Introduction

According to GLOBOCAN estimates for breast cancer, over 2,296,840 new cases were diagnosed in 2022, making it the most prevalent and incident type of tumor among women worldwide [1]. Despite being the most common cancer among women worldwide, the treatment landscape has undergone significant changes in recent years. These changes can be attributed to various factors, including the emergence of molecular markers, advancements in surgical treatments, radiotherapy, chemotherapy, and hormone therapy. Moreover, the introduction of immunotherapy has provided a new perspective for women affected by this tumor type [2].

The use of immunotherapy in breast cancer treatment has been evolving, with research into its various applications steadily expanding. One significant advancement is the development of immune checkpoint inhibitors (ICIs), a form of immunotherapy that targets the programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) pathways. This breakthrough has transformed the treatment of solid tumors and demonstrated promising results in improving patient outcomes [3].

In the field of immunotherapy, dendritic cell (DC) therapy provides an alternative treatment option for patients who have not responded to conventional therapies. This approach leverages the unique ability of DCs to identify and eliminate neoplastic cells through immunological surveillance, which involves the coordination of innate and adaptive immunity, utilizing components such as CD8⁺ T lymphocytes, CD4⁺ Th1 lymphocytes, natural killer (NK) cells, DCs, and phagocytic cells (monocytes and macrophages). Together, these immune cells establish a targeted response specifically directed against neoplastic cells [3–7].

DCs, classified as professional antigen-presenting cells (APCs), can capture tumor antigens, process and present them through the major histocompatibility complex (MHC) classes I and II to CD8⁺ and CD4⁺ T lymphocytes, respectively. The presentation of tumor antigens to CD4⁺ T lymphocytes leads to their differentiation into Th1 lymphocytes, which secrete cytokines that influence other components of the immune system, including macrophages [8–11].

Macrophages, like Th1 and Th2 lymphocytes, exhibit two activation profiles: M1 and M2. M1 macrophages are polarized via the Th1 pathway through IFN-γ, leading to the secretion of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, IL-1, IL-6, IL-12), reactive oxygen species (ROS), and nitric oxide (NO), which are critical for inflammation and antitumor activity [10, 12]. Conversely, Th2 lymphocytes secrete IL-4 and IL-13, polarizing M2 macrophages to produce IL-10, IL-4, arginase, prostaglandin, TGF-β, and VEGF, mediators that support tumor progression [10].

In addition to cytokines, transcription factors significantly impact the tumor process. Although their expression was initially thought to be restricted to hematopoietic lymphoid lineages, the presence of FOXP3, GATA3, and T-bet has been observed in other cells, contributing to immunosuppression and tumor growth [6, 13], as has the modulation of immune-cell infiltration within the tumor microenvironment [14]. RORγT is the lineage-defining transcription factor of Th17 cells, driving IL-17 production [15], which in the breast tumor microenvironment acts predominantly as a pro-tumoral mediator, promoting tumor cell invasiveness [16]. Although most studies of DC-based immunotherapy in breast cancer have focused on the local tumor microenvironment, tumor progression in aggressive models such as 4T1 is shaped by the host’s broader inflammatory state, which extends beyond the tumor site. Myeloid cells, including monocytes and macrophages, are central to this systemic inflammatory compartment and can either support or restrain tumor growth depending on their activation profile. Characterizing how DC immunotherapy modulates these cells outside the tumor itself may therefore reveal effects that are not captured by intratumoral analysis alone. In this context, the present study evaluated the impact of DC-based immunotherapy on the inflammatory profile of CD14⁺ myeloid cells obtained from the peritoneal compartment of mice bearing 4T1-induced breast tumors, with the aim of clarifying how this therapeutic approach reshapes the systemic immune response rather than the tumor microenvironment alone.

Materials and methods

A total of 30 randomly selected 8-week-old female BALB/c wild-type mice were used in a 28-day experimental protocol (Figure 1). Animals were housed in plastic cages under a 12-hour light/dark cycle at 21 ± 3°C, with food and water provided ad libitum. Mice were randomly assigned to three experimental groups: the control group (healthy mice treated with 0.9% saline solution), the tumor group (mice inoculated with 4T1 breast tumor cells), and the tumor + DC group (mice inoculated with 4T1 cells and treated with DC-based immunotherapy). Each group consisted of 10 animals divided equally across two independent experimental replicates (n = 5 per group per replicate). An additional 10 mice were used exclusively for bone marrow extraction to generate DCs. Animals from different experimental groups were housed in separate cages to avoid cross-exposure. All animals were obtained from the Central Animal Facility of the Federal University of Triângulo Mineiro, were not subjected to genotyping, and were of the same genetic background and age-matched.

