This study adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for the reporting of experimental results. Female BALB/c mice (6–8 weeks old) were purchased from the Mossakowski Medical Research Centre of the Polish Academy of Sciences (Warsaw, Poland) and allowed to acclimatize for 14 days before experimentation. Animals were housed individually in polycarbonate cages within a controlled environment. The room was maintained at a constant relative humidity of 50 ± 10% and a temperature of 24 ± 1 °C, with a 12-hour light-dark cycle, where lights were turned on at 7:00 a.m. Food and water were provided ad libitum. All procedures were approved by the Local Bioethical Committee for Animal Care in Bydgoszcz, Poland (permission no. LKE 50/2022). This research was conducted on four groups of mice: untreated (NT), endotoxin-tolerant (ET), breast cancer-bearing (BC), and endotoxin tolerant breast cancer-bearing (ETBC). Figure 1 presents a diagram illustrates the procedure conducted for each group of animals.

Lipopolysaccharide (LPS) from Escherichia coli (0111:B4, Merck, Darmstadt, Germany) was prepared by dissolving LPS in sterile 0.9% sodium chloride. Prior to injection, the stock solution of LPS (2 mg/mL) was warmed to 37 °C, then diluted in warm sterile saline to the desired concentration. To induce ET, mice received daily intraperitoneal (i.p.) injections of LPS (50 μg/kg) four consecutive doses (Figure 1). ET development was assessed by measuring T_b of the mice using biotelemetry.

On the day of the third LPS injection, mice were inoculated subcutaneously (s.c.) with 2.5 × 10⁴ 4T1 breast cancer cells on the right flank (Figure 1). Before the experiments, 4T1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 IU/mL penicillin (all from Merck, Darmstadt, Germany). The cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere. The mice were monitored daily for tumor growth. After approximately four weeks, the mice were euthanized with an overdose of ketamine/xylazine (240 mg/kg and 30 mg/kg, respectively). Blood, tumor, and spleen samples were collected. Tumor and spleen samples were rinsed twice in sterile ice-cold PBS and immediately flash-frozen in liquid nitrogen (Figure 1). These tissue samples were stored at -80 °C for subsequent analysis.

Blood samples were collected through cardiac puncture in EDTA-treated tubes from mice anesthetized with an overdose of ketamine/xylazine (240 mg/kg and 30 mg/kg, respectively). Total blood cell counts of leukocytes, lymphocytes, monocytes, and granulocytes were evaluated using the Auto Hematology Analyzer BC-2800Vet (Mindray, Shenzhen, China).

Spleens were collected following euthanasia of the mice using an overdose of ketamine and xylazine mixture. Immediately after removal, the spleens were weighed using a highly sensitive scale to ensure precise measurement.

Mouse mammary tumor tissues were assessed by pathologists. Tumor tissues were fixed in 10% neutral-buffered formalin for 24 h. Tissue samples were then routinely processed for histopathology and embedded in paraffin wax (FFPE). Paraffin sections of 5 μm thickness were stained for histology with Mayer’s hematoxylin and eosin (H&E). All images were captured using a Nikon Eclipse E800 microscope.

Total RNA extraction from spleen and tumor tissues was performed by lysing them in PureZOL™ RNA Isolation Reagent with mechanical disruption, according to the manufacturer’s protocol. Then, cDNA was obtained using 1 μg of total RNA and the iScript™ cDNA Synthesis Kit, following the manufacturer’s protocol. RT-qPCR was performed in a final volume of 10 μL using SsoAdvanced Universal SYBR^® Green Supermix and PrimePCR™ SYBR^® Green Assays. Table 1 lists the unique assay IDs of the immune-related gene primers. The test samples were analyzed in triplicate using a CFX Connect Real-Time PCR Detection System. The specificity was verified through melt curve analysis. Standard curves were generated for both target and reference genes (actin). Calibrator-normalized quantification was performed using the CFX Manager Software 3.1. Each reaction was performed at least twice. All reagents and software used to analyze cytokine expression were purchased from Bio-Rad (Hercules, CA, USA).

The survival rates of the mice in the BC and ETBC groups were compared. The Kaplan-Meier plot was used to assess the differences, and statistical analysis between these groups was performed using the log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. This analysis was performed using data obtained from an independent experimental set.

