Abstract
Background/Aim: Liver metastases are among the principal mortality causes in cancer patients. Dendritic cell immunotherapies have shown promising results in some tumors by mediating immunological mechanisms that could be involved in liver metastases during primary tumor growth. The present study aimed to evaluate the impact of prophylactic dendritic cell vaccination on the liver of mice with 4T1 mouse breast carcinoma. Materials and Methods: Adult female Balb/c mice were submitted or not to vaccination with dendritic cells before the induction of 4T1 tumor lineage. Liver tissues from mice were analyzed by flow cytometry (markers CD3, CD4, CD8, CD25, IL-10, IL-12, IL-17, TNF-α, IFN-γ, T-bet, GATA3, RORγt, and FoxP3) and hematoxylin-eosin. The dendritic cell vaccine was differentiated and matured ex vivo from the bone marrow. Results: Prophylactic vaccination reduced areas of liver metastases (p=0.0049), induced an increase in the percentage of total T and cytotoxic T lymphocytes (p<0.0001), as well as FoxP3+ (p<0.0001). It also increased the levels of cytokines IL-10 and IL-17 in helper T lymphocytes (p<0.0001). Conclusion: The prophylactic dendritic cell vaccine changed the cell phenotype in the immune response of liver, and it was able to reduce metastases. Cytotoxic T cells and regulatory T lymphocytes were more present, likewise, the production of IL-10 and IL-7 simultaneously, demonstrating that the vaccine can induce a state of control of pro-inflammatory responses, which can provide a less favorable environment for metastatic tumor growth.
Although liver is not considered a classic secondary lymphoid organ, it represents a unique site for the development of adaptive immune responses. The responses are mediated by a diverse repertoire of immune cells and other non-hematopoietic cells. In addition to hepatocytes, there is a considerable wealth of antigen-presenting cells (APCs), natural killer cell (NK), natural killer T cell (NKT), T and B lymphocytes, neutrophils, eosinophils, and components of the complement system (1). However, the full spectrum of immune cells that resides in the liver and their role in different pathophysiological processes is still unclear. In the liver, the generation of an integrated adaptive immune response may not occur as efficiently and directly as in a lymph node or spleen. Some authors considered this to be immune paralysis. However, it could occur due to patterns related to the organ’s anatomy, physiology, and location, which directly affect the way the organ will articulate its immune mechanisms (2).
The liver can activate a robust immune response via T cells in baseline conditions; however, it needs to have an immune-tolerogenic response pattern that leads to the expansion of cognate T lymphocytes, without supporting the effectiveness of cytotoxic effector functions (3, 4). The organ can still provide a hypoimmune environment, with an easing in the processes of immune tolerance, which could justify the success of transplants (1).
Liver is often a target organ for metastases from different types of primary tumors (5). Metastatic development is a multi-step process, supported by both the primary tumor site and the hepatic metastatic microenvironment. Growth factors, cytokines, and other soluble factors are involved in the interactions between the disseminated cancer cells and the resident hepatic cells that control liver metastasis (5-7). The presence of liver metastases represents a poor prognosis, affecting the overall survival of patients (8).
In the fight against neoplasms, immunotherapies can be applied to treat the disease with minimal impact on normal tissues. The cell-based vaccines can be of a prophylactic or therapeutic type. Both types have been shown to generate a specific immune response against the tumor, and in parallel have the capacity to generate cellular immune memory, aiming to protect against possible recurrences (9).
Dendritic cells (DCs) have a fundamental role in antitumor vaccine models for the treatment of cancer, as they have a high potential for stimulation of T lymphocytes (10). In the literature, there are studies demonstrating that the antitumor immune response provided by the DC vaccine-activated effector T lymphocytes ensures an efficient antitumor mechanism. This is due to the fact of DCs being the best antigen-presenting cells, performing the processes of recognition, processing, and presentation of tumor cell antigens, as well as of their potential to stimulate T lymphocytes and establish a bridge between immune mechanisms. Therefore, DC vaccination allows tumor regression and remaining memory cells, hence, protecting recurrences (10).
