Pattern response of dendritic cells in the tumor microenvironment and breast cancer

Abstract

Breast cancer (BC) is the most common malignant neoplasm and the cause of death by cancer among women worldwide. Its development, including malignancy grade and patient prognosis, is influenced by various mutations that occur in the tumor cell and by the immune system’s status, which has a direct influence on the tumor microenvironment and, consequently, on interactions with non-tumor cells involved in the immunological response. Among the immune response cells, dendritic cells (DCs) play a key role in the induction and maintenance of anti-tumor responses owing to their unique abilities for antigen cross-presentation and promotion of the activation of specific lymphocytes that target neoplasic cells. However, the tumor microenvironment can polarize DCs, transforming them into immunosuppressive regulatory DCs, a tolerogenic phenotype which limits the activity of effector T cells and supports tumor growth and progression. Various factors and signaling pathways have been implicated in the immunosuppressive functioning of DCs in cancer, and researchers are working on resolving processes that can circumvent tumor escape and developing viable therapeutic interventions to prevent or reverse the expression of immunosuppressive DCs in the tumor microenvironment. A better understanding of the pattern of DC response in patients with BC is fundamental to the development of specific therapeutic approaches to enable DCs to function properly. Various studies examining DCs immunotherapy have demonstrated its great potential for inducing immune responses to specific antigens and thereby reversing immunosuppression and related to clinical response in patients with BC. DC-based immunotherapy research has led to immense scientific advances, both in our understanding of the anti-tumor immune response and for the treatment of these patients.

Key Words: Breast cancer, Dendritic cells, Tumor microenvironment

Core tip: Breast cancer is a worldwide major public health problem, and dendritic cells are of crucial importance for activating an effective antitumor immune response. Deepening our understanding of the tumor microenvironment can enable the development of new therapies that will make it possible to induce an efficacious antitumor response. Given the search for effective means with which to induce such a response via dendritic cell (DC) immunotherapy, the study of the mechanisms involved in the DC pattern of response in the tumor microenvironment is important.

INTRODUCTION

Breast cancer (BC) is the most common cancer among women and the second most prevalent form of terminal cancer in this population[1]. Cancer is characterized by accelerated and uncontrolled proliferation of normal cells due to specific genetic mutations that alter the cell cycle[2,3]. These genetic changes can culminate in the expression of mutant proteins, that in theory, should be recognized as foreign by the immune system[4]. Within the immune system, dendritic cells (DCs) play a distinct role wherein they are essential both to innate and to acquired immunity[5]. They have the unique ability for antigen cross-presentation for T helper lymphocytes (CD4⁺) and cytotoxic T lymphocytes (CD8⁺)[6] in secondary lymphoid organs, a process that activates a sophisticated and specific immunological response[7].

Although they perform the functions of antigen capture and processing in peripheral tissues, DCs are initially immature. They mature when interactions with Toll like Receptors (TLRs) or inflammatory cytokines convert immature DCs into mature DCs that present a specific antigen to naïve T cells, thereby activating them[8]. DCs with differing maturation stimuli upregulate the expression of the cell surface and intracellular molecules required for their journey to secondary lymphoid tissues. However, the activation and maturation of DCs depend on the local microenvironment and can be blocked or polarized by a combination of specific local factors, resulting in the formation of DCs with tolerogenic or immunosuppressive activities[9].

Various types of cells and factors present in tumor microenvironment generate a range of stimuli for DCs, that affect all aspects of their biology and thus control their functionality and longevity[10]. It’s known that immunotherapy with CDs is currently being studied and used as therapy for a large variety of tumors, solid or not. We have chosen to study the mechanisms involved in the pattern of response of the CDs in the tumor microenvironment, specifically in the BC because it is considered major public health problem. The knowledge of these cells response will be the basis for developing an effective immunotherapy. In this work, we provide an up-to-date review of the pattern of DC response in the BC tumor microenvironment and the use of immunotherapy with a DC vaccine.

