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. 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).

诸如压缩这类物理约束，会影响多种肿瘤中癌细胞的侵袭行为与转录动态变化。肝癌的特征表现为肿瘤细胞在致密的组织基质中快速增殖，这会使癌细胞受到挤压与拥挤压力。这种高度失调的力学微环境，凸显了阐明压缩对肝癌发生发展与演进过程更广泛影响的必要性。
本研究针对肝细胞在长期压缩作用下的独特适应性应答展开了探究与描述。肝细胞因压缩作用出现显著转录改变，包括丢失肝细胞特异性标志物，以及富集上皮间质转化（epithelial-to-mesenchymal transition）相关基因。压缩可提升Rac1活性，进而促进细胞突起形成与YAP核转位，并在机械应力下维持细胞存活能力。此外，压缩会干扰细胞内钙信号通路，使癌细胞产生凋亡抵抗能力。通过抑制Rac1活性或调控细胞内钙信号通路来抵消压缩的作用，可促使适应了压缩环境的细胞发生死亡。本研究表明，压缩是肿瘤组织微环境中的关键生物物理信号，可诱导肝细胞状态转变，并产生驱动肝脏疾病发生的表型。
我们在添加了胎牛血清（FBS）的达尔伯克改良伊格尔培养基（DMEM）的二维培养板中培养HepG2细胞。我们通过在培养基中添加4%（体积比）聚乙二醇300（PEG300）以营造高渗环境，对细胞进行为期5天的压缩处理。对照组为在常规等渗培养基中培养的细胞群。
本研究对肝癌细胞系HepG2与Hep3B开展RNA测序（RNA-seq）转录组分析，所涉样本包括：在常规等渗培养基中于二维培养板（或Transwell膜）培养的细胞；经培养基中4%或3%聚乙二醇（PEG300）诱导渗透压缩的细胞；以及通过施加重物实现机械压缩的细胞。针对HepG2细胞，实验分组包括：培养板上等渗培养组、培养板上4%PEG处理组、Transwell膜上非压缩组，以及Transwell膜上机械压缩组。针对Hep3B细胞，实验分组包括：Transwell膜上非压缩组、机械压缩组，以及3%PEG处理组（均在Transwell膜上培养）。