrosis³⁹. To investigate the effect of IL-20 on the pancreatic fibrosis occurring during PDAC, we evaluated the distribution of alpha-smooth muscle actin (αSMA), a hallmark of fibrosis. IHC analysis showed that αSMA was overexpressed in PBS- and mIgG-treated KPC mice, but αSMA expression was substantially reduced in 7E-treated KPC mice (Fig. 1i).

IL-20 blockade inhibited orthotopic pancreatic tumor growth

We next investigated whether IL-20 blockade by 7E inhibits orthotopic pancreatic tumor growth. To test this, pancreatic tumor cells isolated from KPC mice were transfected with a luciferase reporter gene (KPC/Luc cells). 7E treatment significantly inhibited tumor growth in vivo, as measured on day 28 after inoculation of KPC/Luc cells (Fig. 2a). The tumor sizes and weights in the 7E-treated group were significantly smaller than those in the mIgG- and PBS-treated groups (Fig. 2a, b). In line with the findings in transgenic KPC mice, 7E treatment prolonged survival and decreased αSMA expression compared with PBS or mIgG treatment in the orthotopic model (Fig. 2c, d). Furthermore, 7E treatment inhibited the expression of fibrogenesis-related markers, including αSMA, Collagen, Fibronectin, Vimentin, and transforming growth factor (TGF)-β (Fig. 2e).

**Fig. 2: Tumor growth was inhibited in 7E-treated and IL-20R1-knockdown KPC cell-injected orthotopic models of PDAC.**

For both KPC mice and orthotopic PDAC model mice, we performed enzyme-linked immunosorbent assay (ELISA) analyses to measure the circulating levels of IL-20 and found that the serum levels of IL-20 were significantly higher in the KPC mice and orthotopic PDAC model mice than the corresponding healthy control groups. The serum levels of IL-20 were reduced in 7E-treated mice for both models. These data indicated that IL-20 was not only highly expressed in tumor tissues but also expressed systemically (Supplementary Fig. 1a, b).

Inhibiting IL-20 attenuated cancer cell proliferation

To clarify the cellular sources of IL-20 receptors in the pancreatic tumor microenvironment, we performed IHC staining to analyze the expression of IL-20 receptors (IL-20R1, IL-20R2, and IL-22R1) in tumor and nontumor stromal tissue samples from patients and mice with pancreatic cancer. Positive staining for IL-20 receptors was observed on tumor cells but not on the stromal cells in nontumor pancreatic tissue (Supplementary Fig. 2a). Immunostaining showed that both human and murine pancreatic cancer cell lines (PANC-1, BxPC-3, and PANC-02) expressed IL-20 and its receptors (Supplementary Fig. 2b). A methyl thiazol tetrazolium (MTT) assay showed that an anti-IL-20 mAb (7E) suppressed IL-20-mediated cell proliferation in these three pancreatic cancer cell lines (Supplementary Fig. 2c–e), suggesting that IL-20 is pivotal in pancreatic cancer cell proliferation.

IL-20R1 knockdown in KPC cells inhibited tumor growth

Inhibition of IL-20 by 7E reduced tumor growth in vivo (Figs. 1, 2), which may be attributed to reductions in the autocrine effects of IL-20 within the tumor microenvironment. To further confirm the critical role of IL-20-mediated signaling (IL-20R1) in tumor growth, we generated IL-20R1-knockdown KPC cells (Supplementary Fig. 3) and orthotopically injected these cells into the pancreas of C57BL/6 mice. Tumor volume and weight were significantly decreased in the IL-20R1-knockdown KPC tumor group compared with the parental KPC tumor group (Fig. 2f, g), suggesting that IL-20R1 directly contributes to the tumorigenic activity of KPC cells.

IL-20 blockade inhibited M2 macrophage in vivo and in vitro

TAMs play a major role in tumor progression. To investigate whether blockade of IL-20 affects macrophages in pancreatic tumors, we stained tumor tissue samples for a macrophage marker (F4/80) and an M2-type macrophage marker (CD206). The frequencies of F4/80⁺ macrophages and CD206⁺ macrophages were decreased in 7E-treated mice (Fig. 3a). These results suggested that inhibiting IL-20 might reduce the numbers of M2-type macrophages in pancreatic tumors in the orthotopic model. To evaluate whether IL-20 induces M2 macrophage differentiation in vitro, we incubated bone marrow-derived macrophages (BMDMs) with mIL-20 for 48 h and analyzed macrophage polarization (Fig. 3b). Fluorescence-activated cell sorting analysis showed that IL-20 dose-dependently upregulated the CD206⁺ macrophage frequency and that 7E inhibited IL-20-induced CD206⁺ macrophage polarization (Fig. 3c and Supplementary Fig. 4). Conversely, blockade of IL-20 increased CD86⁺ (M1-type macrophage marker) macrophage differentiation (Fig. 3c).

