Abstract 5678: Hypoxia promotes cell attachment and proliferation due to extracellular matrix compositional and structural changes
Authors: Quiles Conrado Guerrero , Abalos Julia Gonzalez , Humphries Jonathan , Humphries Martin , CHOUDHURY Ananya , West Catharine 
Published: June 2022

Abstract
Background: Hypoxia is a common feature of cancer that promotes hallmarks such as cell migration and sustained proliferation. Hypoxia also has a strong link with extracellular matrix (ECM) remodeling, affecting both composition and structure. ECM changes also affect cancer hallmarks through integrin signaling. However, effects of long-term chronic hypoxia stress on ECM have not been studied in bladder cancer. We aimed to investigate the changes induced by hypoxia in the ECM and how they affected cell attachment and proliferation.
Methods: UMUC3, J82, RT4 and T24 bladder cancer cell lines were cultured for 7 days in 21% (normoxic) or 0.2% (hypoxic) O2. For obtaining the cell-derived ECM (CDM), cell cultures were decellularized with NH4OH. CDM samples were in-gel trypsin digested and tandem mass spectrometry (MS) performed. IF was performed for FN and COL5A1 protein. Hypoxic ECM effects on cancer cell behavior were studied using attachment and proliferation assays. Attachment assays involved incubating cells at 37°C for 20min (T24, J82, RT4) or 2h (UMUC3) on hypoxic or normoxic CDM. Cells were fixed, stained with crystal violet and absorbance measured at 540nm. Proliferation was measured after seeding cells on hypoxic or normoxic CDM, analyzing cell growth up to 7 days using an IncuCyte S3 system.
Results: MS detected 186 ECM proteins, with 66 differentially expressed due to hypoxia in ≥1 cell line (p&lt;0.05). MS data clustering showed variation in CDM composition due to both hypoxia and cell line. Gene Ontology analysis showed hypoxia-induced enrichment (p&lt;0.001) of pathways associated with ECM structure, cell adhesion and integrin adhesion. IF confirmed morphological changes in FN and COL5A1 protein fibres (p&lt;0.05) for 3 of 4 cell lines. All cell lines showed a significant increase in attachment on hypoxic vs normoxic CDM (p&lt;0.05). Initial data showed a significant increase in proliferation of T24 cells grown on hypoxic vs normoxic matrix (p&lt;0.05).
Conclusion: Hypoxia induces ECM remodeling in vitro, significantly affecting the level of one third of the proteins detected by MS. In silico analysis of the MS results correlated compositional changes with structural changes correlated compositional changes with structural changes. Those changes were confirmed morphologically for FN and COL5A1 fibers. Cell and integrin adhesion changes predicted in silico were also observed when performing attachment assays, where hypoxic ECM significantly increased cell attachment. Finally, pilot proliferation data showed that hypoxic ECM promotes T24 cell proliferation. To our knowledge, this is the first comprehensive study to report compositional and structural changes in bladder cancer ECM due to hypoxia stress. Further studies should focus on the role of integrin, whose interaction with hypoxic ECM may be key for the induction of cell proliferation and attachment reported in this study.
Citation Format: Conrado Guerrero Quiles, Julia Gonzalez Abalos, Jonathan Humphries, Martin Humphries, Ananya CHOUDHURY, Catharine West. Hypoxia promotes cell attachment and proliferation due to extracellular matrix compositional and structural changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5678.