Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy

Lisa M Ebert; Wenbo Yu; Tessa Gargett; John Toubia; Paris M Kollis; Melinda N Tea; Brenton W Ebert; Cedric Bardy; Mark van den Hurk; Claudine S Bonder; Jim Manavis; Kathleen S Ensbey; Mariana Oksdath Mansilla; Kaitlin G Scheer; Sally L Perrin; Rebecca J Ormsby; Santosh Poonnoose; Barbara Koszyca; Stuart M Pitson; Bryan W Day; Guillermo A Gomez; Michael P Brown

doi:10.1002/cti2.1191

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy

Clin Transl Immunology. 2020 Oct 14;9(10):e1191. doi: 10.1002/cti2.1191. eCollection 2020.

Authors

Lisa M Ebert^{1

2}, Wenbo Yu¹, Tessa Gargett^{1

2}, John Toubia¹, Paris M Kollis^{1

2}, Melinda N Tea¹, Brenton W Ebert¹, Cedric Bardy^{3

4}, Mark van den Hurk^{3

4}, Claudine S Bonder^{1

2}, Jim Manavis², Kathleen S Ensbey⁵, Mariana Oksdath Mansilla¹, Kaitlin G Scheer^{1

6}, Sally L Perrin^{1

6}, Rebecca J Ormsby⁴, Santosh Poonnoose^{4

7}, Barbara Koszyca⁸, Stuart M Pitson^{1

2}, Bryan W Day^{5

9

10}, Guillermo A Gomez¹, Michael P Brown^{1

2

11}

Affiliations

¹ Centre for Cancer Biology SA Pathology and University of South Australia Adelaide Australia.
² Adelaide Medical School University of Adelaide Adelaide Australia.
³ South Australian Health and Medical Research Institute (SAHMRI) Adelaide Australia.
⁴ College of Medicine & Public Health Flinders University Adelaide Australia.
⁵ Department of Cell and Molecular Biology Sid Faithfull Brain Cancer Laboratory QIMR Berghofer Medical Research Institute Brisbane QLD Australia.
⁶ Clinical and Health Sciences University of South Australia Adelaide Australia.
⁷ Department of Neurosurgery Flinders Medical Centre Bedford Park Australia.
⁸ Department of Anatomical Pathology SA Pathology Adelaide Australia.
⁹ Faculty of Health Queensland University of Technology Brisbane QLD Australia.
¹⁰ Faculty of Medicine The University of Queensland Brisbane QLD Australia.
¹¹ Cancer Clinical Trials Unit Royal Adelaide Hospital Adelaide Australia.

Abstract

Objectives: Targeted immunotherapies such as chimeric antigen receptor (CAR)-T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples.

Methods: Glioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets. Short-term cultures of glioma neural stem (GNS) cells were compared to cultures of healthy astrocytes and neurons using flow cytometry. Glioblastoma tissues were dissociated and analysed by high-parameter flow cytometry and single-cell transcriptomics (scRNAseq).

Results: Compared to normal brain, FAP was overexpressed at the gene and protein level in a large percentage of glioblastoma tissues, with highest levels of expression associated with poorer prognosis. FAP was also overexpressed in several paediatric brain cancers. FAP was commonly expressed by cultured GNS cells but absent from normal neurons and astrocytes. Within glioblastoma tissues, the strongest expression of FAP was around blood vessels. In fact, almost every tumor vessel was highlighted by FAP expression, whereas normal tissue vessels and cultured endothelial cells (ECs) lacked expression. Single-cell analyses of dissociated tumors facilitated a detailed characterisation of the main cellular components of the glioblastoma microenvironment and revealed that vessel-localised FAP is because of expression on both ECs and pericytes.

Conclusion: Fibroblast activation protein is expressed by multiple cell types within glioblastoma, highlighting it as an ideal immunotherapy antigen to target destruction of both tumor cells and their supporting vascular network.

Keywords: blood vessels; fibroblast activation protein; glioblastoma; immunotherapy; scRNAseq; target antigen.