Murine breast carcinoma with 4T1 cells is a highly tumorigenic and invasive model, with a very aggressive profile, with rapid evolution, high risk of recurrence, mostly visceral metastases, high death rates, and significant resistance to immunotherapies (11). Therefore, it is important to have alternatives that control its growth, preventing its accelerated dissemination, focusing on detection and prognosis.
In this work, the aim was to evaluate the impact of prophylactic dendritic cell vaccination on the phenotypic profile of T lymphocytes and metastases in the liver of mice with 4T1 mouse breast carcinoma, submitted to prophylactic vaccine with dendritic cells.
Materials and Methods
Animals. A total of 25 adult female Balb/c mice (6-8-weeks-old, an average weight of 23g±0.8 g) from Biotério Central of Universidade Federal do Triângulo Mineiro-UFTM were used. The animals were kept in plastic cages, in a closed housing system with ventilation and controlled temperature (21±3°C), in a 12-h light-dark cycle, with access to food and water ad libitum.
The animals were randomized into three experimental groups: C (Control; n=7) animals without induction of 4T1 mouse breast tumor cells and without prophylactic vaccination by dendritic cells; group T (Tumor; n=8), with animals submitted to tumor induction with 4T1 cells; and group DC (Dendritic cell vaccine; n=7), with animals that received dendritic cell vaccine and subsequent tumor induction (Figure 1). Three animals were euthanized for the preparation of the dendritic cell vaccine. This study was approved by the Ethics Committee on the Use of Animals - CEUA of the UFTM (registration number 379) and followed the regulations of the Basel Declaration.
Tumor induction of breast cancer model. The animals of T and DC groups were inoculated with 4T1 mouse breast tumor cells (model of syngeneic transplantable tumors) (12, 13); in a single dose of 2.0×105 cells, injected into the lower-left mammary gland. This lineage has tumor growth and metastatic spread similar to stage IV human breast cancer. The strain was obtained by the Rio de Janeiro Cell Bank and maintained in RPMI-1640 medium (Sigma-Aldrich®, St. Louis, MO, USA), supplemented with 0.24% HEPES (C8H18N2O4S), sodium bicarbonate (NaHCO3), 10% fetal bovine serum (FBS), 1% streptomycin, 1% L-glutamine (200 mM), 0,1% sodium pyruvate (C3H3NaO3), 0.1% β-mercaptoethanol (C2H6OS), at 5% CO2 and 37°C. All products used for supplementing the RPMI-1640 medium were obtained from Sigma-Aldrich®, St. Louis, MO, USA.
Dendritic cell vaccine and vaccination protocol. The dendritic cell vaccine was made from the bone marrow of the femur and tibia of the three mice Balb/c, grown in IMDM medium (Sigma-Aldrich®, St. Louis, MO, USA), supplemented with 0.1 mM of vitamins, 2 mM L-Glutamine, 100 μg/ml Gentamicin, 1 mM Sodium Pyruvate and 5% FBS, incubated in a 5% CO2 and 37°C. All products used for supplementing the IMDM medium were obtained from Sigma-Aldrich®.
On the first day, the cells were stimulated with 10 ng/μl of Granulocyte-macrophage Colony-stimulating factor (GM-CSF) and 10 ng/μl of IL-4. In day five, cells were stimulated with TNF-α and tumor lysate from 4T1 cells. GM-CSF, IL-4 and TNF-α was obtained from BD Pharmigen™, BD Biosciences, San Diego, CA, USA. After 48 h of incubation, the differentiated dendritic cells were washed and resuspended in 0.9% saline solution. A single dose of 5.0×106 cells was administered in the animals of the DC group, 7 days before tumor inductions with 4T1 tumor cell line (Figure 1).
Isolation of liver cells and flow cytometry. At the end of experimental period, the livers were removed after euthanasia and necropsy of the animals. Part of the organs was subjected to mechanical rupture, and the cells followed a flow cytometry protocol. All material for flow cytometry analysis was obtained from BD Biosciences, San Diego, CA, USA. The leukocyte cells of liver were obtained by centrifugation after using lysis solution FACS Lysing Solution. The technique was performed according to the protocol suggested by the manufacturer, using BD PharmigenTM antibodies for extracellular labeling (CD3, CD4, CD25, and CD8 for lymphocyte evaluation) and for intracellular marking (T-bet, GATA3, RORγt, FoxP3, IL-12, TNF-α, IFN-γ, IL-10, and IL-17A). The liver samples from the three experimental groups were analyzed using FACS CaliburTM cytometer.