**Fig. 3: 7E decreased macrophage infiltration in an orthotopic model of pancreatic cancer.**

IL-20 upregulated PD-L1 in tumors of the orthotopic model

To examine the relationship between IL-20 and PD-L1, we used co-immunofluorescence staining to analyze the expression of IL-20 and PD-L1. Pancreatic tumors from the orthotopic model showed colocalization of IL-20 and PD-L1 in vivo, which indicated that IL-20 might be involved in PD-L1 regulation (Fig. 4a). We further investigated whether PD-L1 is regulated by IL-20 in vitro. KPC cells were treated with IL-20, and reverse transcription polymerase chain reaction (RT-PCR) showed that PD-L1 expression was upregulated in the IL-20-treated KPC cells and that 7E blocked the activity of IL-20 (Fig. 4b). A recent publication reported that IFN-α is correlated with PD-L1 induction⁴⁰. To elucidate whether IL-20 upregulates PD-L1 expression through activation of IFN-α, RT-qPCR was performed and revealed that IL-20 upregulated the expression of IFN-α and that 7E blocked IL-20-induced IFN-α expression (Fig. 4c, d). Inhibiting the activity of IFN-α with an anti-IFN-α antibody completely inhibited PD-L1 expression in IL-20-treated KPC cells, indicating that IL-20 regulates PD-L1 expression via IFN-α activation (Fig. 4e). Based on our in vitro data, we speculated that blockade of IL-20 would suppress PD-L1 expression in vivo. PD-L1 expression was weaker in 7E-treated mice than in PBS- and mIgG-treated mice (Fig. 4f). Overall, these results are consistent with the in vitro data and confirmed that 7E reduces PD-L1 expression in vivo and in vitro.

**Fig. 4: IL-20 induced PD-L1 expression, and 7E neutralized IL-20-induced PD-L1 expression.**

IL-20 blockade promoted CD8⁺ T cell infiltration

Previous studies demonstrate elevated expression of the checkpoint receptor PD-1 in activated T cells in PDAC^41,42. To address whether the reduction in tumor growth observed in 7E-treated mice is also mediated by T cells, we performed IHC staining with an anti-CD8 antibody, the cytotoxic T cell marker, on tumor tissue samples from orthotopic PDAC model and KPC mice. In these two models, increased CD8 and PD-1 expression was observed in 7E-treated mice but not in PBS- or mIgG-treated mice, indicating that 7E treatment promoted CD8⁺ T cell infiltration into tumor tissues (Fig. 4g, h). To further clarify whether 7E can restrain tumor growth in T cell-deficient mice, we generated an orthotopic model of PDAC in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/YckNarl mice (advanced severe immunodeficiency (ASID)) in which T cells, B cells, and natural killer (NK) cells are absent. We found that the tumor size and tumor weight of 7E-treated mice were still smaller than those of PBS- and mIgG-treated mice (Fig. 4i–k), indicating that IL-20 blockade can directly inhibit tumor growth in a T cell-, B cell-, and NK cell-independent manner.

7E combined with anti-PD-1 antibody showed better efficacy

Based on the findings that inhibiting IL-20 enhanced the infiltration of CD8⁺ and PD-1⁺ cells in orthotopic PDAC model and KPC mice, we postulated that combined treatment with IL-20 blockade by 7E and immune checkpoint inhibition with an anti-PD-1 antibody, which enhances CD8⁺ T cell activity, could produce synergistic efficacy for pancreatic cancer treatment. In the orthotopic PDAC model, we indeed observed that tumor weight was decreased more in mice treated with the combination of 7E plus the anti-PD-1 antibody than in mice treated with either 7E or the anti-PD-1 antibody alone (Fig. 4l–n).

IL-20 blockade alleviated cachexia in the KPC tumor model

7E treatment or knockdown of IL-20R1 signaling profoundly inhibited orthotopic KPC tumor growth (Fig. 2). We therefore hypothesized that blocking IL-20 ameliorates CAC. Tumor-bearing mice reduced their food intake and lost body weight compared with tumor-free healthy mice (Fig. 5a, b). Interestingly, the body weight and food intake of tumor-bearing mice treated with 7E continued to increase over time, as seen in the healthy mice (Fig. 5a, b). In addition, 7E treatment prevented the cancer-induced loss of epididymal fat mass (Fig. 5c), reduced infiltration of macrophages into adipose tissue (Fig. 5d), and preserved adipose tissue size (Fig. 5e, f).

**Fig. 5: 7E reversed fat mass loss in an orthotopic model of pancreatic cancer.**

Most cancer cachexia is accompanied by muscle wasting, in which the expression of the muscle wasting marker β-dystroglycan (DAG-1) is enhanced in muscle tissues⁴³. DAG-1 was prominently expressed in the soleus muscle of mice in the PBS- and mIgG-treated groups in the orthotopic model (Fig. 5g). However, 7E treatment did not decrease DAG-1 expression, and there was no significant difference among these three groups. These data suggest that IL-20 may not be directly involved in cancer-induced muscle wasting.

