*2.5. MDA-MB-231/THP-1 Co-Culture Model*

The co-culture of MDA-MB-231 cells and THP-1 macrophages was performed using a Transwell system (Corning, NY, USA), utilizing both 6-well and 24-well plates. THP-1 cells, previously differentiated into macrophages, were introduced into the upper chamber of the Transwell insert, which had a pore size of 0.4 μm. On the other hand, MDA-MB-231 cells were added to the lower chamber of the Transwell system. The cells added to their respective compartments were cultured and allowed to attach for a period of 24 h at 37 ◦C within a humidified 5% CO2 incubator (Sanyo). Following the attachment phase, mistletoe water extract at various concentrations was introduced, and the co-culture was continued for an additional 24 and 48 h. Subsequently, the MDA-MB-231 cells that were attached to the lower chamber were recovered, and the supernatants were collected for use in the subsequent experimental procedures. A schematic representation of the co-culture of MDA-MB-231 and M0 macrophages is provided in Figure 1.

**Figure 1.** MDA-MB-231/THP-1 M0 macrophage co-culture model using Transwell system. THP-1 monocytes were differentiated with PMA (10 ng/mL) for 48 h before being plated in the upper insert. MDA-MB-231 cells were plated in the bottom chamber. Subsequently, both cell types were incubated in the presence or absence of mistletoe water extracts.

#### *2.6. Wound Healing Assay*

MDA-MB-231 cells were plated in a 24-well plate at a concentration of 2 × <sup>10</sup><sup>5</sup> cells/mL. They were then allowed to culture for 24 h, reaching a cell density of approximately 90%. Following this incubation period, a gap of a certain size was created by gently scratching the central portion of the cell monolayer on the plate using a sterile 200 μL pipette tip. The scratched monolayer was subsequently washed twice with phosphate-buffered saline (PBS). After the creation of the scratch, various concentrations of mistletoe water extract were added to the wells, and the cells were further cultured for 24 and 48 h. During this time, the extent of cell movement into the scratched area was observed and documented using an optical microscope (Nikon, Tokyo, Japan). The resulting gaps caused by the migration of cells were then quantified using Image J software (version 1.53e, National Institutes of Health, Bethesda, MD, USA).

#### *2.7. Migration Assay*

To assess the migration of MDA-MB-231 cells, a 24-Transwell plate with an 8.0 μm pore size was employed. Initially, M0 macrophages or RPMI with 10% FBS were introduced into the Transwell chamber and incubated for 24 h at 37 ◦C in a 5% CO2 incubator (Sanyo). The mistletoe water extract was diluted with serum-free RPMI, and 100 μL of the diluted extract was added to the Transwell insert. MDA-MB-231 cells were suspended in the serum-free RPMI 1640 medium and placed in the Transwell insert, where they were incubated for either 24 or 48 h. For the visualization of cells that migrated through the filter and reached the bottom surface of the insert, Giemsa staining was employed. Briefly, the residual medium within the insert was removed, and the insert was washed twice with PBS (pH 7.4). The cells were fixed at room temperature for 2 min using 3.7% formaldehyde. After two washes with PBS to remove the formaldehyde, 100% methanol was added and incubated for 20 min at room temperature. Following this, the insert was washed twice with PBS, and a 0.4% Giemsa solution (Sigma-Aldrich) was added. The insert was further incubated for 15 min at room temperature. After removing excess reagents with PBS, the cells on the top surface of the insert were gently wiped away using a cotton swab. The insert was then dried, and an optical microscope (Nikon) was used to count the number of cells that had migrated.

#### *2.8. ELISA*

The quantification of secreted IL-6 from MDA-MB-231 cells and THP-1 M0 macrophages was performed using an ELISA kit (BD Biosciences), following the provided instructions. Initially, 100 μL of the capture antibody diluted in a coating buffer

(0.1 M sodium carbonate, pH 9.0) was added to each well of a 96-well EIA/RIA plate (Corning), and the plate was incubated at 4 ◦C for 24 h. Subsequently, the plate was washed three times with a washing buffer containing PBS and 0.05% Tween-20. Then, a blocking buffer consisting of PBS with 1% bovine serum albumin (BSA) was added to the wells and allowed to incubate at room temperature for 1 h. After another three washes, the cell supernatant and the standard solutions were added to the wells and incubated at room temperature for 2 h. After an additional five washes with the washing buffer, the detection antibody was added to the wells, and the plate was incubated in darkness for 1 h. Following this, equal volumes of 3,3- ,5,5- -Tetramethylbiphenyl-4,4- -diamine (TMB) Solution (0.4 g/L, Thermo Scientific, Cat#34021) and Peroxide Solution (0.02% hydrogen peroxide in citric acid buffer, Thermo Scientific, Cat#34021) were mixed, and 100 μL was added to each well; the plate was once again placed in darkness for an incubation of 30 min. After this incubation, 100 μL of 2 M sulfuric acid was introduced into the wells to stop the reaction, and the absorbance was measured at a wavelength of 450 nm.