Gate strategy: lymphocytes were initially selected based on size and granularity (FSC x SSC). In lymphocytes, a gate for CD3+ was designed to mark T cells. From this gate, a CD4 vs. CD8 graph was plotted to separate CD4+ and CD8+ T lymphocytes. Within the CD4+ T lymphocytes, a T-bet graph was plotted to mark helper T lymphocytes type 1 (Th1), another plotting GATA-3 to mark helper T lymphocytes type 2 (Th2), RORγt, to mark helper t lymphocytes type 17 (Th17) and a graph plotting CD25 and FoxP3 was used to outline regulatory T lymphocytes (Treg). Also from the helper T lymphocytes gate (CD3+CD4+), histograms for the intracellular TNF-α, IFN-γ, IL-12, IL-17, and IL-10 markings were plotted.
Analysis of metastases in the liver (H&E) – histology. The livers of T and DC group were fixed in a 10% formalin solution, followed by inclusion in paraffin. Tissue was cut in 5-μm sections in an automated rotating microtome (Leica RM2255) and the sections were stained with the hematoxylin and eosin (H&E). The slides were observed under an optical microscope (Olympus BX41). Qualitative and quantitative measurements were made by blinded researchers. For each organ, three slides were prepared so that the entire organ could be sampled. In each slide, 15 blind fields were evaluated (45 fields per animal of each group). Quantification of metastases was performed using a Nikon Eclipse Ti2 microscope with Nikon Analyzes Software.
Statistical analyses. Graphs and statistical analyses were performed using GraphPad Prism 8.4 (GraphPad Software). One-way ANOVA with Tukey post-test and t-Student was performed with results expressed as mean±SD. Fisher’s exact test was used to establish statistical differences in the association of vaccination and metastasis. For all analyses, p<0.05 was considered statistically significant.
Results
The prophylactic vaccine with dendritic cells reduced the number of liver metastases. Metastatic liver areas were observed in all animals in the two groups, T and DC (Figure 2). In general, the foci of metastases were smaller and rarer in DC than in T group.
First, we evaluated the association between vaccination and the number of fields that were positive or negative for metastases. A number of 45 fields were evaluated per animal (15 fields per slide, 3 slides per animal, totaling 360 fields evaluated for the T-group, and 315 fields for the DC). A reduction in the positive fields was observed in DC (95 fields) compared to the T group (171 fields) (p<0.0001; Figure 2A). Thus, the data showed that approximately 70% of the total evaluated fields were negative in the DC group. In addition, when the total metastasis area was measured, a statistically significant reduction was observed in DC (0.7×106±0.41×106) compared to T (3.8×106±2.3×106) (p=0.0049; Figure 2B). Figures 2C and 2D illustrate metastatic foci found in animals of the T and DC group, respectively. Therefore, prophylactic vaccination with dendritic cells appeared to reduce total number of metastases and total metastasis area in the liver of mice with breast cancer.
Subtypes of intrahepatic lymphocytes and cytokine production by helper T lymphocytes. The characterization of the phenotypic profile of immune cells in the livers of animals was performed by flow cytometry, right after euthanasia. The percentage of total T lymphocytes (CD3+) was higher in the DC group (97.03±1.83) compared to the group T (85.09±2.38) (p<0.0001) (Figure 3A). The percentage of cytotoxic T lymphocytes (CD3+CD8+) was also significantly higher in the same group (DC) compared to T (p<0.0001) (Figure 3C). However, the percentage of helper T lymphocytes (CD3+CD4+) did not show significant changes (p=0.2756; Figure 3B) between the two groups (T and DC).
Regarding lymphocyte populations, the profile of T helper lymphocyte subtypes was also evaluated using the mean fluorescence intensity (MFI) of the key transcription factors of the differentiation process. The transcription factors T-bet, GATA3, and RORγt were significantly reduced in the vaccinated group (p<0.0001), compared to the T group (Figure 4A-C). However, FoxP3 was increased in the DC group compared to the tumor group (p<0.0001), while was decreased compared to the control group (p<0.0001; Figure 4D).