Tumor samples obtained from mice with BC and ETBC were measured using Vernier calipers. The tumor volume was calculated using the following formula:

where L represents the average tumor length and W represents the average width of the tumors. A comparison analysis between the BC and ETBC mice groups was conducted using nonlinear regression analysis.

The relative gene expression levels were evaluated and compared using the 2^−ΔΔCt method. Multiple group comparisons were performed using one-way analysis of variance (ANOVA), followed by the Sidak test. An unpaired t-test was used to evaluate gene expression between the two groups. For the evaluation of tumor growth rate, nonlinear regression with an exponential growth model was used. Tumor volume was calculated as (π/6) × width × length. Measurements were initiated once tumors were measurable in all mice, and follow-up measurements were taken after a few days to capture clear differences in growth between groups. The number of time points was limited to capture meaningful differences in tumor growth. GraphPad Prism version 8 software (GraphPad Software Inc., La Jolla, CA, USA) was used for calculations, analysis, and visualization of results. Statistical significance was set at p < 0.05.

Mice are nocturnal animals with low daytime and high nighttime T_b. Using four consecutive doses of LPS, we observed a progressive change in response to LPS. The first injection of LPS induced fever, which started within 1 h of LPS injection. The occasional transient increase in the T_b of mice at 9 a.m. (injection time) was mainly caused by stress related to the injection and handling of the mice. Fever onset was achieved and maintained for approximately one hour, with the highest body temperature reaching 38.02 ± 0.33 °C, compared to 36.72 ± 0.21 °C in NT mice (p < 0.001). This was followed by a fever duration of roughly 1.5 hours before the gradual return of body temperature to baseline levels. Repeated daily injections of LPS over several days resulted in progressive attenuation of the febrile response, culminating in the establishment of the ET model in mice. By the fourth dose of LPS, the maximum temperature in the treated group was 37.03 ± 0.33 °C, compared to 38.02 ± 0.33 °C following the first dose of LPS (p < 0.001) (Figure 2A).

Another important aspect of ET is sickness behavior, such as a decrease in motor activity. Indeed, a significant reduction in locomotor activity was observed in mice exhibiting ET compared to that in NT animals. The average motor activity value recorded was 2.66 ± 0.67 counts in ET mice and 2.6 ± 0.52 counts in LPS mice when compared to 5.14 ± 1.37 counts in NT mice after the LPS injection (p < 0.05) (Figure 2B). The reduction in activity, typically associated with fever, was evident in LPS-treated mice and persisted throughout the development of ET.

The absence of a systemic febrile response, a hallmark of ET, indicates successful immune reprogramming, thereby validating the robustness of this approach. In subsequent experiments, breast cancer was induced in ET mice, and its progression was compared to that in non-endotoxin-tolerant mice.

Six days after 4T1 cell injection, palpable breast tumors were observed in mice. These tumors were allowed to develop in the mice until the study’s conclusion on day 28; subsequent histological analysis showed solid sheets of proliferating epithelial cells, polygonal to oval-shaped, with poorly demarcated margins and scant cytoplasm. Nuclei are vesicular, with coarse chromatin, and a single central basophilic nucleus. There is a very fine fibrovascular connective tissue that rarely subdivides the neoplasm into lobules. Tubular differentiation was not observed, and the neoplastic cells were pleomorphic with numerous mitoses. The majority of the ETBC mice were consistent with solid carcinoma. Both the ETBC and BC groups showed numerous cells with large and small vacuoles in the cytoplasm and nucleus, which are often located at the periphery of the cell. These results are consistent with those of lipid-rich carcinoma (Figures 3A, B). Necrotic areas were present in all samples in different amounts and were surrounded by a small number of macrophages and very few neutrophils (Figures 3C–F).

Having established a stable model of ET manifested by the absence of fever, we studied its role in modulating tumor progression and overall survival outcomes in an experimental model of breast cancer. We observed a difference in the survival capacity of ETBC mice compared to that of BC group animals (Figure 4A). Although the difference was not statistically significant (p = 0.0710), mice with ETBC reached an advanced stage of the experiment more rapidly. Notably, after reaching the advanced stage of cancer, their condition deteriorated at a faster rate than that in BC mice (data not shown). None of these mice allowed for the continuation of observation beyond 40 days from the induction of breast cancer. In contrast, BC mice exhibited slower disease progression, and their condition allowed observation for up to 45 days (Figure 4A).

