Clinical Effects of CpG-Based Treatment on the Efficacy of Hepatocellular Carcinoma by Skewing Polarization Toward M1 Macrophage from M2

Abstract

Objective:

This study aims to explore the clinical efficacy of CpG-based therapy for treating hepatocellular carcinoma (HCC) by skewing polarization toward M1 macrophage from M2.

Methods:

Pulmonary metastasis rate, overall survival time, and remission rate of 10 patients with HCC treated with transcatheter arterial chemoembolization (TACE) combined with CpG therapy and 10 age-, gender-, and TNM₀-matched patients treated with TACE (control group) were compared.

Results:

No pulmonary metastasis rate was 70% in the combined treatment group and 40% in the control group, respectively; and the differences between the two groups were statistically significant (p < 0.05). Median overall survival time was 22 months in the combined treatment group, compared with 6.65 months in the control group (p < 0.05). Remission rate in the combined treatment group (70%) was higher than in the control group (30%), but the differences between these two groups were not statistically significant (p > 0.05).

Conclusion:

Compared with TACE, CpG combined with TACE can decrease the pulmonary metastasis rate. This combined therapy can also improve the overall survival time of patients.

Introduction

Hepatocellular carcinoma (HCC) is a cancer that has rapid progression and extremely high mortality worldwide. It can easily metastasize and recur in patients who have received therapy within 1 year. The extracellular matrix (ECM) plays an important role in the first step process of tumor metastasis. Inflammatory cells influence angiogenesis and tissue remodeling, which could accelerate the growth, local invasion, and metastasis of cancer.^1
–3 Recently, researchers have focused on the correlation of cancer and inflammation.

The study of Jenkins et al.⁴ has shown that tissue macrophages can proliferate in response to IL-4, suggesting that monocyte recruitment and definitive hematopoiesis may not be required for macrophage expansion in type-2 immunity. In addition, the macrophage microenvironment in different stimulating environments can polarize toward either macrophage 1 (M1) or macrophage 2 (M2).^5,6 M1 is characterized for releasing many pro-inflammatory factors such as IL-12, TNF, and IL-6. M1 can stimulate the immune system, inhibiting the invasion and metastasis of tumor cells. However, M2 can suppress the immune system, which is characterized by high IL-10, promoting tissue reconstruction and angiogenesis, and wound healing.^7,8 Hence, macrophages can alter their phenotype toward M1 or M2 in different environment conditions due to their plasticity.

In tumor tissues, macrophages evolve into M2 polarized macrophages, which are also called tumor associated macrophages (TAMs).⁷ TAMs are present in various kinds of cancers. It is one of the main indicators of worse prognosis. TAM can degrade ECM, which accelerates the growth, local invasion, and metastasis of cancer via secreting inflammatory mediators.^9,10 M2 macrophage plays a key role in promoting tumor growth, invasion, and metastasis as the dominant phenotype in TAMs.^11

–14 M2 macrophage can make the endothelium more susceptible for tumor cell invasion by producing proteases, such as matrix metalloproteinase (MMP), which can break down the basement membrane around the endothelium.¹⁵ Under these mechanisms, M2 macrophage contributes largely to tumor invasion and metastasis, which usually lead to worse prognosis in both mice and humans.¹⁶

CpG-DNA is immunostimulatory DNA sequences which can induce both innate and adaptive cellular immune responses and has great potential clinical applications.¹⁷ CpG-DNA is a synthetic TLR9 ligand expressed by dendritic cells, macrophages, and myeloid dendritic cells.¹⁸ Through TLR9, CpG-DNA is capable of inducing immune cells to produce type I interferons and pro-inflammatory cytokines.¹⁹ Therefore, the inflammatory stimulus is the one most important factor that contributes to converting M2 macrophages into the M1 phenotype.

The macrophage microenvironment in different stimuli can polarize toward either macrophage 1 (M1) or macrophage 2 (M2).²⁰ Cytokines such as IL-10 and IL-4 can polarize toward M2.²¹ Chihara et al.²² found that HIV-1 proteins, particularly Nef proteins, derived M2-like to M1-like phenotypic shift. Ryu et al.²³ found factors such as synthetic oligonucleotides containing CpG motifs (CpG-ODN), which modulate the phenotype of renal macrophages toward M1. Guiducci et al.²⁴ reported that CpG plus anti-IL-10 receptor antibody promptly redirected in vivo elicited tumor infiltrating macrophages from M2 to M1 and triggered an innate response that debulked large tumors.