We also evaluated the impact of prophylaxis on the expression of cytokines IL-12, IFN-γ, TNF-α, IL-10, and IL-17 in helper T lymphocytes. IL-12, IFN-γ, and TNF-α were significantly reduced in the vaccinated group, compared to the T group (p<0.0001). The cytokines IL-10 and IL-17 were significantly increased in the DC group compared to the T group (p<0.0001; Figure 5).
Discussion
Liver is a target organ for metastases in several types of cancers. In breast cancer, metastases could target various organs, such as lymph nodes, liver, lungs, brain, and bones (14). The hepatic metastases represent poor prognosis in breast cancer, affecting survival of patients (8, 15).
In the immune context, liver represents a fundamental place for adaptive immune responses, even though it is not considered a typically secondary lymphoid organ, as spleen, for example (1, 16). Furthermore, it is known that the interactions between the immune system and the tumor can mediate the progression or regression of cancer; hence, the same immune mechanisms may facilitate or not the implantation of metastatic colonies (17). Thus, this study sought to assess the impact of prophylactic DC vaccination, evaluating the metastatic area and profile of T lymphocytes in the liver of animals with breast cancer.
Our data showed reduced metastatic areas in the livers from vaccinated animals, suggesting that DC-vaccination may be an important instrument to manipulate the antitumor and metastatic response. Previous studies have also demonstrated the antitumor and antimetastatic effect of prophylactic DC vaccination in melanoma and pancreatic cancer (18-20). Ηowever, there is little data on its effectiveness in target organs for metastases, such as liver.
Some studies have shown that the DC vaccine could act on both growth of the tumor and antimetastatic response by mechanisms of the antitumor immune response. They demonstrated that autologous DCs loaded with tumor antigens increased the polarization of Th1 immune responses and reduced Treg, in addition to the cytotoxicity generated by DC. Moreover, the vaccine induced responses and cytotoxic T lymphocytes, both in vitro and in vivo. Thus, they suggested that immunotherapy with DC in the context of breast cancer not only is safe, but it also reduces the risk of relapse and improves immune parameters, and can be a strategic approach to combating metastasis. Generally, this activity may be associated with immune surveillance generated by vaccination, specifically prepared for antitumor activity. Th1 cells and an increase of cytotoxic T lymphocytes mediate this antitumor mechanism. However, these studies did not used the DC vaccine as a prophylactic intervention (21, 22).
The liver, as a secondary lymphoid organ, activates T cells locally and this activation occurs in an environment that is biased towards immune tolerance (23). Immune tolerance could be attributed to the low expression of the major histocompatibility complex (MHC) and IL-10 expression (24-26). Even though the liver is a tolerogenic organ, its immune function can generate rapid and controlled responses to tumor cells or pathogenic organisms that have the liver as their target (27, 28).
Despite the metastatic reduction due to prophylactic vaccination, our data indicated an increase in cytotoxic T lymphocytes and Treg in the liver tissue. The liver is rich in lymphocytes. Even though they are populations similar to those present in the peripheral circulation, these lymphocytes differ in proportions and performance standards, and their roles remain uncertain (1). The composition and location of cell populations can change liver inflammatory conditions. Associated with anatomy and the vasculature liver, these conditions can exchange immunological information continuously due to communication with splenic venous system and inferior vena cava, lymphatic drainage (17, 29).
In the liver, T helper lymphocytes play a crucial role in the production of cytokines that act on hepatocytes and other immune response cells. Cytotoxic T lymphocytes, activated or memory cells, are also present in the liver and their activity is associated with apoptosis and exclusion of T cells (30). Some studies have pointed out liver-mechanisms that actively prevent the generation of cytotoxic T lymphocytes, preventing immunity mediated by functional T cells and inducing T cell tolerance (3, 31). However, this information is still uncertain. The liver T helper lymphocyte subtypes are in balance, Th1/Th2 and Th17/Treg. The Th1 cells are involved in pro-inflammatory cell-mediated responses, while the Th2 cells promote tolerance (32). An imbalance, however small, leads to the production of dominant cytokines for one of these profiles.