Anticachexia effect of 7E in Lewis lung carcinoma (LLC) model

We next explored whether IL-20 is a critical and common mediator in another mouse model of CAC, which is induced by inoculation of LLC cells¹⁹. Mice inoculated with LLC tumor cells lost significantly more body weight than sham control mice, while 7E treatment prevented cancer-induced body weight loss (Fig. 6a) and preserved the average food intake (Fig. 6b) compared with PBS or mIgG treatment. Intriguingly, 7E treatment did not reduce the LLC tumor mass (Fig. 6c) or cancer-induced muscle wasting in this model (Fig. 6d, e). Consistent with the findings in the orthotopic KPC tumor model, the epididymal fat mass was significantly preserved in the 7E-treated mice compared with the mIgG-treated mice (Fig. 6f). For LLC tumor-bearing mice, the size of adipocytes in epididymal fat was maintained in the 7E-treated group but not in the PBS- or mIgG-treated groups (Fig. 6g, h).

**Fig. 6: 7E treatment alleviated LLC tumor-induced cachexia symptoms in mice.**

7E prevented fat loss and reduced serum triglyceride (TAG) levels

Browning of WAT, in which brown adipocytes are induced within WAT depots, and increased lipolysis are two major causes of CAC-induced fat loss^21,44. The expression of the adipose browning-related genes Ucp1, Pgc1a, Dio2, and Prdm16 was upregulated in the epididymal fat of LLC tumor-bearing mice⁴⁵, whereas no difference in the expression of these genes was detected between the 7E- and mIgG-treated groups (Supplementary Fig. 5a–d). Thus we next analyzed whether 7E alleviates fat loss in the LLC-induced CAC model via inhibition of lipolysis. Increased lipolysis is associated with activation of the lipolytic enzymes HSL and ATGL in adipose tissue^46,47. 7E treatment significantly reduced CAC-induced HSL and ATGL gene expression in epididymal fat following LLC cell inoculation (Fig. 7a, b). IHC staining confirmed that the increased number of HSL-positive cells in the epididymal fat of tumor-bearing mice was reduced by 7E treatment compared with PBS or mIgG treatment (Fig. 7c). Elevated levels of TAG and cholesterol are often detected in patients suffering from CAC⁴⁸. Murine models of CAC also show increased serum levels of fatty acids and glycerol, suggesting increased TAG catabolism and delipidation of fat reserves¹⁹. In our LLC tumor-bearing mice, the level of TAG was significantly higher in the PBS- and mIgG-treated groups than in the sham control group, but the level of TAG was reduced in the 7E-treated group (Fig. 7d). The results suggest that IL-20 is involved in abnormal lipid metabolism during CAC but this activity can be neutralized with the anti-IL-20 antibody.

**Fig. 7: 7E alleviated LLC tumor-induced cachectic lipolysis in mice, and IL-20R1 deficiency mitigated LLC tumor-induced cachexia symptoms.**

7E reduced F4/80⁺ macrophage infiltration to adipose tissue

Macrophages play an important role in the pathogenesis of CAC. Therefore, we explored whether IL-20 is involved in the infiltration of macrophages into adipose tissue. Immunofluorescence staining revealed that IL-20 colocalized with F4/80⁺ macrophages in adipose tissue following tumor inoculation (Fig. 7e). 7E treatment significantly reduced the F4/80⁺IL-20⁺ macrophage frequency in the epididymal fat tissue (Fig. 7e). We examined whether IL-20 directly induces lipolysis in adipocytes in vitro. Epididymal fat adipocytes from C57BL/6 mice were isolated and cultured in the presence of IL-20 for 1 week. The expression of the lipolytic genes ATGL and HSL, however, was not affected by IL-20 in the adipocyte cultures (Fig. 7f, g), suggesting that IL-20 may indirectly promote lipolysis via the induction of tumor-derived lipolytic factors, such as TNF-α⁴⁷. To test this hypothesis, we treated LLC cells with mIL-20 for 24 h and then cultured the cells in fresh medium without IL-20 for another 24 h. The primary adipocytes were then incubated in conditioned medium in the presence of 7E or an anti-TNF-α antibody for 24 h. The conditioned medium from IL-20-treated LLCs increased the expression of ATGL and HSL in the adipocytes (Fig. 7h, i). Notably, the anti-IL-20 antibody was more effective than the anti-TNF-α antibody in inhibiting the paracrine effect of IL-20 driving lipolysis (Fig. 7h, i). The results suggest that IL-20 may be involved in lipolysis through the induction of TNF-α.

