Hyaluronan nanoscale clustering and Hyaluronan synthase 2 expression are linked to the invasion of child fibroblasts and infantile fibrosarcoma in vitro and in vivo

Infantile fibrosarcoma is a rare childhood tumour that originates in the fibrous connective tissue of the long bones for which there is an urgent need to identify novel therapeutic targets. This study aims to clarify the role of the extracellular matrix component hyaluronan in the invasion of child fibroblasts and Infantile fibrosarcoma into the surrounding environment. Using nanoscale super-resolution STED (Stimulated emission depletion) microscopy followed by computational image analysis, we observed, for the first time, that invasive child fibroblasts showed increased nanoscale clustering of hyaluronan at the cell periphery, as compared to control cells. Hyaluronan was not observed within focal adhesions. Bioinformatic analyses further revealed that the increased nanoscale hyaluronan clustering was accompanied by increased gene expression of Hyaluronan synthase 2, reduced expression of Hyaluronidase 2 and CD44, and no change of Hyaluronan synthase 1 and Hyaluronidases 1, 3, 4 or 5. We further observed that the expression of the Hyaluronan synthase 1, 2 and 3, and the Hyaluronidase 3 and 5 genes was linked to reduced life expectancy of fibrosarcoma patients. The invasive front of infantile fibrosarcoma tumours further showed increased levels of hyaluronan, as compared to the tumour centre. Taken together, our findings are consistent with the possibility that while Hyaluronan synthase 2 increases the levels, the Hyaluronidases 3 and 5 reduce the weight of hyaluronan, resulting in the nanoscale clustering of hyaluronan at the leading edge of cells, cell invasion and the spread of Infantile fibrosarcoma.