Introduction. Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a five-year relative survival rate (after diagnosis) of only 36%. GBM is characterized for its fast proliferation rate and angiogenic character, making it the leading cause of cancer-related deaths in young adults. During the onset and evolution of GBM, the composition of its microenvironment, in particular the extracellular matrix (ECM), plays a critical role on the invasiveness of the tumour. An example is the glycosaminoglycan Hyaluronic Acid (HA, comprising 30-50% of GBM’s ECM), which is the dominant component of the GBM microenvironment. Of note, HA of low molecular weight (Mw) has been correlated to the invasive character of cancer cells, while long HA chains are associated with cancer latency. Here, we explored the role of HA Mw on the migration and invasive behaviour of U-87 glioma cells with a 3D ECM model using a hydrogel composed by Alginate and HA of different Mw, 5.6 and 1450 kDa.
Methodology. Hydrogels were prepared by combining alginate (Alg - Pronova VLVG, 20 mg/mL) and HA (Mw= 5.6, and 1450 kDa, 1mg/mL), crosslinked with CaCl2 (100mM). U-87 spheroids (with 0.05x106 cells) were encapsulated into the Alg/Alg-HA hydrogels during their preparation. Live imaging was performed to follow in real time the cancer cells’ motility when in contact with the 3D ECM model. Glioma cells were immunostained for CD44, the main receptor for HA and cortactin (a key player in the modulation of cellular motility), to evaluate the effect of HA Mw on the invasive character of U-87 cells.
Results. Time-lapse images showed a correlation between the hydrogel composition and the migratory behaviour of U-87 cells: HA of 5.6 kDa induces the invasive character of glioma cells promoting the migration of individual cells from the U-87 spheroid to the surrounding hydrogel. This behaviour is not observed on the hydrogels presenting HA of 1450 kDa. Moreover, immunostaining showed an increase expression of CD44 on U-87 cells in the presence of HA, which co-localizes with cortactin with particular emphasis in the migratory cells present on Alg-HA of 5.6 kDa.
Conclusions. Our data clearly demonstrate that the HA of low Mw influences glioma cells’ migration and invasiveness by triggering the CD44 expression that, in turn, activates cortactin promoting its invasive character. The GBM 3D model was able to mimic the tumour microenvironment and allowed to assess the influence of the biochemical features of specific components of the cancer ECM, in particular of HA (and its Mw) on glioma cells’ behaviour.
Acknowledgements. We acknowledge the financial support from the European Commission (#668983-FORECAST, #964342-ECaBox) and the Portuguese FCT (PTDC/NAN-MAT/28468/2017-CancerCage, PTDC/CTM-REF/0022/2020-OncoNeoTreat and CEECINST/00077/2018).
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