IL-20R1 deficiency protected against LLC-induced CAC

We next investigated whether IL-20R1 deficiency also protects against CAC symptoms in an LLC model. LLC cells were injected subcutaneously (s.c.) into the right flank of IL-20R1^−/− and WT mice. Twenty-six days later, the IL-20R1^−/− mice showed less body weight loss than the WT mice (Fig. 7j). There was no significant difference in tumor weight between the groups (Fig. 7k). However, the IL-20R1^−/− mice had a greater epididymal fat mass and lower triglyceride levels than the WT mice (Fig. 7l, m). In addition, the IL-20R1^−/− mice were protected against adipose atrophy and immune cell infiltration into the epididymal fat pad in the LLC-induced CAC model (Fig. 7n). These results further confirm the potential therapeutic effect of IL-20 signaling blockade on CAC.

Discussion

In this study, we demonstrated that relatively high IL-20 expression was correlated with poor survival in patients with PDAC, especially those with late-stage PDAC. IL-20 expression was also significantly correlated with PD-L1 expression in patients with PDAC. Thus IL-20 could be used as a biomarker in the clinic.

To investigate whether IL-20 directly targets tumor cells, IHC staining was performed and confirmed that IL-20 and its receptors were expressed in the tumor glands of both human and mouse PDAC specimens. In vivo, IL-20 expression was correlated with increased tumor cell proliferation and tissue fibrosis. In addition, relatively high IL-20 levels were found in the serum of both KPC mice and orthotopic model mice, indicating that IL-20 is not only involved locally in neoplastic tissue but also elevated systemically in the circulation, which may become involved in CAC.

The anti-IL-20 antibody significantly prolonged the survival of tumor-bearing mice. Human and mouse pancreatic cancer cell lines (PANC-1, BxPC-3, and PANC-02) expressed endogenous IL-20 and its receptors, and 7E inhibited the proliferation of these cell lines in vitro. In addition, IL-20R1 knockdown in KPC cells reduced tumor growth in the orthotopic model. These data indicated that IL-20 secreted by pancreatic cancer cells could shape the tumor microenvironment to favor tumor progression in an autocrine manner.

In addition to targeting tumor cell growth, M2-like macrophages were recently suggested to comprise approximately 85% of TAMs in PDAC and promote tumor immune escape by enhancing tumor fibrosis and excluding cytotoxic T cells^49,50. In vitro, IL-20 promoted M2 polarization, which was attenuated by 7E. Blocking IL-20 with 7E also decreased the number of CD206⁺ TAMs in orthotopic tumors compared with control PBS or mIgG treatment. Furthermore, 7E could reduce fibrosis and inhibit fibrogenesis-related gene expression (αSMA, Collagen 1, Fibronectin, Vimentin, and TGF-β)^51,52,53,54 in the orthotopic PDAC model, which implies that the paracrine function of IL-20 allows this molecule to contribute to PDAC progression as a profibrotic factor. According to our previous study, IL-20 effectively activates stellate cells, which promote tissue remodeling in liver fibrosis³⁹. This finding suggests that IL-20 is a crucial profibrogenic factor and promotes the M2 polarization of TAMs to shape the tumor microenvironment toward conditions favorable to the tumor. IL-20 is also a potent angiogenic factor and promotes tumor metastasis^51,52,53,54. Targeting IL-20 could effectively inhibit tumor growth, reduce the M2 polarization of TAMs, and alleviate fibrosis, which are all considered to contribute to unfavorable conditions for PDAC progression.

PD-L1 expression in PDAC is associated with tumor progression since PD-L1 promotes immune suppression. In PDAC tumors from patients and KPC mice, we observed that IL-20 expression significantly correlated with PD-L1 expression. In addition, IL-20 upregulated PD-L1 expression via IFN-α, which is an upstream regulator of PD-L1 produced by some types of cancer cells^40,55. Blocking the activity of IFN-α with an antibody attenuated IL-20-induced PD-L1 expression in KPC tumor cells in vitro, indicating that IL-20 mediated PD-L1 upregulation via IFN-α. In our animal models, neutralizing IL-20 activity also decreased the expression of PD-L1 in tumors from KPC mice and orthotopic PDAC model mice.

Of note, 7E treatment also increased the numbers of CD8⁺ and PD-1⁺ T cells in tumor tissue samples from both animal models, which likely contributed to the antitumor function of 7E in restraining tumor masses. Although the results from the orthotopic PDAC model established with ASID mice revealed that 7E could inhibit tumor growth independently from T cells, we did not exclude the possibility that IL-20 blockade disrupts the immunosuppressive microenvironment of PDAC tumors by increasing cytotoxic CD8⁺ T cell accumulation. Indeed, the combination of IL-20 blockade with anti-PD-1 therapy showed remarkable antitumor efficacy, as significantly reduced tumor weights were observed for mice treated with the combination of 7E and the anti-PD-1 antibody compared to mice treated with either 7E or the anti-PD-1 antibody alone, demonstrating synergistic efficacy in PDAC. Immune checkpoint therapy targeting PD-L1 has been shown to be ineffective, as the response rate was <3.1% and there was no response to monotherapy for PDAC¹⁰. Therefore, combined therapies with an anti-PD-1 antibody and other antibodies or chemotherapeutic agents have been investigated and shown to be more effective than monotherapies^56,57,58. Our study provided evidence that our anti-IL-20 antibody could be a potential candidate for combination therapy with an anti-PD-1 antibody in PDAC.