ROLE OF DCs IN TUMOR IMMUNITY

DCs are key regulators of immunity by B and T lymphocytes because of their superior ability to capture, process and present tumor-associated antigens[11]. They are derived from a hematopoietic line with cell subsets that differ in terms of their morphology, phenotype and function depending on the particular conditions and the tissue in which they reside. However, the various DC subsets generally have the ability to stimulate T cells[12]. DCs have different co-stimulatory molecules, such as B7, intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), lymphocyte function-associated antigen-3 (LFA -3) and CD40 on their cell surfaces. As a set, these proteins generate a chain of secondary signals essential to reinforcing the T cell activation, with the resultant immune response initiation, programming and regulation being directed specifically at the tumor[13].

The DC maturation process determines the presentation of processed tumor antigens and the subsequent activation of CD4⁺ and CD8⁺ T cells[14]. Their migration from tissues to the lymph nodes is fundamental to this process, since it provides the period during which expression levels of co-stimulatory molecules as class II MHC molecules, adhesion molecules and the C-C motif chemokine receptor (CCR7) are increased. This process is accompanied by a reduction in CCR6[15,16], which coincides with increase in the expression of particular C-C motif chemokine ligands (CCLs)-namely CCL19 (a.k.a. EBI1 ligand chemokine) and CCL21 (a.k.a. secondary lymphoid-tissue chemokine)-within the lymph nodes as well as increased CCR7 binding[17]. The expression of molecules on the DC membrane and the migration of DCs to T-cell populated areas in the lymph nodes, where drainage occurs, during this process are fundamental to specific immune response by the T lymphocytes[7,18]. Nevertheless, depending on microenvironment conditions, which involve the cells present in the tissue as well as the presence of mediators, DCs can present alternative phenotypes that are implicated directly in an immunogenic to tolerogenic change of function[19].

PROGRESSION OF BC, ITS RELATIONSHIP WITH THE TUMOR MICROENVIRONMENT AND THE PROCESS OF DC MATURATION AND ACTIVATION

If found in an early stages, BC has a good cure rate, though the metastatic or recurrent form of the disease has a poor prognosis. BC consists of a heterogeneous group of neoplasms derived from the ductal epithelium. The majority of BCs (80%) are ductal (including medullary, papillary, tubular and mucinous subtypes), and the remaining 20% are lobular cancers. The normal mammary gland consists of a layer of luminal epithelial cells, myoepithelial cells and a continuous basal membrane. The stroma contains fibroblasts, immune system cells (e.g., macrophages, DCs and lymphocytes) and vasculature surrounded by the extracellular matrix and adipocytes, which maintain the structure of normal tissue[20].

The evolution of malignant breast tumors beings with epithelial hyperproliferation and progressing to in situ, invasive ductal and metastatic carcinomas[21]. In in situ ductal carcinoma, epigenetically and phenotypically changed myoepithelial cells, which are surrounded by a basal membrane that is still mostly continuous, are incapable of contributing to the polarization and organization of normal cellular processes. In invasive ductal carcinoma, there has been a loss in the continuity of the basal membrane, loss of function of the myoepithelial cells and an invasion of the epithelial cells into the stroma and vasculature. During conversion from in situ to invasive carcinoma, there is an increase in the production of extracellular proteases that degrade the stromal matrix and cells[22]. Concomitantly, tumor cells in the stroma produce proteolytic enzymes; while chemokines and cytokines continue to attract leukocytes, modulate tumor remodeling, and facilitate tumor cell invasion of distant organs, leading to metastasis. Infiltrating fibroblasts and leukocytes are induced to increase the secretion of growth factors, cytokines, chemokines and matrix metalloproteinases (MMPs) which produce an immune-evasion mechanism that enables the tumor to progress and achieve angiogenesis[23].

We now know that the tumor microenvironment is the main regulator of carcinogenesis and is responsible for the course of tumor progression, including how tumors respond to various types of treatment[24,25]. The development and progression of a tumor to a malignant phenotype is highly dependent on interactions between the tumor cells and normal cells present in the tumor microenvironment[23,26]. There are extensive changes in genetic expression in all cell types during BC progression. Genetic changes detected in cancerous epithelial cells have been tied to the overexpression of particular C-X-C motif ligand (CXCL) chemokines, namely CXCL14 by myoepithelial cells and CXCL12 by myofibroblasts. These chemokines bind receptors in cancerous epithelial cells, resulting in increases in their proliferation, migration, and invasion, thus favoring tumor remodeling, as well as their ability to migrate to and induce metastasis in distant organs. Chemokines and cytokines produced by tumor cells and infiltrating cells continue to attract more leukocytes, further facilitating tumor progression[27]. Abnormal matrix remodeling in BC tumor progression is attributable to MMPs, which can also activate chemokines, cytokines, adhesion molecules, and growth factors that support tumor progression. Angiogenic factors are activated by MMPs 1, 2, 9 and 14[28].

