Xinbo Gao ¹ Xiangqin Zhao ¹ Xuesong Li ² Jin Zhang ³ Hui Zhao ⁴ Ying Ma ^1*

• ¹ Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
• ² Shandong Cancer Hospital, Shandong University, Jinan, Shandong Province, China
• ³ Department of Orthopedics, First Affiliated Hospital, Nanjing Medical University, Nanjing, Liaoning Province, China
• ⁴ University of Texas MD Anderson Cancer Center, Houston, Texas, United States

Tertiary lymphoid structures (TLSs) (1,2) are organized clusters of immune cells that develop within non-lymphoid tissues under specific conditions, including autoimmunity, chronic infections, and cancer. These structures resemble lymphoid follicles, typically featuring a core of B cells surrounded by T cells, along with dendritic cells, a supporting network of extracellular matrix, and specialized high endothelial venules facilitating lymphocyte entry. TLSs are thought to recruit and activate naive T and B cells within the tumor microenvironment (TME) via chemokine signaling, contributing significantly to the complex interplay of immune cells and tumor cells within the TME.The TME in solid tumors comprises a complex ecosystem of tumor cells, stromal components, blood vessels, and immune cells. This environment plays a crucial role in tumor progression and its interaction with surrounding tissues. Tumor-infiltrating lymphocytes (TILs) exert a powerful influence within the TME, with cytotoxic TILs inhibiting tumor growth while certain suppressive or exhausted lymphocyte populations can promote it. TLS have been recognized as a significant source of TILs, and their presence often correlates with improved patient prognosis. However, our understanding of TLS function within the TME remains incomplete. Factors like TLS location, density, and maturity likely influence clinical outcomes, including survival and treatment response, across different cancer types. Furthermore, research into methods of manipulating TLS for therapeutic benefit is an area of active investigation, exploring their potential as immune niches to enhance existing and future cancer therapies. This editorial introduces a collection of articles in our Research Topic focused on TLS in solid tumors, exploring their anatomy, key features, immunological roles, and future research directions. These studies collectively highlight three key areas: the significance of TLS in predicting immunotherapy response and patient prognosis; the importance of assessing TLS maturity and density across different tissue types and spatial locations; and the crucial link between immune checkpoint pathways and TLS formation and maturation, with implications for understanding the mechanism of immune checkpoint inhibitors. (1) TLS and Prognostic Value in Cholangiocarcinoma and Pancreatic Cancer Cholangiocarcinoma (CCA), a malignancy of the biliary epithelium, carries a poor prognosis, hampered by the lack of reliable biomarkers for predicting treatment response and survival.Recognizing the role of tertiary lymphoid structures (TLS) as crucial microenvironments for anti-tumor immunity, Shang et al. investigated their prognostic value in a cohort of 471 CCA patients. Using H&E and immunohistochemical (IHC) staining to assess TLS maturity and composition, they observed varying degrees of TLS maturity and identified a four-gene signature (PAX5, TCL1A, TNFRSF13C, and CD79A) strongly expressed within TLS regions. High intratumoral TLS density correlated with improved overall survival (OS), while, interestingly, high peritumoral TLS density was associated with shorter OS.Similarly, a previous study (3) analyzed pancreatic cancer samples, identifying TLSassociated marker genes and developing a risk score model. This model stratified patients into high-and low-risk groups, with the low-risk group exhibiting increased immune cell infiltration and improved prognosis.(2) TLS in Colorectal Cancer: Challenges and Opportunities While TLS are generally associated with favorable outcomes in several cancers, their role in colorectal cancer (CRC) is more nuanced. Although some studies have linked TLS presence to improved OS, progression-free survival (PFS), disease-free survival (DFS), and recurrence-free survival (RFS), Yu et al. highlight the lack of significant association between TLS and OS in CRC-specific subgroup analyses. This discrepancy may stem from the presence of pre-existing lymphoid tissues like GALT or Peyer's patches, which could be misidentified as TLS. This approach could potentially enhance the prognostic utility of TLS in CRC. Furthering this line of inquiry, Xu Z. et al. developed a 14-gene TLS-related prognostic risk model, validated in TCGA and GEO datasets. They identified TLS-related subclusters and characterized hub genes, including PRRX1, a potential immunomodulatory factor and therapeutic target, whose expression was elevated in the TLS-positive CRC group.Their work showcases the combined power of bioinformatics, IHC, and multiplex immunofluorescence (MIF) for characterizing TLS and identifying clinically relevant markers. Characterizing TLS at a higher resolution, considering their functional, compositional, and spatial heterogeneity, is crucial for understanding their impact on patient survival. (1) TLS Formation, Maturation, and Characterization TLS development is a multi-stage process involving fibroblast activation, immune cell recruitment, and maturation, as detailed by Gao et al.