A recent study demonstrated that the antibody-mediated neutralization of IL-1β significantly enhanced the antitumor activity of an anti-PD-1 antibody, which was accompanied by increased tumor infiltration by CD8⁺ T cells, indicating that targeting IL-1β has a beneficial effect on PDAC therapy outcomes⁵⁹. We previously reported that IL-1β expression was upregulated by IL-20 in glioblastoma cells and oral cancer cells^30,35. Whether targeting IL-20 also inhibits IL-1β-mediated pancreatic oncogenesis awaits further investigation.

In the orthotopic KPC tumor model, 7E treatment or IL-20R1 knockdown not only reduced tumor growth but also alleviated cachexic symptoms, including producing reductions in the loss of body weight and fat mass. The anticachexia effect of 7E was further confirmed in an LLC-induced cachexia model, implying that 7E has therapeutic potential for treating PDAC and alleviating cachexic symptoms.

CAC reflects the activation of the host metabolism with loss of muscle mass and adipose tissue. Only a few murine tumor models have been reported to induce a cachexia-like syndrome, such as PDAC, LLC, melanoma, and prostate tumor models^60,61,62. In a prostate cancer-induced cachexia study, an IL-6 antagonist alleviated weight loss but did not inhibit tumor growth⁶¹. In addition, in a melanoma model, a significant reduction in body weight loss was observed with IL-6 antagonist treatment⁶¹. By contrast, 7E not only mitigated weight loss but also attenuated tumor growth in the orthotopic PDAC model. Although IL-20 blockade did not significantly alleviate muscle wasting, it preserved adipose tissue in the animal models of CAC studied. Therefore, an IL-20 antagonist may be a relatively effective drug for treating PDAC and CAC.

In patients with CAC, the increased catabolism of lipid stores causes exhaustive loss of WAT. Inhibition of lipolysis has been shown to prevent CAC in a mouse model¹⁹. In the current study, mice inoculated with KPC or LLC tumor cells exhibited symptoms of cachexia, including muscle wasting and excessive loss of body weight and epididymal fat. By contrast, treatment of KPC or LLC tumor-bearing mice with IL-20 blockade prevented CAC-associated WAT lipolysis and maintained the adipose fat mass and body weight. Intriguingly, cancer-induced muscle wasting was not prevented by 7E treatment in either model, suggesting that IL-20 contributed to CAC mainly by promoting lipolysis in adipose tissues.

Our results demonstrate that IL-20 blockade by 7E is a promising treatment for PDAC with several advantages: (1) IL-20 is a prosurvival factor, and targeting IL-20 with 7E alone attenuated pancreatic cancer cell growth. (2) IL-20 blockade also shaped the microenvironment toward conditions unfavorable for tumors in multiple aspects, including the inhibition of protumorigenic M2 macrophage infiltration and alleviation of fibrosis. Aggressive fibrosis causes a hardened pancreas inaccessible to drugs. Thus the alleviation of fibrosis by IL-20 blockade might overcome this issue to improve the penetration of cytotoxic T cells or other chemotherapeutic drugs to allow killing of tumor cells. (3) IL-20 induced PD-L1 expression on PDAC tumor cells, and the combination of IL-20 blockade with anti-PD-1 therapy exhibited synergistic tumor-suppressive efficacy. (4) Targeting IL-20 not only limited tumor growth but also alleviated CAC symptoms, which profoundly improved the overall survival of mice with orthotopic PDAC tumors. Collectively, our results showed that IL-20 blockade is a potential treatment for PDAC according to the multiple protumorigenic roles of IL-20, including tumor cell proliferation, fibrosis, metastasis²⁸, angiogenesis⁶³, and M2 macrophage polarization. Blockade of IL-20 could lead to potentially more efficacious outcomes than targeting a single immune checkpoint (PD-L1) and overcome the limitations of currently available immunotherapies for the treatment of PDAC.

In conclusion, we demonstrated that IL-20 played pivotal roles in PDAC and that the anti-IL-20 antibody 7E retarded tumor progression, inhibited fibrosis in the pancreas, downregulated PD-L1 expression, mitigated CAC, and synergized with anti-PD-1 therapy to inhibit tumor growth. Therefore, the anti-IL-20 antibody is a potential therapeutic for PDAC.