In their studies recently²⁵ demonstrated that CpG-DNA could reverse macrophage polarization, converting M2 to M1 both in vivo and in vitro in BALB/c mice. Moreover, reversing the M2 polarization of tumor infiltrating macrophages could inhibit MMP9 and vascular endothelial growth factor (VEGF) expression, as well as tumor metastasis. These findings may support the development of novel therapeutic strategies in many types of cancer patients through monitoring TAM phenotypes.

The aim was to change the macrophage polarization after their maturation from M2 to M1 by CpG, to assess its potential in inhibiting HCC migration and its clinical effects. The authors included 20 HCC patients admitted from January 2011 to December 2015 in their hospital. This study demonstrated that CpG combined with transcatheter arterial chemoembolization (TACE) could inhibit tumor metastasis and prolong the survival time of cancer patients by skewing polarization toward M1 macrophage from M2.

Materials and Methods

Patients and study design

The ethics committee of their institution approved this study. Twenty patients, who were admitted into The Affiliated Zhongshan Hospital of Dalian University from January 2011 to December 2015, were selected as the research object. All patients were diagnosed with HCC by histology or cytology. Ten HCC patients treated with TACE combined with CpG therapy (treatment group) and 10 patients treated with TACE alone during the same period (control group) were compared. The age of these patients ranged between 58 and 89 years (mean: 70 ± 5 years).

In each group of patients, seven were male and three were female. Two T₁N₀M₀ patients, three T₂N₁M₀ patients, three T₃N₂M₀ patients, and two T₄N₄M₀ patients were included into the treatment group; and TNM-matched patients were assigned in the control group. Twenty patients who were estimated to live for at least 3 months provided a written informed consent. All patients did not receive any treatment before receiving TACE therapy. The clinical characteristics of the patients included in the present study are summarized in Table 1.

Table 1.

Clinical Characteristics of Patients with Hepatocellular Carcinoma

Characteristic	Cases	Controls
Number of individuals	10	10
Gender
Male	Seven	Seven
Female	Three	Three
Age
(Mean ± SD)	70 ± 5	68 ± 3
Tumor stage
T1	2	2
T2	3	3
T3	3	3
T4	2	2
Lymph node stage
N0	2	2
N1	3	3
N2	3	3
N3	0	0
N4	2	2
Metastasis
M0	10	10
M1	0	0
Tumor size (cm)
TZ <2	2	2
2 < TZ <5	3	3
TZ >5	5	5
Unknown	0	0

SD, standard deviation.

Therapeutic method

The first and second TACE therapies were given in 4-week intervals for all patients. The treatment interval could be extended according to the condition of the patients. CpG was operated once during the TACE treatment period in the treatment group. Both groups received at least five sessions of CpG therapy.

Indicators of observation

Lung metastasis was observed after therapy in these two groups. According to the World Health Organization (WHO), effects on solid tumors could be divided as follows: complete remission (CR), partial remission (PR), stable disease, and progressive disease. Among these, CR+PR were applied to calculate efficiency. At the same time, patients were followed up via telephone every 3 months, which lasted for 3 years; and patient survival was recorded.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 5.0 software. Kaplan–Meier survival analysis was used to estimate survival, and comparisons between groups were performed with the log-rank test. p < 0.05 was considered statistically significant.

Results

No pulmonary metastasis rate of the two methods

No pulmonary metastasis rate in the treatment group (70%) was significantly higher than in the control group (40%), and there was a statistical difference in the two groups of patients with no pulmonary metastasis (χ² = 4.632, p < 0.05). Results are shown in detail in Figure 1.

FIG. 1.

No pulmonary metastasis rate of two methods.

Comparison of overall survival time between the two methods

Median overall survival time was 6.65 months in the control group and 22 months in the treatment group, and patient survival follow-up results between these two groups were statistically different (χ² = 5.103, p < 0.05). Detailed results are shown in Figure 2.

FIG. 2.

Overall survival time comparison of two methods.

Curative effect evaluation of the two methods

Twenty patients were treated with the combination of TACE with CpG or TACE only. There was no statistical difference in solid tumor curative evaluation results between these two groups of patients (p > 0.05). Detailed results are shown in Figure 3.

FIG. 3.

The recent curative effect evaluation of two methods.

Discussion

The tumor microenvironment has been shown to play an important part in cancer development, which comprises of many components, including endothelial cells, immune cells, TAM, and the ECM. In particular, TAM has a large proportion in the tumor microenvironment and plays a major role in HCC development.

Recent studies have revealed TAM and its functions in tumor invasion and metastasis.^26

–30 Once TAMs are activated around tumor cells, cytokines that could affect tumor stroma and cells are released. Some researchers have identified two main kinds of macrophage phenotypes based on its adaption to its environment.^28,31

Macrophages account for a large component of leukocytes, which infiltrate most tumors.³⁰ The increased infiltration of TAMs is associated with poor prognosis, which has been shown by many studies.^30

–33 Macrophages play an important role in innate and adaptive immunity. For example, engulfing and destroying damaged tissues, starting the healing process, and inflammation regulation.