The tumor microenvironment promotes BC initiation, growth, migration, metastasis, and therapeutic resistance. The mediators and enzymes produced can induce important genetic changes in tumor-associated cells, such as fibroblasts, endothelial cells, adipocytes, and leukocytes. These cells are critical components of the tumor stroma and can damage the microenvironment, enabling malignancy as shown in Figure 1[25]. Tumor occurrence has been shown previously to be associated with DNA methylation and histone modifications in fibroblasts[29]. We also know that germinal mutations in BRCA1 and BRCA2 confer increased risk of BC and ovarian cancer and reduced risk of other types of cancer; TP53 and PTEN mutations are also found in BC cells[30-32]. Mature adipocytes influence tumor behavior by producing hormones, growth factors, and cytokines, as well as a heterogeneous group of molecules known as adipokines, which can change the phenotype of the epithelial cell, increasing its mobility. Interestingly, these molecules provide a link between obesity and BC risk[33,34].

Figure 1 Changes to the tumor microenvironment seen in a mammary gland with breast cancer, consisting of a layer of luminal epithelial cells surrounded by myoepithelial cells, bordered by a continuous basal membrane, with adipocytes external to the mammary gland. In the stroma, there are tumor-associated fibroblasts immune cells surrounded by the extracellular matrix that maintains the tissue structure. In ductal carcinoma, the myoepithelial cells are changed epigenetically and phenotypically. The number of fibroblasts, monocytes and macrophages associated with the tumor are increased in the stroma, elevating secretion of growth factors, cytokines and chemokines, which promotes tumor progression. Dendritic cells (DCs) have plastic characteristics, showing distinct phenotypes depending on their mature state.

The endothelial cells that form tumor neovessels are distinct from normal endothelial progenitor cells[35,36]. Under the influence of proteins in the tumor microenvironment, such as macrophage colony-stimulating factor (M-CSF), monocytes can differentiate into endothelial cells that provide angiogenesis within the tumor[37]. This process, which can also be mediated by components of the extracellular matrix (e.g., fibronectin), leads to the phenomenon of monocyte-epithelial cell differentiation[38].

Tumor-associated macrophages are a major component of the leukocyte infiltrate; when activated, they exercise a tumoricidal action[39]. However, the presence of tumor-associated vascular leukocytes has been shown to be linked directly with tumor growth through increased expression of the cytokine tumor necrosis factor (TNF)-alpha[38]. Release of TNF-alpha in the tumor microenvironment promotes the differentiation of tumor-associated monocytes towards a myeloid-epithelial pro-angiogenic phenotype via positive regulation of the fibronectin receptor α5β1.

In a study of tumors obtained from patients with invasive BC[40], we found that differences in T and B lymphocytes, in terms of peritumor/intratumor infiltrate, were related to tumor size. Specifically, small tumors (≤ 2 cm) had relatively lower levels of intratumor B lymphocytes, whereas large tumors (2-6 cm) had relatively low levels of peritumor T lymphocytes. We know that tumor-infiltrating B cells can subserve an antibody response to breast tumors. In a study analyzing the presence of B lymphocytes (CD20⁺) in 1470 invasive breast carcinomas, Mahmoud and colleagues documented that B lymphocytes were diffuse in areas at a marginal distance from the carcinoma (average distance, 12 cells) compared to their more dense presence within the carcinoma and in stromal compartments adjacent to the tumor. Total B-cell counts correlated with a higher tumor grade and a basal phenotype, as well as with estrogen receptor (ER)/progesterone receptor (PR) negativity[41].

Regulatory T lymphocytes (Tregs) and cytokines have also been implicated in the immune cell infiltration of tumors. Invasive BC tumors have been reported to exhibit elevated levels of intratumoral Tregs, with ER/PR negativity and HER2 overexpression being associated with an unfavorable prognosis[42]. Furthermore, enrichment of Tregs in invasive ductal carcinoma of the breast correlates with upregulation of interleukin (IL)-17A expression and augmented invasive ability[43].

