*3.1. Cell Viability*

Different concentrations of mistletoe extract were introduced to the MDA-MB-231 human triple-negative breast cancer cell line and THP-1 human mononuclear cells that had been differentiated into macrophages. Subsequently, the cells were incubated for either 24 h or 48 h, and their viability was assessed using the MTT assay (Figure 2). Through this analysis, we determined that THP-1 macrophages exhibited a survival rate of over 80% when treated with concentrations of 2 μg/mL or lower for both the 24 and 48 h groups. As a result, we selected two specific doses for further experimentation.

**Figure 2.** Cytotoxic effect of mistletoe water extracts in (**a**) MDA-MB-231 and (**b**) THP-1 cells for 24 and 48 h. Both cell lines were exposed to various concentrations of mistletoe water extracts. Cell viability was assessed using the MTT assay. Untreated control cell viability was set at 100%. Results represent the means ± standard deviation (S.D.) of triplicate experiments.

#### *3.2. Wound Healing and Migration Assay*

For the wound healing assay, we first cut an arbitrary wound into the plate, treated it with mistletoe water extract, then monitored cell movement towards the wounded area over time (Figure 3). As a result, 61.1 ± 3.4% and 74.8 ± 3.8% of the wound area were moved in the MDA-MB-231 cells treated with mistletoe at 0.1 μg/mL concentrations for 24 and 48 h, respectively, whereas the MDA-MB-231/THP-1 co-culture group was significantly inhibited by 38.2 ± 3.4% and 57.9 ± 4.4% at 24 and 48 h, respectively. In addition, 57.6 ± 3.3% and 68.9 ± 2.9% of the wound area were moved in the MDA-MB-231 group treated with 2 μg/mL mistletoe for 24 and 48 h, respectively, whereas the MDA-MB-231/THP-1 co-culture group was significantly inhibited by 36.7 ± 1.6% and 45.9 ± 4.8% at 24 and 48 h, respectively (Figure 3d). Using the Transwell migration assay method, the MDA-MB-231 or MDA-MB-231/THP-1 groups were added into the insert membranes with pore sizes that cells can penetrate, and the number of breast cancer cells moving through the membranes under mistletoe extract treatment was observed (Figure 4). As a result, when mistletoe at a concentration of 0.1 μg/mL was treated for 48 h, 71.5 ± 5.3% of cells migrated through the membrane in the MDA-MB-231 group compared to the control, whereas 60.0 ± 3.2% of cells migrated through the membrane in the MDA-MB-231/THP-1 co-culture group compared to the control. When 2 μg/mL mistletoe was added for 48 h, 64.4 ± 2.8% of cells migrated through the membrane in the MDA-MB-231 group compared to the control, whereas 48.3 ± 2.4% of cells migrated through the membrane in the MDA-MB-231/THP-1 co-culture group.

**Figure 3.** Effects of Korean mistletoe water extracts on wound healing assay in MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells. Following the creation of scratches on both cell groups, various concentrations of Korean mistletoe water extracts (**a**) 0, (**b**) 0.1, and (**c**) 2 μg/mL were applied immediately. Representative images from the wound healing assay were captured at 0, 24, and 48 h post-treatment. The closure of the wound area was observed under a light microscope (×40). Additionally, (**d**) a quantitative bar graph of the closed wound area was generated using an image analysis software (Image J). The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.001).

**Figure 4.** Effect of Korean mistletoe water extracts on Transwell migration assay in (**a**) MDA-MB-231 and (**b**) MDA-MB-231/THP-1 co-cultured cells. Mistletoe water extracts were applied at various concentrations (0, 0.1, and 2 μg/mL) for 24 and 48 h. Migrated cells were observed using a light microscope (magnification, ×100; scale bar, 100 μm). The number of migrated cells was quantified using Image J software. Scale bars: 20 μm. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\* *p* < 0.01, \* *p* < 0.001, ns: not significant).

