An exploratory study of cell stiffness as a mechanical label-free biomarker across multiple musculoskeletal sarcoma...

Cancer cells are characterized by changes in cell cytoskeletal architecture and stiffness. Despite advances in understanding the molecular mechanisms of musculoskeletal cancers, the corresponding cellular mechanical properties remain largely unexplored. The aim of this study was to investigate the changes in cellular stiffness and the associated cytoskeleton configuration alterations in various musculoskeletal cancer cells.

Cell lines from five main sarcoma types of the musculoskeletal system (chondrosarcoma, osteosarcoma, Ewing sarcoma, fibrosarcoma and rhabdomyosarcoma) as well as their healthy cell counterparts (chondrocytes, osteoblasts, mesenchymal stem cells, fibroblasts, skeletal muscle cells) were subjected to cell stiffness measurements via atomic force microscopy (AFM). Biochemical and structural changes of the cytoskeleton (F-actin, β-tubulin and actin-related protein 2/3) were assessed by means of fluorescence labelling, ELISA and qPCR.

While AFM stiffness measurements showed that the majority of cancer cells (osteosarcoma, Ewing sarcoma, fibrosarcoma and rhabdomyosarcoma) were significantly less stiff than their corresponding non-malignant counterparts (p < 0.001), the chondrosarcoma cells were significant stiffer than the chondrocytes (p < 0.001). Microscopically, the distribution of F-actin differed between malignant entities and healthy counterparts: the organisation in well aligned stress fibers was disrupted in cancer cell lines and the proteins was mainly concentrated at the periphery of the cell, whereas β-tubulin had a predominantly perinuclear localization. While the F-actin content was lower in cancer cells, particularly Ewing sarcoma (p = 0.018) and Fibrosarcoma (p = 0.023), this effect was even more pronounced in the case of β-tubulin for all cancer-healthy cell duos. Interestingly, chondrosarcoma cells were characterized by a significant upregulation of β-tubulin gene expression (p = 0.005) and protein amount (p = 0.032).

Modifications in cellular stiffness, along with structural and compositional cytoskeleton rearrangement, constitute typical features of sarcomas cells, when compared to their healthy counterpart. Notably, whereas a decrease in stiffness is typically a feature of malignant entities, chondrosarcoma cells were stiffer than chondrocytes, with chondrosarcoma cells exhibiting a significantly upregulated β-tubulin expression. Each Sarcoma entity may have his own cellular-stiffness and cytoskeleton organisation/composition fingerprint, which in turn may be exploited for diagnostic or therapeutic purposes.