Poster Presentation 36th Lorne Cancer Conference 2024

Surfaceome and extracellular matrix analyses of diffuse midline glioma and implications for immune-oncology therapies (#150)

Bryce Thomas 1 2 , Mika L Persson 1 2 , Dilana E Stuadt 1 2 , Evangeline R Jackson 1 2 , Zacary P Germon 1 2 , Ryan J Duchatel 1 2 , Izac J Findlay 1 2 , Holly McEwen 1 2 , Cameron J Fish 1 2 , Alicia M Douglas 1 2 , Birgit Kobbe 3 , Raimund Wagner 3 , Martin R Larsen 4 , Pouya Faridi 5 , Jemma Mayall 6 , Jay Horvat 6 , Matthew D Dun 1 2
  1. Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  2. Cancer Signalling Research Group, School of Biomedical Sciences & Pharmacy, College of Health, University of Newcastle, Callaghan, NSW, Australia
  3. Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany
  4. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
  5. Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Viewbank, VIC, Australia
  6. Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, Australia

Diffuse midline glioma (DMG) is a paediatric brain cancer of the midline structures of the brain including the pons (Diffuse intrinsic pontine glioma (DIPG)), brainstem, thalamus, and spinal cord. The disease has a dismal survival of only 9-11 months. Conventional therapies typically fail to provide any therapeutic benefit. The tumour immune microenvironment (TIME) is immunologically “cold”, characterised by a dominant microglial compartment. The extracellular matrix (ECM) plays a significant role in mediating the immunological niches of DMG through regulation of immunosuppressive cytokines and immune cell infiltration. These mechanisms remain poorly understood and have significant implications for immune-oncology-based treatments such as CAR T cells. This project aims to understand mechanisms of ECM-driven immunosuppression as well as to highlight pathways that could be targeted to improve existing/novel immune-oncology approaches.

Membrane proteomic profiling was performed using TMT-labelled mass spectrometry on patient-derived DMG nuerospheres and compared to an endothelial cell line (hCMEC/D3) derived from the blood brain barrier. Proteins of interest were validated using flow cytometry, Western Blotting, immunohistochemistry, and immunofluorescent microscopy, and correlated against online databases.

Proteomic surface expression of DMG tumours revealed “Protein-X” to be the most upregulated protein (3.95-Log2 Fold-change, p<0.0001) across DMGs. Protein-X is mostly absent from the healthy human proteome with the exception to the testes and ovaries, as a constituent of the ECM. Immunofluorescent microscopy demonstrates the translocation of Protein-X from the Golgi to the cell membrane is dependent on neurosphere formation, hence formation of an ECM. Further, Protein-X also interacts with TGF-β and influences macrophage differentiation, suggestive of an immune-modulatory role. Predictive modelling suggests Protein-X expression is under the control of SMARCA4, a known driver of gliomagenesis. Finally, bioinformatic analyses revealed high-expression of Protein-X across several paediatric brain tumours, with increased expression correlating with poor overall survival.

Resistance to conventional therapies and a poor understanding of the TIME and ECM remains a significant challenge to the development of effective DMG therapies. The identification of Protein-X elucidates a constituent of the ECM. Further, the implication of SMARCA4 in disease initiation/progression highlights additional signalling pathways modulating the ECM, which remains under explored, and may reveal novel therapeutic strategies.