Two distinct polarized macrophage phenotypes have been shown by many studies as follows: M1 and M2. Lipopolysaccharides (LPS) and interferon-γ polarize macrophages toward the M1 phenotype. IL-4, a kind of Th2 cytokine, can polarize macrophage toward M2. Generally, M2 is involved in the inflammatory response of Th2 and promote tumor invasion.

After patients received TACE therapy, tumor cell necrosis occurred; and TAMs accumulated in necrotic areas and vascularized poorly. These necrotic areas trigger the pro-angiogenic program due to hypoxia.

Necrosis cells can induce macrophages to proliferate. Their previous study revealed³⁴ that necrotic cells induce nonadherent peritoneal exudate cells to proliferate and differentiate into macrophage-like cells. Jenkins et al.⁴ found that the large increase in M2 macrophages observed during infection was the result of the expansion of the resident population rather than blood cell recruitment. Generally, M2 macrophages have an immunoregulatory function that helps with parasite clearance, suppresses inflammation, and promotes tissue remodeling, particularly, tumor progression.

The M2 phenotype appears to be the dominant macrophage phenotype in tumors. Reorientating M2 TAMs to M1 TAMs is a more sophisticated strategy and is a new therapeutic strategy for HCC that targets TAMs. Furthermore, it is reversible for the phenotype of TAMs in most cancers. CpG-ODN is a tumor vaccine adjuvant that has been applied in several clinical trials. Some studies have shown that CpG-ODN can improve both cellular and humoral responses to antigens. Compared with the standard regimen, in a phase III clinical trial, CpG-ODN did not prolong median survival time in the treatment of stage IIIB-IV nonsmall cell lung cancers.

In one experimental study, the combination of CpG immunostimulatory oligonucleotide plus an anti-IL-10 receptor antibody switched infiltrating macrophages from the M2 to the M1 phenotype; and this triggered an innate response that debulked large tumors.²⁴ In animal trails, the authors found that CpG-DNA could convert M2 macrophage into the M1 phenotype,²⁵ and the conversion of TAM from M2 phenotype into M1 effectively inhibits tumor metastasis. M2 macrophage produces MMPs, which break down the basement membrane surrounding the tumor, thereby making the environment more suitable for tumor cell invasion.³⁵ M2 macrophage expresses higher levels of VEGF, which promote angiogenesis in tumor tissues and take an active part in tumor growth and metastasis initiation.

The density of M2 macrophages in tumors is often correlated with metastasis.^36,37 However, macrophages can change their phenotype in response to the microenvironment. Their previous results showed that reversely converting the phenotype of tumor infiltrating macrophages from M2 to M1 could significantly inhibit the expression of MMP9 and VEGF in macrophages, which contributed largely to the suppression of primary tumor growth and metastasis. Therefore, reversing TAM phenotype may be useful in prolonging survival of tumor-bearing individuals to some extent.

Their study showed that no pulmonary metastasis rate was 70% in the combined treatment group and 40% in the control group, respectively; and the differences between these two groups were statistically significant (p < 0.05). These results implied that after TACE therapy, tumor cell necrosis induced M2 macrophages to proliferate in situ. Furthermore, M2 macrophages promoted tissue remodeling and repair, activated angiogenesis, and promoted tumor invasion. After providing CpG, CpG promptly switched the infiltrating macrophages to infiltrate from M2 to M1. M1 macrophages are cytotoxic, which stimulates the immune system and inhibits tumor progression. Finally, no pulmonary metastasis rate was higher in the treatment group than in control group. Median overall survival time was longer in the combined treatment group than in the control group.

In the near future, new concepts in adjuvant immunotherapy through monitoring TAM phenotypes would be carried out for tumors. This topic is worth further studying. Based on the animal tests, the authors carried out the study and wanted to assess its potential in inhibiting HCC migration and its clinical effects. Even though the results were optimistic, only the small cohort of patients was admitted for the study. The authors should conduct wide-scale clinical trials in the future.

In their study, no pulmonary metastasis rate was higher in the treatment group than in control group, after all there was pulmonary metastasis. The recurrence and metastasis of cancer are complicated; many factors still need to be addressed. One side, the authors demonstrated that CpG-DNA inhibited tumor metastasis by skewing polarization toward M1 macrophage from M2. These findings may support the development of novel therapeutic strategies in many kinds of cancer patients through monitoring TAM phenotypes.

Ethic Statement

Complied with ethical standards.

Footnotes

Disclosure Statement

No competing financial interests exist.

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