Session Item

The extracellular matrix (ECM) is a key component regulating the tumour microenvironment. The ECM composition and its protein structures affect cancer hallmarks such as cell migration, proliferation and survival. Hypoxia can induce ECM remodelling, strongly influencing its composition and structure. Consequently, hypoxia induced ECM remodelling can have an important effect on cancer hallmarks. We aimed to investigate how hypoxia affects ECM composition and structure, and how these changes affect cell migration in vitro.

UMUC3, J82, RT4 and T24 bladder cancer cell lines were cultured for 7 days at 21% (normoxic) or 0.2% (hypoxic) O₂. Hypoxia phenotype was confirmed by western blotting of both cell lysates and cell derived matrix (CDM). CDM was collected using 2X SDS buffer after decellularization with NH₄OH. CDM samples were then analysed using mass spectrometry (MS). Immunofluorescence was performed for FN and COL5 proteins after cell fixation in a solution of 8% paraformaldehyde and 0.5% glutaraldehyde. Primary FN and COL5 antibodies were incubated overnight, and secondary antibodies for 2h at room temperature. For the migration assay, UMUC3, J82 and T24 cells were seeded onto hypoxic and normoxic CDM and migration assessed using the scratch assay over 24h or 48h.

MS analysis of CDM samples identified 186 proteins. The levels of 66 of the 186 detected proteins changed significantly (p<0.05) in at least one cell line. There was variability in the proteins affected by hypoxia between cell lines, but in silico Gene Ontology analysis showed changes led consistently to an enrichment of proteins affecting ECM structural functions. Immunofluorescence of FN and COL5 confirmed structural changes in the amount and length of CDM fibres. Hypoxia increased FN fibres in 3 cell lines (T24, J82 and RT4) and decreased COL5 fibres in all 4 lines (p<0.05). Importantly, UMUC3 did not produce FN in its normoxic or hypoxic CDM. There was no significant difference in migration on hypoxic and normoxic CDM (J82, T24), but migration was faster on normoxic vs hypoxic UMUC3 CDM. Under radiation stress (2-8 Gy) differences in UMUC3 cell migration on normoxic vs hypoxic CDM disappeared suggesting a potential protective effect on migration promoted by more organised ECM COL5 fibres.

Hypoxia induces significant changes in the composition and structure of bladder cancer ECM in vitro, which may affect cell migration depending on the nature of the ECM produced by each cell line. In T24 and J82 there was no change in cell migration, as COL5 destruction in hypoxia was compensated by FN fibre production. In UMUC3, migration was faster on normoxic CDM as cells lacked the capacity to produce FN. The work highlights cell heterogeneity in the effects of hypoxia on the ECM. It also shows that radiation can impair migration on ECM with high COL5 fibre organisation.