Abstract 3649: Stromal netrinG1-ligand (NGL1) constitutes a new modulator of pancreatic cancer immunosuppression
Authors: Vendramini-Costa Débora B. , Francescone Ralph , Franco-Barraza Janusz , Luong Tiffany , Martinez Esteban , Sykes Stephen , Steele Nina G. , Allen Benjamin L. , Pasca di Magliano Marina , Zhigarev Dmitry I. , Ogier Charline , Astsaturov Igor , Cai Kathy Q. , Klein-Szanto Andres J. , Wang Huamin , Campbell Kerry , Cukierman Edna 
Published: June 2022

Abstract
Pancreatic cancer (PC), is currently the third and predicted to soon become the second deadliest cancer in the US. A unique feature of PC is its fibrous tumor microenvironment (TME), marked by the expansion of cancer-associated fibroblasts (CAFs), highly bundled collagen I, and absence or inactivation of antitumor immune cells. As this TME is a physical and biochemical therapeutic barrier, a better understanding of how pro-tumor immunosuppression is modulated constitutes a highly sought-after goal. Herein we report that in addition to the identified ectopic expression of NetrinG1-Ligand (NGL1) in PC cells, NGL1 is detected in both immune cells and CAFs, with increased levels in CAFs associated with short PC patient overall survival. Further, in tumor-bearing mice, fibroblastic NGL1 expression correlates with PC progression while assorted immune cells express high levels of NGL1. To question the pro-PC role of stromal NGL1, we evaluated tumor progression in NGL1 knockout (KO) mice, using orthotopic PC allografts, and observed that tumors were significantly smaller. Single-cell RNA sequencing pointed to downregulation of pro-tumor transcripts in key assorted KO TME cells (e.g., T Cells). We also noted that tumor-bearing KO tissues included a TME with limited immunosuppressive cytokines, increased CD8+ and CD4+ T cells expressing low levels of tumor-promoting factors, and a limited amount of desmoplastic bundled collagen, all suggestive of a TGFβ-deficient milieu. In order to further dissect between the NGL1-dependent contributions of hematopoietic/immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow KO/WT chimeras. Results suggested that loss of NGL1 in one of the two compartments fails to phenocopy the full-body loss of NGL1, and was thus insufficient to hinder PC tumorigenesis. Further, depletion of selected immune cell subsets informed on NGL1-dependent T- and myeloid-cell contributions. Functional assays showed that KO macrophages released limited pro-tumor factors while KO T cells displayed increased proliferation compared to the WTs of both cell types. Fibroblastic NGL1-dependent immuno-regulatory effects were confirmed with human immune cells collected from healthy donors. Mechanistically, while immune cells and CAFs deficient in NGL1 are both tumor-suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the chimera and full-body KO results. Finally, KO effects were mirrored in WT CAFs treated with an NGL1-neutralizing molecule. Overall, our data suggest that stromal NGL1 is a novel and targetable modulator of PC immunosuppression
Citation Format: Débora B. Vendramini-Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina G. Steele, Benjamin L. Allen, Marina Pasca di Magliano, Dmitry I. Zhigarev, Charline Ogier, Igor Astsaturov, Kathy Q. Cai, Andres J. Klein-Szanto, Huamin Wang, Kerry Campbell, Edna Cukierman. Stromal netrinG1-ligand (NGL1) constitutes a new modulator of pancreatic cancer immunosuppression [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 3649.