Intracellular mechanics and TBX3 expression jointly dictate the spreading mode of melanoma cells in 3D environments.

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma.

Cytoskeletal tubulin competes with actin to increase deformability of metastatic melanoma cells.

The formation of membrane protrusions during migration is reliant upon the cells' cytoskeletal structure and stiffness. It has been reported that actin disruption blocks protrusion and decreases cell stiffness whereas microtubule disruption blocks protrusion but increases stiffness in several cell types. In melanoma, cell migration is of concern as this cancer spreads unusually rapidly during early tumour development. The aim of this study was to characterise motility, structural properties and stiffness of human melanoma cells at radial growth phase (RGP), vertical growth phase (VGP), and metastatic stage (MET) in two-dimensional in vitro environments. Wound assays, western blotting and mitochondrial particle tracking were used to assess cell migration, cytoskeletal content and intracellular fluidity. Our results indicate that cell motility increase with increasing disease stage. Despite their different motility, RGP and VGP cells exhibit similar fluidity, actin and tubulin levels. MET cells, however, display increased fluidity which was associated with increased actin and tubulin content. Our findings demonstrate an interplay between actin and microtubule activity and their role in increasing motility of cells while minimizing cell stiffness at advanced disease stage. In earlier disease stages, cell stiffness may however not serve as an indicator of migratory capabilities.

Effect of paclitaxel treatment on cellular mechanics and morphology of human oesophageal squamous cell carcinoma in 2D and 3D environments.

During chemotherapy, structural and mechanical changes in malignant cells have been observed in several cancers, including leukaemia and pancreatic and prostate cancer. Such cellular changes may act as physical biomarkers for chemoresistance and cancer recurrence. This study aimed to determine how exposure to paclitaxel affects the intracellular stiffness of human oesophageal cancer of South African origin in vitro. A human oesophageal squamous cell carcinoma cell line WHCO1 was cultured on glass substrates (2D) and in collagen gels (3D) and exposed to paclitaxel for up to 48 h. Cellular morphology and stiffness were assessed with confocal microscopy, visually aided morpho-phenotyping image recognition and mitochondrial particle tracking microrheology at 24 and 48 h. In the 2D environment, the intracellular stiffness was higher for the paclitaxel-treated than for untreated cells at 24 and 48 h. In the 3D environment, the paclitaxel-treated cells were stiffer than the untreated cells at 24 h, but no statistically significant differences in stiffness were observed at 48 h. In 2D, paclitaxel-treated cells were significantly larger at 24 and 48 h and more circular at 24 but not at 48 h than the untreated controls. In 3D, there were no significant morphological differences between treated and untreated cells. The distribution of cell shapes was not significantly different across the different treatment conditions in 2D and 3D environments. Future studies with patient-derived primary cancer cells and prolonged drug exposure will help identify physical cellular biomarkers to detect chemoresistance onset and assess therapy effectiveness in oesophageal cancer patients.