Adaptation to Volumetric Compression Drives an Apoptosis-Resistant and Invasive Phenotype in Liver Cancer

Physical constraints like compression influence cancer cell invasion and transcriptional dynamics in various tumors. Liver cancer is characterized by the rapid proliferation of tumor cells within a densely packed tissue matrix, subjecting the cancer cells to crowding and compression. The highly dysregulated mechanical environment highlights the need to elucidate the broader impact of compression on liver cancer development and evolution. In this study, we investigated and described a unique adaptive response of liver cells to prolonged compression. Liver cells presented significant transcriptional changes due to compression, including the loss of liver-specific markers and enrichment of epithelial-to-mesenchymal transition genes. Compression elevated Rac1 activity, which promoted cellular protrusions and YAP nuclear translocation and maintained cell viability under mechanical stress. Furthermore, compression disrupted intracellular calcium signaling, leading to resistance to apoptosis. Counteracting the effects of compression by inhibiting Rac1 or manipulating intracellular calcium facilitated death of compression-adapted cells. This study highlights compression as a critical biophysical signal in the tissue microenvironment that can induce cell state transitions and disease-driving phenotypes in the liver. Overall design: We cultured HepG2 cells on 2D culture plates with FBS supplemented DMEM. We used 4% (v/v) polyethylene glycol 300 (PEG300)-induced hyperosmolarity in the medium to compress cells for five days. The control group is the cell population cultured in the regular, isotonic medium. RNA-seq profiling of liver cancer cell lines HepG2 and Hep3B cultured on 2D plates (or transwell membranes) in the regular isotonic culture medium, under osmotic compression induced by 4% or 3%polyethylene glycol (PEG 300) in culture medium, and under mechanical compression induced by weights applied on cells. For HepG2, conditions are Isotonic on dish, 4% PEG on dish, non-compressed on transmembranes, and mechanically compressed on transmembranes. For Hep3B, conditions are non-compressed, mechanically compressed, and 3% PEG (all on transmembranes).