Abstract PR017: Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer
Authors: Costa Debora Barbosa Vendramini , Francescone Ralph , Franco-Barraza Janusz , Luong Tiffany , Martinez Esteban , Sykes Stephen , Steele Nina , di Magliano Marina Pasca , Zhigarev Dmitry , Ogier Charline , Wang Huamin , Astsaturov Igor , Campbell Kerry , Cukierman Edna 
Published: November 2022

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is soon to be the second deadliest types of cancer, with a 5-year survival of only 10%. The unique features of PDAC are the expansion of cancer-associated fibroblasts (CAFs), presence of a dense fibrous stroma with high bundled collagen I and immunosuppression, representing a challenge for therapies. Ways to overcome these unique pro-tumor features will be key for the development of better therapeutic strategies for PDAC. Herein we report that, in addition to the identified ectopic expression of NGL-1 in PDAC cells, it is also expressed in the stroma (CAFs and immune cells) of PDAC patients, inversely correlating with overall survival. Stromal NGL-1 was important for tumorigenesis in vivo, as NGL-1 knockout mice (KO) orthotopically allografted with pancreatic cancer cells presented less tumor burden. Further analysis showed that the tumors from KO mice presented more CD8+ T cells and less immunosuppressive cytokines, such as TGFβ. Single cell RNAseq analysis of these tumors showed decreased expression of pro-tumor factors, such as immune checkpoint molecules in T cells and TGFβ related genes across different cellular compartments (epithelial, immune and fibroblasts). In accordance with these results, these tumors presented a limited amount of desmoplastic bundled collagen, suggestive of a TGFβ-deficient environment. In order to further dissect between the NGL-1-dependent contributions of immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow chimeras and performed orthotopic injections to generate tumors. The loss of NGL-1 in each cellular compartment alone failed to phenocopy the full body loss of NGL-1, suggesting that NGL-1 in both immune cells and local stromal are important for tumorigenesis. Functionally, NGL-1 deficient CAFs failed to support the survival of starved PDAC cells in vitro, downregulated important myofibroblastic molecules such as p-smad, and produced less immunosuppressive cytokines, suggesting a role for NGL-1 in key pro-tumor features of CAFs. In fact, the fibroblastic NGL-1 dependent immunomodulatory effects were confirmed with human CD8+ T cells from healthy donors, which lost their cytotoxic profile in the presence of conditioned media (CM) from NGL-1+ CAFs, but were able to keep this profile when exposed to CM from NGL-1 deficient CAFs. Bone marrow derived macrophages from KO mice produced less pro-tumor cytokines and CD8+ T cells lacking NGL-1 proliferated more than those from wild type animals, when stimulated in vitro. Mechanistically, while immune cells and CAFs deficient in NGL-1 are both tumor suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the need for a global modulation of NGL-1 expression for an anti-tumor effect. Finally, NGL-1 KO mice orthotopically allografted with PDAC cells responded better (smaller tumors) to chemotherapeutical regimen (FOLFIRINOX) compared to WT animals. All these results point to NGL-1 as a potential new target to modulate immunosuppression and tumorigenesis in pancreatic cancer.
Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina Steele, Marina Pasca di Magliano, Dmitry Zhigarev, Charline Ogier, Huamin Wang, Igor Astsaturov, Kerry Campbell, Edna Cukierman. Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR017.