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While triple-negative breast cancer (TNBC) is known to be heterogeneous at the genomic and transcriptomic levels, spatial information on tumor organization and cell composition is still lacking. Here, we investigate TNBC tumor architecture including its microenvironment using spatial transcriptomics on a series of 92 patients. We perform an in-depth characterization of tumor and stroma organization and composition using an integrative approach combining histomorphological and spatial transcriptomics. Furthermore, a detailed molecular characterization of tertiary lymphoid structures leads to identify a gene signature strongly associated to disease outcome and response to immunotherapy in several tumor types beyond TNBC. A stepwise clustering analysis identifies nine TNBC spatial archetypes, further validated in external datasets. Several spatial archetypes are associated with disease outcome and characterized by potentially actionable features. In this work, we provide a comprehensive insight into the complexity of TNBC ecosystem with potential clinical relevance, opening avenues for treatment tailoring including immunotherapy. Triple-negative breast cancer (TNBC) is a heterogenous disease with several molecular subtypes previously described. Here the authors perform a spatial transcriptomics analysis on a series of 92 patients, providing additional insights into the heterogeneity of TNBC, with implications for clinical outcomes and therapy.

Background We previously proposed an immune cell score (tumour node metastasis (TNM)-Immune cell score) classifier as an add-on to the existing TNM staging system for non-small cell lung cancer (NSCLC). Herein, we examined how to reliably assess a tertiary lymphoid structure (TLS) score to refine the TNM staging system. Methods Using immunohistochemistry (CD8/cytokeratin), we quantified TLS in resected NSCLC whole-tumour tissue sections with three different scoring models on two independent collections (total of 553 patients). In a pilot setting, NanoString gene expression signatures were analysed for associations with TLS. Results The number of TLSs significantly decreased in stage III patients as compared to stage II. The TLS score was an independent positive prognostic factor, regardless of the type of (semi)-quantification strategy used (four-scale semi-quantitative; absolute count of total TLS; subpopulation of mature TLS) or the endpoint (disease-specific survival; overall survival; time to recurrence). Subgroup analyses revealed a significant prognostic impact of TLS score within each pathological stage, patient cohort and main histological subtype. Targeted gene expression analysis showed that high TLS levels were associated with the expression of B cell and adaptive immunity genes/metagenes including tumour inflammation signature. Conclusions The TLS score increases the prognostic power in each pathological stage and hence has the potential to refine TNM staging in resected NSCLC.

High endothelial venules (HEVs) are specialized postcapillary venules composed of cuboidal blood endothelial cells that express high levels of sulfated sialomucins to bind L-Selectin/CD62L on lymphocytes, thereby facilitating their transmigration from the blood into the lymph nodes (LN) and other secondary lymphoid organs (SLO). HEVs have also been identified in human and murine tumors in predominantly CD3 + T cell-enriched areas with fewer CD20 + B-cell aggregates that are reminiscent of tertiary lymphoid-like structures (TLS). While HEV/TLS areas in human tumors are predominantly associated with increased survival, tumoral HEVs (TU-HEV) in mice have shown to foster lymphocyte-enriched immune centers and boost an immune response combined with different immunotherapies. Here, we discuss the current insight into TU-HEV formation, function, and regulation in tumors and elaborate on the functional implication, opportunities, and challenges of TU-HEV formation for cancer immunotherapy.