Th17 are closely related to Treg; the reciprocal relationship between these two represents a delicate balance between tolerance and induction of inflammatory responses or immune tolerance (33). A Th17/Treg imbalance may be responsible for liver-pathological processes (34). In other words, it seems that these two cells populations make it possible to maintain a subtle balance in liver homeostasis, while a slight imbalance is associated with diseases and injuries (35).
In hepatocellular cancer, an imbalance of Th17 and Treg seems to be associated with the process of carcinogenesis. The isolated increase in intratumoral Th17 is indicative of tumor activity and angiogenesis. It also affects the prognosis and is associated with low survival rates (36, 37). Thus, Th17 and Treg and their cytokines play a double role in the liver. The lack of control of the immune system in this organ can generate favorable conditions for metastatic development. Therefore, considering that our data showed that there is an association between an increase in cytotoxic T lymphocyte and Treg and an increase in IL-10 and IL-17, the combined effects seem important for the hepatic immune responses acting on metastasis. Anyway, the presence of Treg is fundamental in regulating a balance on hepatic inflammatory responses and seems to have substantial effects that need to be further evaluated (35).
Another point that leads us to assume a relative tolerance is the low levels of IL-12 observed in the vaccinated group. This environment promotes change in Th1 to Th2 responses and maintenance of Treg (38), which would justify the increase of this phenotype in our study. At the same time, other cells, such as the myeloid lineages, could create new modulations of the immune responses (39).
In addition to the increase in IL-10, we also found that IL-17 was increased in the vaccinated group. IL-10 is an important anti-inflammatory and immunosuppressive cytokine that acts by inhibiting the synthesis of other cytokines, such as IL-12, IFN-γ, and TNF-α, as well as inhibiting proliferation and Th1 at the expense of Th2. Simultaneously, it can also be an important differentiating factor for cytotoxic T lymphocytes, even if it is not its primary function (40).
It is worth mentioning that the healthy adult liver tends to have an active and complex environment composed of cytokines, either pro-inflammatory, such as IL-3, IL-7, IL-12, IL-15, and IFN-γ, or anti-inflammatory, such as IL-10, IL-13, and TGF-β (41). This cytokine environment exists in the absence of any pathogen or pathological inflammation and arises through habitual and physiological processes in the liver (42), which emphasizes and supports the idea of why healthy liver is described as immunologically tolerogenic. However, it is still evident that rapid and robust immune responses are successfully generated in this environment (23, 27).
Our study allowed a description of immune cell populations in the liver tissue involved in the antitumor immune response. We also demonstrated the importance of prophylactic immunotherapy beyond the tumor microenvironment. Other phenotyping and genotyping methods could provide a better knowledge of the possible interactions in this tissue.
The prophylactic DC vaccine altered the cell phenotype in the liver immune response, suggesting that the liver functions as an important regulator of systemic immunity. Our data reveal that the liver mediates distinct functional processes to the preventive response induced by the vaccine, acting as sentinel, which can lead to the interference of metastatic processes. These findings demonstrate that the DC vaccine controls the pro-inflammatory environment, promoting the increase of cytotoxic T cells and regulatory T lymphocytes and increases the production of IL-10 and IL-17, simultaneously. However, additional research is needed in order to comprehend these mechanisms and their relationships.
The literature has mentioned the DC vaccine as a mechanism to control cancer recurrence and as an enhancer of the immune response. Therefore, these data provide evidence of timely activation of immune surveillance in the absence of tumor burden, as well as new perspectives about the immunotherapies scenario. Besides, it can be associated in the future with the study of control metastases cancer.
Acknowledgements
The Authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico, the Fundação de Amparo à Pesquisa do Estado de Minas Gerais and the Fundação de Ensino e Pesquisa de Uberaba.
Footnotes
Authors’ Contributions
Conception and design of the study: Jéssica F. Vieira, Eddie F. C. Murta and Márcia A. Michelin. Acquisition of data: Jéssica F. Vieira, Ana P. Peixoto and Márcia A. Michelin. Analysis and interpretation of data: Jéssica F. Vieira and Márcia A. Michelin. All authors were involved in drafting or revising manuscript. All Authors have approved the final article.
Conflicts of Interest
The Authors declare no potential conflicts of interest.
- Received March 10, 2021.
- Revision received May 3, 2021.
- Accepted May 21, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.