Methods

Human pancreatic tissue samples

Tumor samples were taken from 72 patients with primary PDAC between January 1991 and January 2017 (stage I, n = 3; stage II, n = 38; stage III, n = 16; stage IV, n = 15). The clinicopathological variables evaluated are listed in Supplementary Table 1. Biopsies were taken after obtaining informed consent from the participants. This retrospective study was approved by the National Cheng Kung University Hospital Institutional Review Board (IRB No: A-ER-106-027). The following clinical practice guidelines for pancreatic cancer were applied: In principle, resectable diseases are treated by surgery (Whipple operation or pylorus preserving pancreatoduodectomy) with or without adjuvant chemotherapy and radiotherapy. Unresectable tumors (locally advanced or metastatic disease) are treated with chemotherapy or supportive care.

Histological analysis and IHC staining

Hematoxylin and eosin (H&E) staining and IHC staining were performed following the standard protocols. The primary antibodies used were anti-IL-20 (7E; diluted to 5 µg/ml), anti-IL-20R1 (Catalog number: ab203196; Abcam; 1:200 dilution), anti-IL-20R2 (Catalog: 14-1206, clone: 20RNTC; eBioscience™, Thermo Fisher Scientific; diluted to 5 µg/ml), anti-IL-22R1 (Catalog: MAB42941; Clone: # 496514; R&D Systems, Minneapolis, MN; diluted to 5 µg/ml), anti-Ki-67 (Catalog: ab16667; Cone: SP6; Abcam; 1:200 dilution), anti-F4/80 (Catalog: ab6640; Abcam; 1:200 dilution), anti-CD-206 (Catalog: ab64693; Abcam; diluted to 1 µg/ml), anti-PD-L1 (Catalog: 17952-1-AP; Proteintech; 1:500 dilution), anti-CD8 (Catalog: ab217344; Abcam; 1:2000 dilution), anti-PD-1 (Catalog: ab214421; Abcam; 1:1000 dilution), anti-β-dystroglycan (Catalog: 11017-1-AP; Proteintech; 1:200 dilution), anti-ATGL (Catalog: #2138; cell signaling; 1:500 dilution), and anti-LIPE (Catalog: #OAAF00742; Aviva Systems Biology; 1:100 dilution). The secondary antibodies used were anti-human IgG (Catalog: 109-035-003; Jackson ImmunoResearch; 1:500 dilution), anti-rabbit IgG (Catalog: 111-035-003; Jackson ImmunoResearch; 1:500 dilution), and anti-rat IgG (Catalog: 405405; clone: Poly4054; BioLegend; 1:500 dilution). The anti-IL-20 mAb 7E was generated using the standard protocols⁶⁴. 7E was confirmed to specifically recognize only IL-20, not any other members of the IL-10 family. This antibody was also validated to recognize both human and mouse IL-20 (ref. ⁶⁴). For quantification of Ki-67⁺ proliferating cells in the pancreases of KPC mice, H&E staining and image analysis software (ImmunoRatio; https://www.medfloss.org/node/410) were used. Counterstaining was carried out with hematoxylin, and images were acquired using a digital microscope camera (DP12; Olympus Co., Tokyo, Japan). IHC images were acquired to measure the percentage of positively stained area for IL-20, PD-L1, αSMA, F4/80, CD206, CD8, and PD-1 in clinical samples and animal experiments. The percentage of positively stained area was calculated with the Tissue-Faxs software.

Immunofluorescence staining

Immunofluorescence staining was performed according to the protocols used for IHC analysis. The primary antibodies used were anti-IL-20 (7E) and anti-PD-L1 (Catalog: 17952-1-AP; Proteintech; 1:200 dilution). The secondary antibodies used were AlexaFluor 488-conjugated anti-human (Catalog: 109-545-003; Jackson ImmunoResearch; 1:200 dilution) and AlexaFluor 594-conjugated anti-rabbit (Catalog: 111-585-003; Jackson ImmunoResearch; 1:200 dilution) antibodies.

Isolation of pancreatic tumor cells from KPC mice

Primary pancreatic tumor cells were isolated from KPC mouse tumors. For bioluminescence imaging experiments, a luciferase-expressing derivative of primary KPC tumor cells (KPC-luc) was prepared by stably transfecting cells with pCAG-luc, which has the firefly luciferase gene encoded under the control of the (r)-actin promoter and the CMVIE enhancer, and a stable cell line was established.

Mice and treatment

The KPC mice were maintained in C57BL/6J background with Kras^G12D mutation and Trp53 ablation. KPC/Luc cells (2 × 10⁶) were injected orthotopically into the pancreas of C57BL/6J and NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/YckNarl (ASID) mice, which were obtained from the National Laboratory Animal Center (NLAC; Taiwan). Bioluminescence and fluorescence images were acquired (IVIS 50; Xenogen, Caliper Life Sciences, Hopkinton, MA) to detect luminescence from the KPC/Luc cells. Using the same approaches as those described for the s.c. model, treatments were started on day 4 and ended 4 weeks after tumor inoculation.