In a letter to the editor about an article entitled “cancer stage and local immune response,” the researcher[44] Murta reiterated and defended the idea that the BC stroma and peri-or intratumoral lymphocyte infiltrates are important to the development of and prognosis for BC and that immunotherapy for breast cancer is still under investigation. Moreover, the tumor microenvironment has innumerable escape mechanisms and ways to favor tumor progression, even using the cells themselves and mediators of the immune response in its favor.

INFLUENCE OF THE TUMOR MICROENVIRONMENT ON DENDRITIC CELLS

DCs can change their phenotype in response to tumor microenvironmental factors and contribute to angiogenesis. This cellular plasticity characteristic seems to be exploited by tumors that repress DC maturation, thereby inhibiting specific antitumor immune responses[45]. Tumors can induce the generation and accumulation of immunosuppressive cells (e.g., Tregs) in the tumor microenvironment, improving tumor’s ability to evade immunological defenses[42]. Tumor-associated cytokines such as vascular endothelial growth factor (VEGF), IL-10, and prostaglandin E 2 (PGE2) can affect the phenotype of DCs[46]. Liu et al[47] placed DCs in co-culture with isolated Lewis lung carcinoma cells to simulate the tumor microenvironment. The co-cultured DCs were induced to differentiate into regulatory DCs with a CD11c^low CD11b^high Ia^low phenotype and elevated expression of IL-10, nitric oxide, VEGF, and arginase I. These regulatory DCs inhibited T-cell proliferation both in vitro and in vivo, with PGE2 being the major inducer of arginase I in the regulatory DCs[47]. Corroborating this idea, several studies have shown that tumor-associated DCs can induce Treg expansion and become not only incapable of inducing specific immune responses but also immunosuppressive[45,48,49]. Furthermore, BC tumors have been reported to promote the differentiation of DCs into a phenotype that expresses IL-10 and tumor growth factor (TGF-beta), which, in turn, induce the expansion of Tregs (CD4⁺CD25⁺FOXP3⁺)[50].

Tumor-associated DCs can produce pro-angiogenic factors in the tumor microenvironment. Rapid tumor growth factors are associated with the infiltration of immature DCs that promote angiogenesis and tumor growth, whereas mature DCs are known to suppress angiogenesis[51]. BC cell-secreted IL-6 determines whether monocytes in the tumor stroma will differentiate into DCs or macrophages. In vitro, activated monocytes placed in contact with fibroblasts (common in tumor stroma) induce the fibroblasts to release IL-6. IL-6 regulates the expression of M-CSF receptors in functional monocytes and enables them to become reactive to autocrine M-CSF. Interaction between IL-6 and M-CSF favors monocyte differentiation into macrophages, rather than DCs. Like fibroblasts, Hs578T BC cells modulate monocyte differentiation into macrophages vs DCs depending on the presence of IL-6[52].

DCs are a specialized group of antigen-presenting cells with extraordinary functional plasticity. They have the potential to stimulate or suppress immunity, depending on the sequence and combination of microenvironmental stimuli present[12]. DCs cultivated in vitro in the presence of tumor factors can have some characteristics of epithelial cells, such as low-density lipoprotein absorption, leptin binding[53], and even suffer a process of endothelialization characterized by loss of CD14/CD45, loss of the endothelial markers CD31 and CD34, and loss of the von Willebrand factor, vascular endothelial growth factor receptor 2 (VEGFR-2), and vascular endothelial (VE)-cadherin[54].

By means of a literature review, Curiel[55] defends the need of a reduction in the function of Treg cells in the microenvironment tumor recurrence in patients with cancer, either individually or in combination with other therapies. According to this study the reduction may be clinically beneficial and potentially effective as an immunotherapy against cancer. In his work, he highlights other studies in which the function of Treg cells can be decreased, as an indirect effect, after the use of other immunotherapeutic agents, as the use of Denileukin diftitox (DAB389IL-2), a fusion protein of interleukin 2 (IL-2) and diphtheria toxin[56], and doses of diftitox denileukin, in combination with a recombinant vaccine poxviral can increase the immune responses in an antigen-specific normal murine model. The authors noted that Treg cells in spleen, peripheral blood and bone marrow of the animals were variously reduced after a single intraperitoneal injection of denileukin diftitox, evident reduction in 24-h effect after around 10 d. Similarly, in another study[57] examined the effects of a single dose of low dose cyclophosphamide (CTX) on immunogenicity of DC vaccines in animals with tumors of the colon and melanoma. As a result there was an increase in IFN-γ produced by lymphocytes in the spleen and significantly reduce CD4⁺ CD25⁺FoxP3⁺ Treg cells, thus establishing a proposal for an immunotherapy strategy by combining low dose CTX with DC.

