Shuyue Xin ¹ Shuang Wen ² Xiaopei He ¹ Yulong Zhao ¹ Hui Zhao ^1*

• ¹ Second Affiliated Hospital of Dalian Medical University, Dalian, China
• ² Dalian Municipal Friendship Hospital, Dalian, Liaoning Province, China

Background: Tertiary lymphoid structures (TLS), ordered structure of tumor-infiltrating immune cells in tumor immune microenvironment (TIME), play an important role in the development and anti-tumor immunity of various cancers, including liver, colon, and gastric cancers. Previous studies have demonstrated that the presence of TLS in intra-tumoral (IT), invasive margin (IM), and peri-tumoral (PT) regions of the tumors at various maturity statuses. However, the density of TLS in different regions of non-small cell lung cancer (NSCLC) has not been extensively studied.Methods: TLS and tumor-infiltrating immune cells were assessed using immunohistochemistry (IHC) staining in 82 NSCLC patients. Tumor samples were divided into three subregions as IT, IM and PT regions, and TLS were identified as early/primary TLS (E-TLS) or secondary/follicular TLS (F-TLS). The distribution of TLS in different maturity statuses, along with their correlation with clinicopathological characteristics and prognostic value, was assessed. Nomograms were used to predict the probability of 1-, 3-, and 5-year overall survival (OS) in patients with NSCLC.The density of TLS and proportion of F-TLS in the IT region (90.2%, 0.45/mm 2 , and 61.0%, respectively) were significantly higher than those in the IM region (72.0%, 0.18 /mm 2 , and 39.0%, respectively) and PT region (67.1%, 0.16 /mm 2 , and 40.2%, respectively). A lower density of TLS, especially E-TLS in the IM region, was correlated with better prognosis in NSCLC patients. CD20+ B cells, CD3+ T cells, CD8+ cytotoxic T cells, and CD68+ macrophages were significantly overexpressed in the IM region. CD20+ B cells and CD3+ T cells in the IM region were significantly correlated with the density of E-TLS, while no statistically significant correlation was found with F-TLS. The E-TLS density in the IM region and TNM stage were independent prognostic factors for NSCLC patients. The nomogram showed good prognostic ability.Conclusions: A higher density of E-TLS in the IM region was associated with a worse prognosis in NSCLC patients, potentially due to the inhibition of TLS maturation caused by the increased density of suppressive immune cells at the tumor invasion front.