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Figure 1. Experimental design. BALB/c mice were divided into three groups: control (tumor-free), tumor (4T1 cell-induced), and tumor + DC (treated with dendritic cell-based immunotherapy). Bone marrow-derived DCs were generated in vitro and administered to the treatment group. Peritoneal lavage was performed and cells were analyzed by flow cytometry. Created in BioRender. Arantes, L. (2026) https://BioRender.com/hydw939.

At the end of the experimental period, animals were euthanized by an overdose of ketamine (50 mg/kg) and xylazine (15 mg/kg). Peritoneal lavage was then performed on all groups by injecting 5 mL of 0.9% saline solution into the peritoneal cavity to recover resident monocytes/macrophages.

Results

DC immunotherapy and longitudinal tumor volume assessment

Tumor growth monitoring revealed that mice treated with DC immunotherapy (tumor + DC) showed a trend toward lower tumor volume compared to untreated tumor-bearing mice (tumor group); however, this difference was not statistically significant. Two-way ANOVA showed a significant effect of time (F(6,53) = 3.00; P = 0.013), reflecting tumor growth, but no significant effect of treatment (F(1,53) = 0.74; P = 0.392) and no significant treatment × time interaction (F(6,53) = 0.12; P = 0.994); per-timepoint comparisons were also non-significant (all P > 0.27) (Figure 2). One animal in the tumor group died after day 10 and was analyzed using its available time points.

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Figure 2. Tumor volume progression in 4T1 breast cancer-bearing mice. Mice treated with dendritic cell immunotherapy (tumor + DC) showed a non-significant trend toward lower tumor volume compared to the untreated tumor group (two-way ANOVA, effect of treatment P = 0.392 (n = 5 per group). DC: dendritic cell.

Immunotherapy with DCs altered the cytokine profile

Immunotherapy with DCs altered the cytokine profile of CD14⁺ monocytes/macrophages obtained from the peritoneal wash after euthanasia, analyzed by flow cytometry immediately after collection. Cytokine expression was evaluated using the MFI. A significant reduction was observed in IL-12 (P < 0.0005), IL-17 (P < 0.0001), and TNF-α (P < 0.0001) in the tumor-treated group compared to the untreated group (Figure 3; Figure S2).

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Figure 3. Expression of pro-inflammatory cytokines. Median fluorescence intensity (MFI) of IL-12 (A), IL-17 (B), and TNF-α (C) in CD14⁺ monocytes/macrophages from peritoneal lavage. Tumor-bearing mice treated with DCs showed significantly decreased cytokine expression compared to the untreated tumor group (***: P < 0.001). DC: dendritic cell.

Immunotherapy with DCs reduced GATA3 and RORγT expression

In the same samples, the expression of the transcription factors GATA3 and RORγT was evaluated based on MFI values. A significant reduction in their expression was observed in the group treated with DC immunotherapy compared to the untreated tumor group (P < 0.0001) (Figure 4; Figure S3).

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Figure 4. Expression of transcription factors. MFI of RORγT (A) and GATA3 (B) in CD14⁺ cells. Treatment with DC immunotherapy resulted in significantly lower expression of these transcription factors compared to the untreated tumor group (****: P < 0.0001). DC: dendritic cell; MFI: median fluorescence intensity.

Discussion

After several years of decline, breast cancer mortality has shown an upward trend, currently occupying the second position among the leading causes of cancer-related deaths. This increase may be associated with the COVID-19 pandemic, which reduced screening exams crucial for early detection [20, 21]. Consequently, diagnoses were often made at more advanced stages, resulting in poorer clinical outcomes [21]. Given this scenario, studies using experimental models play a fundamental role in understanding advanced-stage breast cancer behavior and in developing new therapeutic approaches for patients who do not respond to conventional therapies [22]. ICIs have emerged as an alternative to conventional modalities such as chemotherapy, radiotherapy, and surgery, employing strategies focused on immune system modulation [23, 24].