Mice in the ETBC group exhibited a more rapid increase in tumor volume, indicating that ET may enhance tumor progression. This accelerated tumor growth was accompanied by an earlier onset of advanced disease stages, which required humane termination of the experiment. In contrast, tumor progression in the BC group was slower, with some mice maintaining stable conditions for a longer duration. The following data were derived from three time points recorded during our observational study, with the final measurement being day 28. The tumor volume of the BC group on day 22 was observed to be 18.4 ± 1.8 mm³ and it reached 33 ± 2.8 mm³ by day 28. On the other hand, the mean tumor size in the ETBC group was 40.1 ± 7.5 mm³ on day 22, which then reached 67.4 ± 21.4 mm³ by day 28. The comparison of nonlinear regression curves revealed a significant difference between the groups (p < 0.001) (Figure 4B). These findings suggest that ET can exacerbate tumor growth dynamics, potentially through alterations in the immune response.

To better understand how ET influences immune system function, white blood cell (WBC) analysis was conducted in four groups of mice. We observed that the general leukocyte counts in NT and ET mice were almost similar (p > 0.99), whereas a significant increase was observed in the BC and ETBC groups (p < 0.001) (Figure 5A). A detailed analysis of individual leukocyte populations revealed that this increase was observed across all measured leukocyte populations, including lymphocytes, monocytes, and granulocytes (Figures 5B–D, respectively). Interestingly, leukocyte counts in cancer-bearing mice were significantly lower in ETBC animals than in BC mice (p < 0.001).

The spleen is a major reservoir of immune cells and plays a crucial role in the regulation of immune response. We observed distinct differences in spleen size between groups, which further underscored the impact of ET on immune function. Spleen samples collected from BC mice were noticeably larger in size, indicating splenomegaly. Notably, spleens from ETBC animals weighed significantly less than those from the BC group (p < 0.01). Additionally, spleen weight in BC mice was significantly higher than that in the NT and ET groups (p < 0.01) (Figure 6).

The blood morphology results showed some abnormalities i.e., the elevated leukocyte count in both BC and ETBC groups (Figure 5); therefore, gene expression analysis of immune-related genes in the spleens was carried out. We examined the expression of proinflammatory genes, including IL-6 (Figure 7A), IL-1β (Figure 7B), COX-2 (Figure 7C), VEGF (Figure 7D), IFN-γ (Figure 7E), and NOS2 (Figure 7F). Additionally, we investigated the expression of genes associated with immune regulation or inflammation: IL-10 (Figure 7G), STAT6 (Figure 7H), and CSF-1 (Figure 7I). The results showed a significant decrease in the expression of IL-6 and IFN-γ in mice with ETBC compared primarily to that in BC animals (p < 0.001 and p < 0.01, respectively). However, compared to the ET group, the expression of IL-6 and IFN-γ in the ETBC group remained significantly increased (p < 0.001).

Moreover, an increase in the expression of NOS2 (p < 0.001), IL-1β (p < 0.001), STAT6 (p < 0.001), CSF1 (p < 0.001), and COX-2 (p < 0.001) was observed in the ETBC group, notably when compared to the BC group. The expression of NOS2, IL-1β, and COX-2 also remained significantly higher in the ETBC group than in the NT (p < 0.001) and ET groups (p < 0.05, p < 0.001, and p < 0.01, respectively). Additionally, IL-10 expression, though insignificant, has been observed to be higher in ETBC group when compared to NT group (p > 0.6) and significantly higher when compared to ET group (p < 0.01). Similarly, STAT6 and CSF-1 expression levels were significantly higher in the ETBC group than in the NT group (p < 0.001) (Figure 7).

Finally, we wanted to determine whether ET affected the expression of immune-related genes within the tumor itself. We analyzed the expression of IL-10, NOS2, IL-1β, VEGF, COX-2, CSF-1, CD206, and STAT6 (Figures 8A–H, respectively) in tumor tissues from BC and ETBC mice. We observed a significant increase in the expression of IL-10, NOS2, IL-1β, VEGF, and COX-2 in ETBC mice when compared to BC mice (p < 0.01, p < 0.001). Although not statistically significant, the expression of CSF-1, CD206, and STAT6 was higher in the ETBC group than in the BC group (Figure 8).