In the study[58] an antiangiogenic therapy was used in clinical trials of patients with BC through Sunitinib and Bevacizumab and demonstrated that there is a significant induction of hypoxia in the tumor microenvironment , with increased of stem cell cancer population.

MATURE DCs AND THE TUMOR MICROENVIRONMENT IN BC

As shown in Figure 1, the BC tumor microenvironment is highly immunosuppressive due to the presence of growth factors (e.g., VEGF) and cytokines (e.g., IL-10), and the infiltration of Tregs and immature DCs. Infiltration of mature DCs into primary tumor lesions is associated with prolonged patient survival and reduced risk of metastasis[59]. These findings reinforce the importance of DCs in antitumor immunological oversight, including the migration of DCs into regional lymph nodes where they present specific tumor antigens to naïve T cells.

In 2008, Dieu-Nosjean and colleagues reported a study[60] that indicated that the density of tumor-infiltrating lymphocytes (CD4⁺, T-bet⁺ Th1 T cells, in particular) was reduced in tumors that had weak infiltration by mature DCs. The authors interpreted the findings as indicating that the density of mature DCs was a better predictor of clinical evolution than the other parameters tested.

The therapeutic potential of mature-DC vaccines remains of great interest. Research probing the benefits of DC immunotherapy has demonstrated safety with few averse secondary effects as well as interesting results in terms of efficacy for stimulating immune cells[61,62]. Current strategies to curtail immune evasion by tumors include a combination of DC vaccination with Treg depletion by way of administration of anti-CD25 antibodies[63], and blockade of endothelin receptors in endothelial cells to facilitate the infiltration of cytotoxic T cells into the tumor microenvironment[64]. Stimulation of an immune response by DC immunotherapy has been evidenced by increases in the percentages of IL-2-, TNF-alpha-, and IL-10-expressing T-helper (CD4⁺) cells. A similar result was observed for IL-2 expression by cytotoxic T (CD8⁺) cells. The percentage of total T (CD3⁺) cells remained elevated during immunotherapy. Counts of Treg (CD25⁺/FOXP3⁺) cells remained significantly lower than the pre-treatment baseline count throughout the treatment period[65].

Vaccines made from DCs pulsed with autologous tumor lysates induced secretion of Th1 cytokines and an increase in natural killer cells and CD8⁺ IFN-gamma⁺ T lymphocytes in the peripheral blood of BC patients. This finding suggests that a vaccine of DCs pulsed with tumor lysates may be an effective source of tumor antigens, capable of inducing effective immune responses[66]. In another study, a vaccine of DCs pulsed with HER-2 was reported to be well-tolerated and to attenuate expression of HER-2/neu, enabling an immune response to invasive cancer to be mounted[67]. Finally, in a clinical trial, an antitumor DC vaccine administered together with IL-2 was found to induce specific cellular immunity in patients with BC that was accompanied by a reduction in TGF-beta levels, an increase in IL-12 secretion, and a reduction in CD4⁺ CD25⁺ T cells[68]. Based on these promising findings, we believe that DC-based therapies designed to disrupt a tumor-promoting microenvironment should be investigated with the aim of developing more powerful DC vaccines that can generate an intense and long-lasting anti-BC immune response[69].

In conclusion, growing evidence shows that the BC tumor microenvironment is immunosuppressive, at least in part through modification of DCs toward a Treg phenotype, i.e., mature DCs are not functional in the tumor microenvironment in patients with BC. DC maturation is critical for enabling specific molecular identification of BC antigens and DC vaccine immunotherapies have the potential to induce specific immunity against tumor antigens. Importantly, changes in immunological parameters that favor a specific antigen immune response, together with a reduction in immunosuppression, correlate with a positive clinical response in patients treated with a DC vaccine. Clarification of the microtumor environment, the mediators involved, interactions with immune response cells, and immune-evasive mechanisms may lead to new forms of immunotherapy.