Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB087

Poster Communications

The role of the glycocalyx in cell-cell interactions in human melanoma

J. H. Polmann1, V. Hofschröer1, A. Schwab1

1. Institute of Physiology II, WWU Münster, Münster, Germany.


Melanoma is responsible for most skin cancer related deaths and metastasis is a major factor in patient prognosis. Migration importantly contributes to the metastatic cascade. It is affected by the extracellular pH (pHe) of the tumour stroma and the pericellular pHe of individual cells, which is maintained by the glycocalyx. The aim of this project is to study whether the impact of pHe on migration is modulated by the glycocalyx. The glycocalyx of human melanoma cells (MV3) was targeted with heparinase and neuraminidase, enzymes which degrade different N-glycosidic bonds. The effects of the enzymes on the glycocalyx were quantified by fluorescence microscopy with TRITC-labeled wheat germ agglutinin (WGA). Height and stiffness of the glycocalyx were determined with atomic force microscopy (AFM). The functional influence of the treatment at different pHe values was assessed in migration wound assays with time-lapse video microscopy. Values are presented as means ± S.E.M., compared by ANOVA, N=3-4, n=30-40 for all experiments. After 4 hours of heparinase or neuraminidase treatment WGA staining is reduced by 18 ± 0.5% or 31 ± 0.5%, respectively (both p<0.05). The stiffness of the glycocalyx slightly decreases upon neuraminidase treatment (from 0.22 ± 0.01nm/pN to 0.19 ± 0.01nm/pN, p<0.05). The combination of both enzymes elicits a stronger effect (0.15 ± 0.01nm/pN, p<0.05). The height of the glycocalyx is also reduced (p<0.05) by both enzymes individually and in combination (control: 210 ± 3nm, heparinase: 186 ± 4 nm, neuraminidase: 170 ± 5nm, both enzymes: 162 ± 2nm). Collectively, fluorescence microscopy and AFM suggest a pruning of the glycocalyx. Migration was observed at pH 6.4 and 7.4 for 10 hours in wound assays following 4 hours of enzymatic pretreatment. The velocities of MV3 cells at 6.4 and 7.4 are not different from each other (~ 0.45µm/min). Under control conditions, MV3 cells have the highest directionality (0.53 ± 0.04) at pH 6.4. Directionality is lower following enzyme treatment (0.29 ± 0.04) and at pH 7.4 (0.30 ± 0.03, both p<0.05). We further analysed how far two cells migrate together before losing their cell-cell-contact. While vastly different at the two pH levels (pH 6.4: 94 ± 4µm, pH 7.4: 54 ± 4µm, p<0.05) heparinase aligned the distance at both pH values (75 ± 6µm and 76 ± 5µm). Adding 20mM HEPES to the bulk solution as an additional buffer lowers the velocity of the cells (0.23 ± 0.01µm/min, p<0.05) while the distance to separation remains unchanged even after heparinase treatment. The data suggest a modulation of the pH-dependent effects on migration by the glycocalyx. The buffering capacity of the glycocalyx and the concentration of protons in the bulk solution both change various parameters in single cell and collective cell migration. We propose that this is due to alterations of the pericellular pH homeostasis.

Where applicable, experiments conform with Society ethical requirements