ZEB1 expression in NEUROD1-positive and combined YAP1-dominant pulmonary neuroendocrine carcinomas is associated with a locally immunosuppressive microenvironment

Background: Pulmonary neuroendocrine carcinomas (pNECs), comprising small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), are classified into four molecular subtypes based on the expression of ASCL1, NEUROD1, POU2F3, and YAP1. In non-small cell lung cancer, Zinc finger E-box binding homeobox 1 (ZEB1), a key regulator of epithelial-mesenchymal transition (EMT), is more frequently expressed in poorly differentiated carcinoma (large cell and pleomorphic carcinoma) than in adenocarcinoma and squamous cell carcinoma. ZEB1 expression in cancer cells is linked to the decreased CD8+ T-cells and PD-L1 mediated immunosuppression. However, the features of ZEB1-positive SCLC/LCNEC are poorly understood. Aim: We investigated the expression of ZEB1 in pNECs and its association with tumor microenvironment.Methods: Western blotting was performed on pNEC cell lines (13 SCLC and 1 LCNEC: 8 ASCL1-dominant, 3 NEUROD1-dominant, 1 POU2F3-dominant, and 2 YAP1-domiant). Immunohistochemistry was conducted on surgically resected samples (33 SCLC and 46 LCNEC). Spatial transcriptome analysis was performed on one ZEB1-positive SCLC sample. Results: Western blotting using pNEC cell lines showed EMT-like phenotype in NEUROD1- and YAP1-dominant cell lines (high ZEB1, E-cadherin loss). Vimentin was absent in NEUROD1-dominant but present in YAP1-dominant cell lines. ASCL1- and POU2F3-dominant cell lines were largely epithelial (low/absent ZEB1, E-cadherin retained), although a part of ASCL1-dominant cell lines expressed ZEB1. In the xenograft from a ZEB1-positive ASCL1-dominant cell line (H2081), the expression of ZEB1 and E-cadherin was mutually exclusive; ZEB1-positive area was NEUROD1-positve, whereas ZEB1-negative area was ASCL1-positive. Immunohistochemistry on whole-slide sections from surgically resected samples revealed that most ZEB1-positive pNECs were NEUROD1-positive or combined YAP1-dominant cases (combined with adenocarcinoma or squamous cell carcinoma), with localized ZEB1 expression, whereas POU2F3-dominant cases showed low or no expression of ZEB1. Vimentin was highly expressed in a few combined YAP1-dominant pNECs but negative in most cases. The ZEB1-positive area exhibited significantly reduced CD3+ and CD8+ T cells infiltration and, in spatial transcriptome analysis, increased tumor-associated macrophages (TAMs) infiltration than in the ZEB1-negative area in the same cases. Conclusion: Focal expression of ZEB1 was observed in NEUROD1-positive and combined YAP1-dominant pulmonary neuroendocrine carcinomas. ZEB1-positive area was poor in CD3+ and CD8+ T cells and enriched in TAMs, suggesting that ZEB1-mediated EMT is associated with a locally immunosuppressive microenvironment.