IL-20R1-deficient (IL-20R1^−/−) mice were maintained on the C57BL/6J background⁶⁵. To evaluate the effects of 7E, KPC mice (n = 16 per group) and orthotopic model mice (n = 5 per group) were randomly assigned to the indicated groups and treated with PBS, 7E (6 mg/kg, intraperitoneal (i.p.)), mIgG (6 mg/kg, i.p.), an anti-PD-1 mAb (200 μg/per mouse, i.p.; Catalog number: BE0146, BioXCell), or 7E (6 mg/kg, i.p.) plus the anti-PD-1 mAb (200 μg/per mouse) twice per week for the duration of the experiment.

LLC cells (2 × 10⁶ per mouse) were s.c. injected into the right flank of C57BL/6J WT mice. Six days after inoculation, the tumor-bearing mice were treated with PBS, 7E (6 mg/kg, i.p.), or mIgG isotype control (6 mg/kg, i.p.) twice per week for the duration of the experiment. Four weeks after inoculation, the tumors were harvested and weighed.

All animal experiments and animal care were performed according to institutional guidelines at the Laboratory Animal Center of National Cheng Kung University (NCKU) and approved by the Affidavit of Approval of Animal Use Protocol of National Cheng Kung University (IACUC Approval No: 108118, 109071).

Enzyme-linked immunosorbent assay

IL-20 concentrations in mouse serum from tumor-bearing and healthy mice were measured using a mouse IL-20 ELISA Kit (Cat. Number: DY1204; R&D Systems) according to the manufacturer’s instructions.

Cell proliferation assay

BxPC-3 and PANC-1 cells (4 × 10³) were treated with human (h) IL-20 (200 ng/ml) for 72 h in medium containing 0.5% fetal bovine serum. To confirm the specific activity of IL-20, 7E (2 μg/ml) was added to cultures, either alone or with IL-20 at a 10:1 (7E:IL-20) ratio. The cells were then incubated with 1 mg/ml MTT (Sigma-Aldrich) for 2 h, and the MTT–formazan crystals formed were dissolved in dimethyl sulfoxide (Sigma-Aldrich). Absorbance was measured at 550 nm.

Generation of a stable IL-20R1-knockdown KPC cell line

KPC cells were infected with a lentivirus containing a pLKO1 plasmid that carried an IL-20R1-specific short hairpin RNA (shIL-20R1) sequence or nonsilencing shRNA construct (Ctrl) from RNA Technology Platform and Gene Manipulation Core (Academia Sinica, Taiwan) according to the manufacturer’s instructions. Forty-eight hours after the addition of the virus, stably transduced cells were selected by adding 2 mg/ml puromycin to the growth medium for 1 week, and then the surviving cells were collected for further applications.

Culture of macrophages

BMDMs were isolated from the tibias of WT C57BL/6 mice⁶⁶ and treated with monocyte colony-stimulating factor (100 ng/ml) (PeproTech, Catalog number: 315-02) for 5 days to differentiate them into macrophage-lineage cells (M0-type macrophages). Macrophages (1 × 10⁶) were treated with IL-20 (500–1000 ng/ml) for 48 h. To verify the neutralization of IL-20, 7E was added to the culture system, either alone or together with IL-20 at a 10:1 concentration ratio (7E:IL-20).

Flow cytometry

Single-cell suspensions were prepared in ice-cold PBS containing 1% bovine serum albumin (Catalog: UR-BSA004; Sigma) and 1% sodium azide (Catalog: S2002; Sigma). Cells (1 × 10⁶) were labeled with fluorescein isothiocyanate–anti mouse F4/80 (macrophage marker; Catalog: 123107; clone: BM8; BioLegend; diluted to 2 µg/ml), allophycocyanin–anti CD-86 (M1-type macrophage marker; Catalog: 105011; clone: GL1; BioLegend; diluted to 2 µg/ml), and phycoerythrin–anti CD-206 (M2-type macrophage marker; Catalog: 141705; clone: C068C2; BioLegend; diluted to 5 µg/ml) at 4 °C for 60 min. The cells were washed three times and analyzed using a flow cytometry (FACS Canto II; BD Biosciences). FlowJo_V10 software was used for data acquisition and analysis.

PD-L1 expression on KPC cells

To examine the expression of PD-L1 and IFN-α, KPC cells were incubated with mIL-20 (400 ng/ml) in serum-free Dulbecco’s modified Eagle’s medium for 0, 8, 16, and 24 h. 7E was used to inhibit the activity of mIL-20. KPC cells were treated with mIL-20 (400 ng/ml), mIL-20 (400 ng/ml) plus 7E (4 μg/ml), mIL-20 (400 ng/ml) plus mIgG (4 μg/ml), or mIL-20 (400 ng/ml) plus an anti-IFN-α antibody (4 μg/ml) for 24 h, and then total mRNA was isolated and analyzed.