( 4) Cytokines like IL-13, IL-17, and IL-22 play a role in the initial fibroblast priming by immune cells under inflammatory stress (5,6). Histologically, TLS maturity was primarily distinguished by the presence or absence of germinal centers (GCs), crucial sites for B cell maturation and affinity maturation. Mature TLS, containing GCs, exhibit proliferating B cells, follicular dendritic cells (FDCs) expressing DC-LAMP, and markers like Ki67, AID, and BCL6. More recently, a threetiered maturity model for lung cancer TLS has been proposed, classifying TLS as early (dense lymphocytic clusters without FDCs or GCs), intermediate ("primary follicle-like" with CD21+CD23-FDCs), and mature ("secondary follicle-like" with GCs) (7). This model underscores the importance of B cell maturation and humoral immunity in anti-tumor responses. However, TLS definitions vary across studies, with some relying on basic histological examination (H&E staining) and markers like PNAd or Ki67 (8,9), while others employ more rigorous characterization based on distinct T and B cell zones, FDCs, and high endothelial venules (HEVs) (10,11). This lack of standardization highlights the need for consistent criteria for defining and classifying TLS maturity. The maturation state and density of TLS vary based on tumor type and spatial location, leading to diverse prognostic implications. This spatial heterogeneity is further exemplified in melanoma, where increased peritumoral mature TLS density is associated with improved survival (13). In PDAC, TLS are more frequently found at the invasive margin than in the tumor core (14). While one study showed a predominance of peritumoral TLS in PDAC samples (8) , a more recent study highlighted the enhanced maturity, immune cell infiltration, and pro-inflammatory profile of the less abundant intratumoral TLS, associating them with improved survival (8). The dense, fibrotic stroma characteristic of PDAC may necessitate the close proximity of TLS to tumor cells for effective anti-tumor activity. (15)(16)(17) These findings underscore the complex interplay between TLS location, maturation, and the tumor microenvironment in shaping clinical outcomes. Further research is needed to fully elucidate the factors influencing TLS function and their relationship with tumor progression in different cancer types. (1) The Interplay Between Immune Checkpoints and TLS Formation Immune checkpoint inhibitors (ICIs) targeting pathways like PD-1/PD-L1 and CTLA-4/CD80 have shown promise in cancer treatment. Studies have linked TLS abundance and spatial distribution to ICI response in cholangiocarcinoma (CCA). A previous study (3) stratified pancreatic cancer patients into high-and low-risk groups based on TLS marker gene expression. The low-risk group exhibited higher expression of both co-stimulatory immune checkpoints (e.g., CD28, TNFRSF4, CTLA4, CD40LG, ICOSLG, LAG3, PDCD1, TIGIT) and the inhibitory checkpoint CD276. This suggests that patients with abundant and welldistributed TLS might respond more favorably to ICIs.(2) TLS as a Predictive Biomarker and Target for ICI Therapy Feng et al.'s work supports the link between TLS and ICI response. TLS presence could predict anti-PD-1 immunotherapy response in various cancers, including esophageal carcinoma, bladder cancer, melanoma, and head and neck squamous cell carcinoma (HNSCC), and may even be a direct target of PD-1 blockade (18)(19)(20). The association between high PD-1 expression at the invasive margin and TLS presence further suggests that context-specific PD-1 targeting within the tumor microenvironment may enhance efficacy (21). Xu Z. et al. also discussed a positive correlation between TLS and PD-L1 expression in colorectal cancer (CRC). These findings, along with evidence linking TLS to improved outcomes and immunotherapy efficacy in melanoma and breast cancer (22,23), suggest that TLS can convert "cold" tumors to "hot" by enhancing immune recognition and clearance (24). Furthermore, recent research suggests that combining immunotherapy with strategies to promote TLS formation or maturation could amplify treatment efficacy. Further research with large, prospective cohorts is needed to validate these findings and address limitations of previous retrospective studies, such as limited sample sizes and potential biases. Future studies should also incorporate more immunotherapy subgroups and address the challenge of comprehensively assessing TLS across the entire tumor. Larger sample sizes will help provide robust prognostic data and minimize the influence of individual differences and geographic variation. These efforts will advance the understanding of the complex relationship between TLS and ICI therapy, paving the way for more effective cancer immunotherapies. The articles in this Research Topic provide a meaningful overview of the crucial relationship between TLS and ICI immunotherapy, highlighting the clinical significance of TLS in promoting anti-tumor immunity and predicting its prognostic value in solid tumors. Future mechanistic studies are needed to further explore this complex interplay.