Tertiary lymphoid structures (TLSs), which consist of B cells, T cells, follicular dendritic cells and high endothelial venules, have recently been found to be associated with effective antitumor immune responses in patients with cancer. Tumor-infiltrating T cells and B cells have each been demonstrated to be associated with survival in patients with cancer. We hypothesized that TLSs, an assembly of immune cells, may be important for the initiation and/or maintenance of T cell and B cell responses against tumors. The aim of the present study was to examine the cellular mechanism of B cells in TLSs within gastric cancer and to understand the antitumor immune response of TLSs. Each B cell subset in a tumor was examined using flow cytometry to evaluate B cell differentiation and the functional status of B cells. In addition, B cell clonality was investigated by analyzing the B cell antigen receptor gene using PCR, and the function and formation/maintenance of TLSs were evaluated using reverse transcription-quantitative PCR. Tumor-infiltrating B cells were more differentiated compared with that in distant non-tumor tissues and tumor-draining lymph nodes. The PCR results revealed specific BCR gene expression in tumor-infiltrating B cells. The expression of co-stimulatory factors, CD80 and CD86, was observed, in addition to the constantly expressed major histocompatibility complex molecules (HLA-ABC and HLA-DR). CD70 was expressed in addition to CD27 in both CD20+ B cells and CD8+ T cells, indicating that these factors are activated together through their interaction. The mRNA expression levels of CCL21, CXCL13, PD-L1, perforin and granzyme B in TLSs was significantly higher compared with that in non-TLSs. The majority of tumor-infiltrating B cells in gastric cancer exist in the form of TLSs around the tumor and have been antigen-sensitized and differentiated, and proliferated in TLSs but not in the lymph nodes. In addition, B cells in TLSs might primarily function as antigen-presenting cells and be associated with the induction of cytotoxic T cells.

Tumors and their surrounding area represent spatially organized “ecosystems”, where tumor cells and the immune contextures of the different compartments are in a dynamic interplay, with potential clinical impact. Here, we aimed to investigate the prognostic significance of peritumoral tertiary lymphoid structures (TLS) either alone or jointly with the intratumoral densities and spatial distribution of CD8 + and CD163 + cells in breast cancer (BCa) patients. TLS were identified peritumorally, within the area distancing up to 5 mm from the infiltrative tumor border, counted and further characterized as adjacent or distal, in formalin-fixed, paraffin-embedded tumor tissue samples from a cohort of 167 patients, with histologically confirmed invasive ductal BCa. TLS and tumor-infiltrating immune cells were determined by H&E and immunohistochemistry. Clinical follow-up was available for 112 of these patients. Patients with peritumoral TLS exhibited worse disease-free survival (DFS) and overall survival (OS) as compared to patients lacking TLS. Moreover, the density of peritumoral TLS was found to be crucial for prognosis, since patients with abundant TLS exhibited the worst DFS and OS. By combining the density of adjacent TLS (aTLS) with our recently published intratumoral signatures based on the differential distribution of CD8 + and CD163 + in the tumor center and invasive margin, we created two improved immune signatures with superior prognostic strength and higher patient population coverage. Our observations strengthen the notion for the fundamental role of the dynamic interplay between the immune cells within the tumor microenvironment (center/invasive margin) and the tumor surrounding area (peritumoral TLS) on the clinical outcome of BCa patients.

Tertiary lymphoid structures (TLS) are clusters of immune cells that resemble and function similarly to secondary lymphoid organs (SLOs). While TLS is generally associated with an anti-tumour immune response in most cancer types, it has also been observed to act as a pro-tumour immune response. The heterogeneity of TLS function is largely determined by the composition of tumour-infiltrating lymphocytes (TILs) and the balance of cell subsets within the tumour-associated TLS (TA-TLS). TA-TLS of varying maturity, density, and location may have opposing effects on tumour immunity. Higher maturity and/or higher density TLS are often associated with favorable clinical outcomes and immunotherapeutic response, mainly due to crosstalk between different proportions of immune cell subpopulations in TA-TLS. Therefore, TLS can be used as a marker to predict the efficacy of immunotherapy in immune checkpoint blockade (ICB). Developing efficient imaging and induction methods to study TA-TLS is crucial for enhancing anti-tumour immunity. The integration of imaging techniques with biological materials, including nanoprobes and hydrogels, alongside artificial intelligence (AI), enables non-invasive in vivo visualization of TLS. In this review, we explore the dynamic interactions among T and B cell subpopulations of varying phenotypes that contribute to the structural and functional diversity of TLS, examining both existing and emerging techniques for TLS imaging and induction, focusing on cancer immunotherapies and biomaterials. We also highlight novel therapeutic approaches of TLS that are being explored with the aim of increasing ICB treatment efficacy and predicting prognosis.

