Korean mistletoe (Viscum album L. var. coloratum (Kom.) Ohwi) water extract inhibits viability and matrix invasion of MDA-MB-231 triple-negative breast cancer spheroids in three-dimensional culture

Mistletoe (Viscum album L.) preparations are used clinically as complementary anticancer agents in Europe, yet most mechanistic work relies on lectin-enriched fractions and two-dimensional monolayer culture, which poorly reproduces drug penetration, hypoxic gradients and matrix-dependent invasion. Here we evaluated a crude water extract of Korean mistletoe (Viscum album L. var. coloratum (Kom.) Ohwi, syn. V. coloratum (Kom.) Nakai) in a three-dimensional spheroid model of MDA-MB-231 triple-negative breast cancer cells. The extract was prepared as described previously (Sci. Pharm. 2023;91:48) from dried mistletoe collected on oak trees in Gangwon-do, Korea (voucher VCA101), by threefold aqueous extraction at 4 °C and freeze-drying; it was not chemically characterised in this study. Spheroids were formed in basement-membrane extract and characterised from Day 0 to Day 7, compacting to 42.6 ± 0.4 % of Day 0 projected area by Day 1 and growing to 170.0 ± 4.5 % by Day 7. Viability after five days of treatment, measured by a water-soluble tetrazolium assay, fell dose-dependently from 96.8 ± 4.9 % at 78 µg/mL to 13.3 ± 5.6 % at 2500 µg/mL, giving a half-maximal inhibitory concentration of 504.6 ± 42.2 µg/mL and a twenty-percent inhibitory concentration of 237.5 ± 45.5 µg/mL by four-parameter logistic regression. Acridine orange/propidium iodide staining showed a concentration-dependent rise in propidium-iodide-positive signal. Invasion into basement-membrane extract, quantified as invaded area, did not differ between groups at Day 1 (p > 0.6). By Day 2 invaded area was reduced to 64.6 % and 48.2 % of the untreated control at the twenty-percent and half-maximal inhibitory concentrations respectively, and to 81.0 % and 72.1 % at Day 3; the two concentrations differed significantly at both time points. Korean mistletoe water extract therefore inhibits both viability and matrix invasion of triple-negative breast cancer spheroids, supporting further mechanistic study.