#### *3.3. Secretion of Inflammatory Cytokines*

A quantitative and qualitative study was conducted by ELISA and cytokines array to investigate if Korean mistletoe water extract may modulate the release of inflammationrelated cytokines produced by triple-negative breast cancer cells by activating macrophages. Initially, MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells underwent treatment with or without 100 ng/mL lipopolysaccharides (LPS); this process served to induce the expression of inflammatory cytokines in MDA-MD-231 cells. Following treatment with 0.1 and 2 μg/mL concentrations of Korean mistletoe water extracts for a period of 48 h, MDA-MB-231 cells, THP-1 macrophages, and MDA-MB-231/THP-1 co-cultured cells underwent testing to quantify IL-6 expression through ELISA. Consequently, when cells were not stimulated with LPS, MDA-MB-231 cells secreted 1501.5 ± 110.6 pg/mL and MDA-MB231/THP-1 co-cultured cells secreted 1418.5 ± 80.4 pg/mL of IL-6. However, after exposure to 100 ng/mL LPS, MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells showed an elevated IL-6 expression of to1987.6 ± 65.7 pg/mL and 1847.9 ± 120.5 pg/mL, respectively. There was no significant difference in IL-6 expression between the MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells. Following this, when 0.1 μg/mL mistletoe water extract was administered to MDA-MB-231 cells, we confirmed an IL-6 secretion level of 2053.1 ± 77.9 pg/mL, whereas MBA-MB-231/THP-1 co-cultured cells showed an IL-6 secretion level of 1503.2 ± 89.8 pg/mL. Additionally, treatment of MDA-MB-231 cells with 2 μg/mL mistletoe water extract resulted in an IL-6 secretion of 1993.1 ± 71.6 pg/mL. However, in MDA-MB-231/THP-1 co-cultured cells, the expression of IL-6 was significantly inhibited to 1447.6 ± 74.5 pg/mL (Figure 5). Moreover, through qualitative analysis aimed at determining the regulation of cytokine expression, it was observed that there was an increase in the expression levels of IL-4, TGF-β, and IFN-γ in MDA-MB-231/THP -1 co-cultured cells compared to the MDA-MB-231 group (Figure 6).

**Figure 5.** Effects of mistletoe water extracts on pro-inflammatory cytokine (IL-6) production in LPS-stimulated MDA-MB-231 and MDA-MDA-MB-231/THP-1 co-cultured cells. Cells were treated with or without various concentrations of mistletoe water extracts (0, 0.1, and 2 μg/mL) and LPS (100 ng/mL) for 48 h. Cell supernatant was collected and assessed using an ELISA kit. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \*\* *p* < 0.01, ns: not significant).

**Figure 6.** Effects of Korean mistletoe water extracts on (**a**) IL-4, (**b**) TGF-β, and (**c**) IFN-γ secretion in MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells. Both cell groups were treated with or without mistletoe water extracts (0 or 2 μg/mL) and LPS (100 ng/mL) for 48 h. Cell supernatants were then collected and qualitative relative profiling of cytokine levels was measured using the Multi-Analyte ELISArray kit according to the manufacturer's protocol. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \* *p* < 0.001, ns: not significant).

#### *3.4. Expression of Apoptosis-Related Proteins*

The apoptosis-related protein expressions were assessed in triple-negative breast cancer cells upon administration of Korean mistletoe extract. Mistletoe water extract was administered to MDA-MB-231 cells and MDA-MB-231 cells co-cultured with THP-1 cells for a duration of 48 h, followed by conducting Western blot and immunofluorescence assays. Western blot analysis measured the ratio of Bax and Bcl-2 proteins, which are proteins that induce MOMP in mitochondrial outer membranes. When treated with 0.1 μg/mL mistletoe extract, MBA-MB-231 cells and MBA-MB-231/THP-1 co-cultured cells showed no significant difference in Bax/Bcl-2 protein ratio, compared to the controls. However, when treated with 2 μg/mL mistletoe extract, the MDA-MB-231/THP-1 co-culture group showed a protein ratio of 2.1 ± 0.1 times (Figure 7c). To determine if MOMP induced the activation of caspase-3 protein to cleaved caspase-3, we measured the ratio of cleaved caspase-3 and caspase-3 proteins via Western blot. Upon treatment with 0.1 μg/mL Korean mistletoe water extract, no significant differences were shown in either group when compared to the control. However, when treated with 2 μg/mL mistletoe extract, MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells showed a cleaved caspase-3/caspase-3 protein ratio of 1.6 ± 0.1 and 2.7 ± 0.1 times, respectively (Figure 7d). Therefore, the ratio of cleaved caspase-3/caspase-3 protein was significantly increased in MDA-MB-231 cells co-cultured with THP-1 cells compared to single-cultured MDA-MB-231 cells. We also assessed the influence of cleaved caspase-3 on PARP protein deactivation through its transformation into cleaved PARP. As a result, when treated with 0.1 μg/mL mistletoe extract, the expression of cleaved PARP protein was 0.9 ± 0.1 times and 1.2 ± 0.1 times that of the control in MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells, respectively. In addition, when treated with 2 μg/mL mistletoe extract, the expression of cleaved PARP protein was 0.9 ± 0.2 times and 1.4 ± 0.2 times that of the control in MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells, respectively (Figure 7e). The immunofluorescence staining confirmed the expression level of apoptosis proteins Bax, cleaved caspase-3, and cleaved PARP; interestingly, the protein expression of Bax and cleaved-caspase-3 increased dose-dependently in MDA-MB-231/THP-1 co-cultured cells (Figure 8a,b). However, there were no significant differences in cleaved PARP expression in both MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells, compared to the controls (Figure 8c).

