High-nitrogen artificial gas mixture-induced hypoxia decreases nanoparticle uptake in breast tumor cells with varying malignancy

Introduction. The rapid and disorganized growth of tumors along with poor blood supply generates a hypoxic environment, causing cancer cells to adapt through HIF-1 signaling, which drives glycolysis (Warburg effect), stemness, and increased invasion. Previously, we demonstrated that nanoparticle (NP) uptake correlates with metastatic potential (MP) of breast cancer (BC) cells. Here, we investigated the high-nitrogen artificial gas mixture-induced hypoxia effect on NP uptake in non-tumorigenic mammary epithelial and BC cell lines.

Materials and methods. Breast cancer MDA-MB-231 (high-MP), MCF-7 (low-MP) and non-tumorigenic mammary epithelial MCF-10A cell lines were cultured under normoxic conditions (21% O₂, 5% CO2, 74% N₂) in a standard cell incubator and then subjected to 4 h of artificial gas mixture hypoxic conditions (5% CO₂, 5% O₂, 90% N₂) in specially designed camera. Cells were subsequently incubated with 200 nm carboxylated fluorescent NPs for 1h, then washed with PBS and trypsin and fixed with 4% paraformaldehyde. Nuclei were stained with Hoechst. NP uptake was assessed by high content fluorescence microscopy, and Pearson correlation coefficients of NP colocalization with nucleus signals were calculated. Statistical significance between groups was evaluated using ANOVA.

Results. As expected, under all tested conditions, uptake of NPs was significantly higher in BC cells with high MP compared with low MP (by 1.64 times) and non-tumorigenic mammary epithelial cells (by 7.6 times). Gas mixture-induced hypoxia led to a reduction in nanoparticle uptake by 1.27-fold in cancer cells and by 3-fold in non-tumorigenic cells (p<0.03).

Conclusions. High-nitrogen artificial gas mixture-induced hypoxia decreases nanoparticle uptake, indicating a possible reduction in cells’ metastatic potential. The magnitude of this effect depends on cell malignancy: non-tumorigenic cells demonstrate the highest uptake reduction, whereas both cancer cell lines exhibit lower reductions. This differential response can indicate that increased hypoxia tolerance may represent an adaptive advantage in BC cells.