Introduction. Tumor hypoxia is a major driver of cancer progression and therapy resistance. However, its cellular consequences can vary with metastatic potential (MP) of tumor cells. We compared the effects of chemically induced hypoxia on cell death and morphology of breast cancer lines MCF-7 and MDA-MB-231, having low and high MP, respectively.

Methods. Cells were exposed to CoCl2 (100μM, 20h) and analyzed immediately (T0) and 24h later (T1). Necroptosis was quantified by propidium iodide imaging (≥10 fields, three wells per condition). Morphometric parameters (area, circularity, aspect ratio, solidity, eccentricity, roundness) were extracted from at least 100 cells/condition. HIF-1α expression was assessed by immunofluorescence following fixation, permeabilization, and Hoechst counterstaining.

Results. In both untreated groups, cell death increased from 1±1% (T0) to 16±4% at T1. Under hypoxia, low-MP cells exhibited 33±5%, whereas high-MP cells showed only 4±1% necroptotic cells at T1. In non-treated high-MP cells, HIF-1α expression were higher than in low-MP cells and further increased after CoCl2 treatment. Morphology of low-MP cells was unaffected, while high-MP cells displayed increased circularity and roundness with reduced eccentricity (p<0.05) after CoCl2 treatment. At T1, CoCl2 treatment further decreased area, perimeter, and aspect ratio of high-MP cells, indicating a shift toward less elongated and more spindle-like forms compared with normoxia.

Conclusions. Chemically induced hypoxia elicits distinct subtype-specific responses: low-MP cells undergo necroptosis, whereas high-MP cells are less prone to this type of death exhibiting mesenchymal-like morphological traits. The combined use of HIF-1α detection and quantitative morphological analysis enables a thorough examination of the diverse responses to hypoxia.