Poster Presentation 35th Lorne Cancer Conference 2023

CREB-regulated immunosuppression in the GBM tumour microenvironment (#351)

Samuel S. Widodo 1 , Marija Dinevska 1 , Laura Cook 2 , Miguel Á. Berrocal-Rubio 3 , Christine A. Wells 4 , Alexander D. Barrow 2 , Stanley S. Stylli 1 5 , Theo Mantamadiotis 1 2 4
  1. Department of Surgery (RMH), The University of Melbourne, Melbourne, VIC, Australia
  2. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  3. Department of Anatomy and Physiology, The University of Melbourne, Melbourne, VIC, Australia
  4. The Centre for Stem Cell Systems, The University of Melbourne, Melbourne, VIC, Australia
  5. Department of Neurosurgery, Royal Melbourne Hospital, Parkville, VIC, Australia

Glioblastoma (GBM) is the most common and aggressive type of primary cancer affecting the central nervous system in adults. GBM is characterised by an immunosuppressive tumour microenvironment, which hinders the effectiveness of the patient’s immune system or immunotherapy to efficiently kill cancer cells. The immunosuppression is largely driven by infiltrating tumour-associated macrophages and microglia (TAMs), which constitute up to 40% of cells in the GBM tumor microenvironment. Previous studies show that the expression of some anti-inflammatory/immunosuppressive factors in macrophages is regulated by the kinase inducible transcription factor, cyclic AMP response element binding protein (CREB). However, this has not been confirmed in TAMs in GBM.

 

Therefore, this project aims to investigate whether CREB modulates TAM-mediated immunosuppression in GBM.

 

To investigate CREB activation in TAMs in GBM patient samples, multiplex immunohistochemistry was performed using macrophage and microglial subtype markers, including CD68, TMEM119, HLA-DR (‘M1-like’/pro-inflammatory marker), CD163 and CD206 (‘M2-like’/immunosuppressive markers), and pCREB-specific antibody. My data shows that about 80% TAMs exhibit high pCREB expression, and that the majority of pCREBhigh TAMs are positive for M2-like markers, suggesting a positive correlation between CREB activation and immunosuppressive characteristics in TAMs.

 

To further investigate CREB-regulated immunosuppressive cytokine expression in macrophages in vitro, differentiated THP-1 monocytic cells were treated with GBM-conditioned media collected from several GBM cell lines. CREB activation was observed in THP-1 cells treated with GBM cell conditioned media, as well as increased expression and secretion of immunosuppressive cytokines, including IL-10. This effect could be blocked by a CREB inhibitor, 666-15, suggesting that CREB plays roles in regulating the expression of immunosuppressive cytokines.

 

Future experiments will examine transcriptional profiles of wildtype and CREB knockout macrophages, stimulated with GBM conditioned media and investigate their interactions with GBM cells.