### *3.5. Inhibition of STAT3 Activation*

Unregulated STAT3 protein activation is frequently seen in cancer cells, and it is involved in cell proliferation and differentiation, which leads to tumor malignancy [52]. Therefore, to confirm whether Korean mistletoe extract can inhibit STAT3 protein activation in triple-negative breast cancer cells in the breast cancer microenvironment, Western blot assay was performed by treating MDA-MB-231 cells and MDA-MB-231/THP-1 co-cultured cells for 48 h with 0.1 and 2 μg/mL of Korean mistletoe extract (Figure 9a,b). The ratio of the expression of the total STAT3 protein and the phosphorylated and activated p-STAT3 protein was calculated and compared. When 0.1 μg/mL mistletoe was treated, p-STAT3/STAT3 protein was expressed 0.9 ± 0.1 times and 1.13 ± 0.1 times more than the control in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, and no significant difference was observed between the two groups. However, when 2 μg/mL mistletoe was treated, p-STAT3/STAT3 protein was expressed 1.2 ± 0.1 times and 0.7 ± 0.1 times in the MDA-MB-231 and MDA-MB-231/THP-1 co-culture group, respectively, showing a significant difference in inhibiting the activation of the STAT3 protein (Figure 9c).

(e)

**Figure 7.** Effects of Korean mistletoe water extracts on apoptosis-related protein expression in (**a**) MDA-MDA-231 and (**b**) MDA-MB-231/THP-1 co-cultured cells using Western blot analysis. Both cell groups were treated with mistletoe water extracts (0, 0.1 and 2 μg/mL) for 48 h, and their relative protein expression levels (**c**) Bax/Bcl-2, (**d**) cleaved caspase-3/caspase-3, and (**e**) cleaved PARP were quantified and presented using a bar graph. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.001, ns: not significant).

**Figure 8.** Representative images and quantitative bar graph of pro-apoptosis proteins using immunofluorescence staining. MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells were treated with various concentrations of Korean mistletoe water extracts (0, 0.1, and 2 μg/mL) for 48 h. The expressed proteins (**a**) Bax, (**b**) cleaved caspase-3, and (**c**) cleaved PARP were labeled with Alexa Fluor™ 488 (green), while nuclear DNA was counterstained with DAPI (blue) then observed using fluorescence microscope (magnification ×200). Fluorescence intensity was quantified using Image J software. Scale bars: 20 μm. The results are represented as the mean ± standard deviation (S.D.). The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.001, ns: not significant).

(c)

**Figure 9.** Inhibitory effects of Korean mistletoe water extract on STAT3 activation in (**a**) MDA-MB-231 and (**b**) MDA-MB-231/THP-1 co-cultured cells. After treatment with Korean mistletoe water extracts at concentrations of 0, 0.1, and 2 μg/mL for 48 h, Western blot analysis was conducted. (**c**) The quantification bar graph represents the ratio of p-STAT3/STAT3 protein expression. Each bar represents the mean ± standard deviation (S.D.) of experiments performed in triplicates. The *p*-value indicates significant differences between MDA-MB-231 and MDA-MB-231/THP-1 co-cultured cells (\*\* *p* < 0.01, ns: not significant).