Skeletal muscle and adipose tissue histology

For histopathological analysis, skeletal muscle and adipose tissue samples were fixed in 4% paraformaldehyde, and the cross-sectional area (CSA) of the soleus muscle or the sizes of adipocytes in epididymal fat were analyzed. To quantify the CSA of the soleus muscle or the sizes of adipocytes in epididymal fat, five different fields were randomly chosen and analyzed using ImageJ (https://imagej.nih.gov/ij/).

Isolation of epididymal fat adipocytes and coculture with conditioned medium

The epididymal fat pads of mice were isolated, and adipocytes were cultured following a standard protocol. To test the effect of IL-20 on lipolytic gene expression, the adipocytes were cultured in differentiation medium (1 μg/ml insulin, 0.25 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 2.5 μM rosiglitazone, and 1 nM triiodothyronine) for 7 days and then incubated with mIL-20 (200 ng/ml) or 7E (2 μg/ml) for 24 h.

To evaluate the effect of IL-20 on tumor-derived lipolytic factors, LLC cells were pretreated with mIL-20 (200 ng/ml) for 24 h. Then the medium was replaced with fresh medium without mIL-20. The conditioned medium was collected 24 h later and added to adipocyte cultures with 7E (2 μg/ml), mIgG (2 μg/ml; Invitrogen), rat IgG (2 μg/ml; BioLegend), or an anti-TNF-α antibody (2 μg/ml; BioLegend).

Real-time quantitative polymerase chain reaction

Total RNA was isolated. Reverse transcription was performed with a reverse transcriptase (Thermo Fisher). cDNA was then amplified on a thermocycler (LightCycler 480; Roche Diagnostics, Indianapolis, IN) with SYBR Green I (Roche Diagnostics). The quantitative results for mRNA were normalized to the results for glyceraldehyde 3-phosphate dehydrogenase, the control housekeeping gene. Relative changes in mRNA expression were determined by the 2^−ΔΔCt method. The primer sequences are listed in Supplementary Table 2.

Statistical analysis

Statistical analysis was performed with GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). Experimental data are presented as the mean ± SD/SEM. GraphPad Prism software was employed to process initial data and plot graphs. The endpoints analyzed were the survival of patients with low or high IL-20 expression and that of KPC mice treated with PBS, mIgG, or 7E, which were evaluated using Kaplan–Meier methods. The Cox proportional hazards model was used for univariate analyses of disease prognosis. Pearson correlation analysis was used for evaluation of the relationship between IL-20 and PD-L1 in clinical specimens. Two-group comparisons were analyzed by a two-sided Student’s t test. One-way analysis of variance was used to compare data among groups in experiments including three or more groups. p < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The source data underlying Figs. 1–7 and Supplementary Figs. 1–5 are provided with the paper as a Source data file. All remaining relevant data are available in the article, supplementary information, or from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the grant MOST 108-2320-B-006-025 from the Taiwan Ministry of Science and Technology. We are grateful for support from the Core Research Laboratory, College of Medicine, National Cheng Kung University and the technical services provided by the Bioimaging Core Facility of the National Core Facility for Biopharmaceuticals, Ministry of Science and Technology, Taiwan. We thank the Laboratory Animal Center, College of Medicine, National Cheng Kung University and Taiwan Animal Consortium for the technical support in IVIS system (Xenogen IVISR Spectrum Noninvasive Quantitative Molecular Imaging System). We are grateful to Dr. Kuen-Jer Tsai and Ya-Chun Hsiao for the services of image acquiring and analyzing from the FACS-like Tissue Cytometry in the Center of Clinical Medicine, National Cheng Kung University Hospital. We thank Dr. Yan-Shen Shen and Dr. Po-Hsien Huang from College of Medicine, National Cheng Kung University for providing the KPC mice.

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Authors and Affiliations

Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Shao-Wei Lu & Ming-Shi Chang
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Hong-Chin Pan & Ming-Shi Chang
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Yu-Hsiang Hsu & Kung-Chao Chang
Research Center of Clinical Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Yu-Hsiang Hsu
Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Kung-Chao Chang
Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Li-Wha Wu
Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
Wei-Yu Chen

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Shao-Wei Lu
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Contributions

Conceived and designed the experiments: S-W.L., H-C.P., and M-S.C.; performed the experiments: S-W.L. and H-C.P.; analyzed the data: S-W.L., H-C.P., W-Y.C., and K-C.C.; contributed reagents/materials/analytic tools: M-S.C., K-C.C., and L-W.W.; and wrote the paper: S-W.L., H-C.P., W-Y.C., Y-H.H., and M-S.C.

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Correspondence to Ming-Shi Chang.

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Lu, SW., Pan, HC., Hsu, YH. et al. IL-20 antagonist suppresses PD-L1 expression and prolongs survival in pancreatic cancer models. Nat Commun 11, 4611 (2020). https://doi.org/10.1038/s41467-020-18244-8

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Received: 20 October 2019
Accepted: 28 July 2020
Published: 14 September 2020
DOI: https://doi.org/10.1038/s41467-020-18244-8

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