Metastasising child fibroblasts show increased Hyaluronan at the cell periphery
To understand the role of Hyaluronan in the cell adhesion of metastasising child fibroblasts, we analysed the spatial distribution of Hyaluronan and focal adhesions in metastasizing cells. Both in metastasising and control cells, Hyaluronan showed mainly a cytoplasmic localisation. Hyaluronan was also found in the extracellular environment, it localised where a cell has resided, leaving tracks after migrating cells (Fig. 1). The metastasising cells showed more often Hyaluronan at the cell periphery, and the signal was increased, as compared to control (Fig. 1)
Metastasising cells show increased assembly of nanometre clusters of Hyaluronan at the plasma membrane of the leading edge of migrating cells
Although there are strong links between Hyaluronan and cell motility [32], the spatial distribution of Hyaluronan in cells in the leading edge of cells at the nanometer level is not known. We therefore characterised the nanoscale spatial distribution of Hyaluronan at the lamellipodia of cells, and if this is altered in metastasising cells, as compared to non-metastasising control cells, using super-resolution STED microscopy. In line with previous observations, we observed increased filamentous actin and actin ruffles at the leading edge of metastasising cells, as well as smaller and less distinct cell-matrix adhesions, as compared to normal control [11, 33].
We observed that Hyaluronan was localised in the same subcellular region in the leading front of the cells, but we could not detect a co-localisation to cell-matrix adhesions. Rather, Hyaluronan was not observed within focal adhesions. However, the metastasising cells showed Hyaluronan at the very cell periphery, often in longer aggregates, which was not observed in control cells (Fig. 2A). To further identify differences between the distribution of Hyaluronan in the front of metastasising and control cells, the STED imaging was followed by computational image analysis, as described in the Material and Method section. For this, we first analysed the heterogeneity and the contrast of the signal in the images and observed that the heterogeneity of the pixels was higher and more widely distributed in the metastasising cells, as compared to control, and that the typical contrast in the signal, in the metastasising cells, was significantly higher compared to the control (Fig. 2B, Suppl. Fig. S6 and Suppl. Table. S2).
We further observed that the signal energy, a measure of the homogeneity of the signal of the pixels, was lower in the metastasising cells, as compared to control. Thereafter, we analysed to which degree Hyaluronan was assembled in nanoscale clusters. We observed that the Hyaluronan in the metastasising cells was distributed in a higher number of clusters per cell surface area, and these clusters were 50% larger as compared to clusters in control cells. The average pixel intensity of these clusters did not significantly differ between control and metastasising cells (see Suppl. Fig S4). However, the signal of metastasising cells showed lower pixel intensity per cluster compared to the control. Under the assumption that pixel intensity is proportionally related to the density of Hyaluronan within the pixel boundary, these observations indicate that Hyaluronan tends to be more assembled into larger clusters, that these clusters are closer to each other spatially and individual clusters are less densely packed with Hyaluronan in metastasising cells as compared to in non-metastasising cells (Fig. 2A).
Hyaluronan synthases interact with known regulators of cell motility
With the aim to identify candidate proteins that regulate or mediate the effect of Hyaluronan on cell motility and infantile fibrosarcoma invasion, we performed a systematic literature review, and STRING analysis, as described in the Material and Method section. Thereby, we identified Hyaluronan synthases 1-3, Hyaluronidase 1-5 and their interactors, as shown in Supplementary figure S2. To clarify the signalling pathways involving these proteins, we analysed their known protein-protein interactions between these proteins. As expected, Hyaluronan synthase- and Hyaluronidase- interactors clustered to two separate groups of proteins (Suppl. Fig. S3). The highest confidence of interaction was observed between the Hyaluronan receptor CD44 and Hyaluronidases. To further clarify the molecular mechanism by which Hyaluronan can control cell motility and cancer invasion, we analysed known functions of the identified interactors. In contrast to Hyaluronan synthase 1-3 that showed no strong interactions with any other proteins (Suppl. Fig. S2), Hyaluronidase 1-5 all showed strong interactions with the following proteins: GUSB, ARSB, IDUA, CD44, RHAMM, STAB2, CHP1, SLC9A, LYVE, IDS and GALNS (Suppl. Fig. S3). The analysis further showed interaction between CD44 and all five Hyaluronidases.
Metastasising fibroblasts show increased gene expression of Hyaluronan synthase 2 and reduced expression of Hyaluronidase 2 and CD44
We then wished to identify novel Hyaluronan-related molecular mechanisms by which child fibroblasts can gain the capacity to invade and metastasise. To this end, we compared the gene expression of the genes corresponding to the proteins identified as Hyaluronan-interacting proteins on the analysis above between isogenetically matching metastasising and control child fibroblasts [11, 33]. We observed that Hyaluronan synthase 2 and β-glucuronidase were upregulated in the metastasising fibroblasts with Hyaluronan synthase 2 showing the most statistically significant increase (Table 1).
