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

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. DayGuillermo A. Gomez, Michael P. Brown

College of Medicine and Public Health

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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.

Original language	English
Article number	e1191
Number of pages	24
Journal	Clinical and Translational Immunology
Volume	9
Issue number	10
DOIs	https://doi.org/10.1002/cti2.1191
Publication status	Published - 14 Oct 2020

Bibliographical note

Funding Information:
This work was supported by the Health Services Charitable Gifts Board (Adelaide) and a SA Cancer Council Beat Cancer Project Hospital Research Support Package (MB); the Neurosurgical Research Foundation (LE, MB, TG, MT, SP, GG); generous donations from the Sid Faithfull Family (KE, BD); a Senior Research Fellowship (1156693) from the National Health and Medical Research Council of Australia (SP); Flinders Foundation (CB); Cure Brain Cancer Foundation (CB, MB, GG, BD); Perpetual Impact Philanthropy (CB); the Brain Foundation (CB); Rebecca L. Cooper Foundation (CB); the Cancer Council SA Beat Cancer Project (to MPB and GAG); the Australian Research Council (FT160100366 to GAG); and a Netherlands Organisation for Scientific Research Rubicon Fellowship 019.163LW.032 (MvdH). SLP is supported by an Australian Government Research Training Program Scholarship. The authors would like to thank Samantha Escarbe and Nathalie Nataren for excellent technical assistance, Erica Yeo and Natansh Modi for assisting with the development of FAP immunostaining and Mark DeNichilo for HUVEC culture. We also acknowledge the support and generosity of the patients and medical and technical staff, including from SA Pathology and the SA Neurological Tumour Bank (supported by Flinders University, Flinders Foundation and The Neurosurgical Research Foundation), who made collection of tissue specimens possible.

Publisher Copyright:
© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.

Keywords

blood vessels
fibroblast activation protein
glioblastoma
immunotherapy
scRNAseq
target antigen

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Ebert, L. M., Yu, W., Gargett, T., Toubia, J., Kollis, P. M., Tea, M. N., Ebert, B. W., Bardy, C., van den Hurk, M., Bonder, C. S., Manavis, J., Ensbey, K. S., Oksdath Mansilla, M., Scheer, K. G., Perrin, S. L., Ormsby, R. J., Poonnoose, S., Koszyca, B., Pitson, S. M., ... Brown, M. P. (2020). Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy. Clinical and Translational Immunology, 9(10), Article e1191. https://doi.org/10.1002/cti2.1191

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

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",

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

note = "Funding Information: This work was supported by the Health Services Charitable Gifts Board (Adelaide) and a SA Cancer Council Beat Cancer Project Hospital Research Support Package (MB); the Neurosurgical Research Foundation (LE, MB, TG, MT, SP, GG); generous donations from the Sid Faithfull Family (KE, BD); a Senior Research Fellowship (1156693) from the National Health and Medical Research Council of Australia (SP); Flinders Foundation (CB); Cure Brain Cancer Foundation (CB, MB, GG, BD); Perpetual Impact Philanthropy (CB); the Brain Foundation (CB); Rebecca L. Cooper Foundation (CB); the Cancer Council SA Beat Cancer Project (to MPB and GAG); the Australian Research Council (FT160100366 to GAG); and a Netherlands Organisation for Scientific Research Rubicon Fellowship 019.163LW.032 (MvdH). SLP is supported by an Australian Government Research Training Program Scholarship. The authors would like to thank Samantha Escarbe and Nathalie Nataren for excellent technical assistance, Erica Yeo and Natansh Modi for assisting with the development of FAP immunostaining and Mark DeNichilo for HUVEC culture. We also acknowledge the support and generosity of the patients and medical and technical staff, including from SA Pathology and the SA Neurological Tumour Bank (supported by Flinders University, Flinders Foundation and The Neurosurgical Research Foundation), who made collection of tissue specimens possible. Publisher Copyright: {\textcopyright} 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.",

year = "2020",

month = oct,

day = "14",

doi = "10.1002/cti2.1191",

language = "English",

volume = "9",

journal = "Clinical and Translational Immunology",

issn = "2050-0068",

publisher = "Nature Publishing Group",

number = "10",

}

Ebert, LM, Yu, W, Gargett, T, Toubia, J, Kollis, PM, Tea, MN, Ebert, BW, Bardy, C, van den Hurk, M, Bonder, CS, Manavis, J, Ensbey, KS, Oksdath Mansilla, M, Scheer, KG, Perrin, SL, Ormsby, RJ , Poonnoose, S, Koszyca, B, Pitson, SM, Day, BW, Gomez, GA & Brown, MP 2020, 'Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy', Clinical and Translational Immunology, vol. 9, no. 10, e1191. https://doi.org/10.1002/cti2.1191

TY - JOUR

T1 - Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy

AU - Ebert, Lisa M.

AU - Yu, Wenbo

AU - Gargett, Tessa

AU - Toubia, John

AU - Kollis, Paris M.

AU - Tea, Melinda N.

AU - Ebert, Brenton W.

AU - Bardy, Cedric

AU - van den Hurk, Mark

AU - Bonder, Claudine S.

AU - Manavis, Jim

AU - Ensbey, Kathleen S.

AU - Oksdath Mansilla, Mariana

AU - Scheer, Kaitlin G.

AU - Perrin, Sally L.

AU - Ormsby, Rebecca J.

AU - Poonnoose, Santosh

AU - Koszyca, Barbara

AU - Pitson, Stuart M.

AU - Day, Bryan W.

AU - Gomez, Guillermo A.

AU - Brown, Michael P.

N1 - Funding Information: This work was supported by the Health Services Charitable Gifts Board (Adelaide) and a SA Cancer Council Beat Cancer Project Hospital Research Support Package (MB); the Neurosurgical Research Foundation (LE, MB, TG, MT, SP, GG); generous donations from the Sid Faithfull Family (KE, BD); a Senior Research Fellowship (1156693) from the National Health and Medical Research Council of Australia (SP); Flinders Foundation (CB); Cure Brain Cancer Foundation (CB, MB, GG, BD); Perpetual Impact Philanthropy (CB); the Brain Foundation (CB); Rebecca L. Cooper Foundation (CB); the Cancer Council SA Beat Cancer Project (to MPB and GAG); the Australian Research Council (FT160100366 to GAG); and a Netherlands Organisation for Scientific Research Rubicon Fellowship 019.163LW.032 (MvdH). SLP is supported by an Australian Government Research Training Program Scholarship. The authors would like to thank Samantha Escarbe and Nathalie Nataren for excellent technical assistance, Erica Yeo and Natansh Modi for assisting with the development of FAP immunostaining and Mark DeNichilo for HUVEC culture. We also acknowledge the support and generosity of the patients and medical and technical staff, including from SA Pathology and the SA Neurological Tumour Bank (supported by Flinders University, Flinders Foundation and The Neurosurgical Research Foundation), who made collection of tissue specimens possible. Publisher Copyright: © 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

PY - 2020/10/14

Y1 - 2020/10/14

N2 - 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.

AB - 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.

KW - blood vessels

KW - fibroblast activation protein

KW - glioblastoma

KW - immunotherapy

KW - scRNAseq

KW - target antigen

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U2 - 10.1002/cti2.1191

DO - 10.1002/cti2.1191

M3 - Article

AN - SCOPUS:85094149597

SN - 2050-0068

VL - 9

JO - Clinical and Translational Immunology

JF - Clinical and Translational Immunology

IS - 10

M1 - e1191

ER -

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

Abstract

Bibliographical note

Keywords

UN SDGs

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