However, despite advances in immunotherapy, some patients exhibit little to no response to ICIs. These non-responders present a significant challenge [25], making it essential to pursue alternative immunotherapies capable of modulating the immune system [26].

In this context, cellular therapies have gained prominence as a promising option for patients who do not respond to ICIs [27]. Among these, DC immunotherapy stands out for its role in modulating the immune system, and it can synergize with ICI treatment [28].

DC vaccines function by priming the immune system to recognize and attack tumor cells. The cells are previously induced to differentiate into DCs with an anti-tumor profile, and upon administration they stimulate the immune system to fight the tumor [29].

In this preclinical study conducted on BALB/c mice, we observe a significant reduction in IL-12, IL-17, and TNF-α levels in the tumor-treated group compared to the untreated group. The overall reduction of these cytokines is important considering the concept of the tumor escape mechanism associated with chronic inflammation [30]. High levels of IL-17A are associated with more aggressive and invasive breast cancer; the reduction of IL-17 may contribute to antitumor effects, as a decrease in this cytokine reduces associated processes such as angiogenesis, tumorigenesis, inhibition of apoptosis, and migration [16, 30, 31]. Although there was no statistically significant difference in tumor growth between the tumor-only group and the tumor + DC group, the immunological findings were robust. The immunological findings were substantial, providing insight into the systemic behavior of CD14⁺ cells from peritoneal lavage following DC immunotherapy. These findings corroborate those from other cell types and tissues that point to improved immune system performance when DC immunotherapy is used to prevent or treat 4T1-induced tumors [7, 8, 32].

Similarly to IL-17, TNF-α expression was reduced by DC-based immunotherapy. This reduction also contributes to decreased proliferation, angiogenesis, and migration, as these processes are partly regulated by TNF-α [16, 30, 31, 33].

The regulation of the immune system by DCs can also lead to reduced expression of IL-12, a cytokine classically associated with antitumor activity [34]. However, reduced expression does not necessarily indicate a compromised immune response [35, 36]; it may reflect attenuation of the local inflammatory milieu while systemic levels remain elevated [37]. In the present study, the peritoneal lavage of BALB/c mice was examined. We note that peritoneal cells represent a systemic/peritoneal compartment rather than the tumor microenvironment itself, and the cytokine findings should be interpreted accordingly. The reduction in IL-12, along with the decrease in other cytokines, highlights the importance of controlling chronic inflammation [38, 39], which in turn reduces tumor-induced immunosuppressive mechanisms and reflects the balance necessary for antitumor activity [39].

The critical balance between acute and chronic inflammation can also be illustrated by the transcription factors RORγT and GATA3, which showed a significant reduction in treated mice. RORγT is the lineage-defining transcription factor of Th17 cells, driving IL-17 production [15]. Although not classically associated with the myeloid compartment, it was deliberately assessed here, and its significant reduction in the CD14⁺ compartment following DC-based immunotherapy merits further investigation.

Similarly, GATA3 is a lineage-defining transcription factor of the T-cell/Th2 compartment [40] and is therefore not typically regarded as a monocyte factor; nonetheless, its expression was significantly reduced in the CD14⁺ population of the DC-treated group. This reduction in GATA3 expression may be associated with decreased macrophage polarization toward the M2 profile and lower production of cytokines involved in tumor progression, such as IL-4 and IL-13 [41]; however, this link remains associative, as macrophage polarization was not directly assessed here.

In addition to the flow-cytometry approach employed in this study, the incorporation of complementary methodologies in future research would facilitate a more comprehensive characterization of the systemic effects of DC-based immunotherapy. Specifically, the implementation of functional cellular assays and proteomic profiling of CD14⁺ myeloid cells holds potential in elucidating the cellular response to treatment at the functional and protein-expression levels, extending beyond the cytokine and transcription-factor signals that have been assessed in this study. Furthermore, the potential of spectral and hyperspectral imaging techniques, which have demonstrated efficacy in computer-aided detection of breast cancer [42], to provide complementary, label-free assessments of the host response warrants further investigation. A collaborative effort between these approaches would facilitate the delineation of the systemic immunomodulatory footprint of DC immunotherapy and the role of peripheral CD14⁺ cells in this response.