Table 1. Up-regulated Hyaluronan-related genes in metastasising (Bj-metastasising) relative to control child fibroblasts (Bjhtert).
Protein Name
|
Protein
Abbreviation
|
Bj-metastasising vs Bjhtert (log2 change)
|
p-value
|
β-glucuronidase
|
GUSB
|
1.347
|
6.00 x 10-3
|
Hyaluronan synthase 2
|
HAS2
|
1.532
|
2.17 × 10-38
|
The gene expression corresponding to the following proteins was downregulated in the metastasising cells, as compared to control: Hyaluronidase 2, CD44, Iduronate 2-Sulfatase, Galactosamine (N-Acetyl)-6-Sulfatase, Alpha-L-Iduronidase, CD44, Calcineurin Like EF-Hand Protein 1, Solute Carrier Family 9 Member A1. Of these, CD44 showed the most statistically significant down-regulation (Table 2). The Hyaluronan-related genes that did not show a statistical change in expression are shown in Supplementary table S1. The genes coding for the identified Hyaluronan-related proteins Hyaluronidase 5/SPAM1, RHAMM, STAB2, KY, LYVE1 were not expressed in these cells [33] .
Table 2. Down-regulated Hyaluronan-related gene expression in metastasising (Bj-metastasising) relative to control child fibroblasts (Bjhtert).
Protein Name
|
Protein
Abbreviation
|
Bj-metastasising vs Bjhtert (log2 change)
|
p-value
|
1 Duronate 2-Sulfatase
|
IDS
|
-0.320
|
1.66 × 10-4
|
Galactosamine (N-Acetyl) 6-Sulfatase
|
GALNS
|
-0.793
|
1.13 × 10-6
|
Alpha-L-Iduronidase
|
IDUA
|
-2.257
|
4.56 × 10-13
|
CD44
|
CD44
|
-1.306
|
6.73 × 10-68
|
Calcineurin Like EF-Hand Protein 1
|
CHP1
|
-0.673
|
2.16 × 10-8
|
Solute Carrier Family 9 Member A1
|
SLC9A1
|
-0.461
|
2.00 x 10-3
|
Hyaluronidase 2
|
HYAL2
|
-0.389
|
1.10 x 10-2
|
Expression of Hyaluronidases 3 and 5 in fibrosarcoma tumours is linked to increased mortality
To determine the importance of hyaluronidases on the progression of fibrosarcoma, we analysed survival of patients expressing the genes encoding for Hyaluronidase 3 and 5 using the Cancer Genome Atlas, as described in the Material and Method section. Patients with fibrosarcoma tumours expressing genes encoding for Hyaluronidase 3 or 5 showed a worse prognosis when compared to the overall patient cohort. The three-year survival rate for patients which tumour expressed the genes corresponding to Hyaluronidase 3 and 5 was 89% and 36%, respectively. This should be compared to that of 93% for the overall patient cohort (Table 3). All Hyaluronan synthases and Hyaluronidases, with the expection of Hyaluronidase 1, was linked to reduced survival of patients diagnosed with fibrosarcoma (Suppl. Fig. 7). As hyaluronidases are responsible for cleaving high molecular weight hyaluronan into low molecular weight variants, an adverse outcome on survival rates supports the hypothesis that low molecular weight hyaluronan promotes metastasis. Due to lack of bioinformatic data, Hyaluronidase 5 is absent from this analysis. Taken together, these results indicate that expression of Hyaluronidases can contribute to the progression and invasion of fibrosarcoma tumours.
Table 3. Mortality of child fibrosarcoma expressing Hyaluronidases and the overall patient cohort [15].
Protein Name
|
Gene name
|
3-year patient survival rate (%)
|
Hyaluronidase 3
|
HYAL3
|
89
|
Hyaluronidase 5
|
HYAL5
|
36
|
|
Overall
|
93
|
Hyaluronan levels are increased at the periphery of Infantile fibrosarcoma tumours
We then wished to determine if Hyaluronan can promote the invasion of child fibrosarcoma tumours into the surrounding tissue. For this, we compared the levels of Hyaluronan in the central and peripheral regions of tumour sections from patients with infantile fibrosarcoma. The Paediatric oncology team considered the tumour to be aggressive, and to have a definite potential to spread prior to excising the tumour (oral communication). The levels of hyaluronan at the peripheral areas of the tissue appeared to be increased, as compared to the central regions of the tumour (Fig. 3 and Suppl. Fig. S5).