Tertiary lymphoid structures (TLSs) are defined as lymphoid aggregates formed in non-hematopoietic organs under pathological conditions. Similar to secondary lymphoid organs (SLOs), the formation of TLSs relies on the interaction between lymphoid tissue inducer (LTi) cells and lymphoid tissue organizer (LTo) cells, involving multiple cytokines. Heterogeneity is a distinguishing feature of TLSs, which may lead to differences in their functions. Growing evidence suggests that TLSs are associated with various diseases, such as cancers, autoimmune diseases, transplant rejection, chronic inflammation, infection, and even ageing. However, the detailed mechanisms behind these clinical associations are not yet fully understood. The mechanisms by which TLS maturation and localization affect immune function are also unclear. Therefore, it is necessary to enhance the understanding of TLS development and function at the cellular and molecular level, which may allow us to utilize them to improve the immune microenvironment. In this review, we delve into the composition, formation mechanism, associations with diseases, and potential therapeutic applications of TLSs. Furthermore, we discuss the therapeutic implications of TLSs, such as their role as markers of therapeutic response and prognosis. Finally, we summarize various methods for detecting and targeting TLSs. Overall, we provide a comprehensive understanding of TLSs and aim to develop more effective therapeutic strategies.

Tertiary lymphoid structures (TLS) in the tumor microenvironment (TME) are emerging solid-tumor indicators of prognosis and response to immunotherapy. Considering that tumorigenesis requires metabolic reprogramming and subsequent TME remodeling, the discovery of TLS metabolic regulators is expected to produce immunotherapeutic targets. To identify such metabolic regulators, we constructed a metabolism-focused sgRNA library and performed an in vivo CRISPR screening in an orthotopic lung tumor mouse model. Combined with The Cancer Genome Atlas database analysis of TLS-related metabolic hub genes, we found that the loss of Acat1 in tumor cells sensitized tumors to anti-PD1 treatment, accompanied by increased TLS in the TME. Mechanistic studies revealed that ACAT1 resulted in mitochondrial protein hypersuccinylation in lung tumor cells and subsequently enhanced mitochondrial oxidative metabolism, which impeded TLS formation. Elimination of ROS by NAC or Acat1 knockdown promoted B cell aggregation and TLS construction. Consistently, data from tissue microassays of 305 patients with lung cancer showed that TLS were more abundant in non-small cell lung cancer (NSCLC) tissues with lower ACAT1 expression. Intratumoral ACAT1 expression was associated with poor immunotherapy outcomes in patients with NSCLC. In conclusion, our results identified ACAT1 as a metabolic regulator of TLS and a promising immunotherapeutic target in NSCLC.

Tertiary lymphoid structures (TLS) are acquired ectopic lymph follicle‐like structures observed inside and around tumors, in which clusters of CD20‐positive B lymphocytes are surrounded by CD3‐positive T lymphocytes. In many cancers, the existence of TLS is a useful biomarker for better prognosis and better response to immune checkpoint inhibitors (ICI) and plays important roles in activating anti‐tumor immunity. In order to induce TLS and enhance the therapeutic effect of ICI, we attempted to induce TLS using multiple chemokines in malignant melanoma, for which there have been no reports of TLS induction previously. Immunohistochemical analysis of tumor samples from 41 melanoma patients treated with ICI revealed TLS in 63.4% of cases. Patients with ≥ 5 TLS exhibited significantly improved disease‐specific survival compared to those with fewer or no TLS. Plasma chemokine profiling in 46 samples from 18 melanoma patients showed elevated CC motif chemokine ligand 21 (CCL21) in TLS‐positive samples before and after ICI treatment and CXC motif chemokine ligand 13 (CXCL13) significantly increased pre‐ to post‐ICI treatment in paired samples from TLS‐positive patients. In a mouse melanoma model, co‐administration of CXCL13 and CCL21 alongside anti‐programmed death ligand‐1 (PD‐L1) antibody therapy significantly increased TLS formation and improved tumor growth suppression. Gene expression analysis of human melanoma samples demonstrated that high CXCL13 and CCL21 expression correlated with upregulation of immune response, particularly B cell activation. These findings highlight the potential of chemokine‐based therapies. TLS induction using CXCL13 and CCL21 in combination may be useful for enhancing the effects of ICI therapy in melanoma. Plasma CCL21 levels were significantly increased in tertiary lymphoid structures (TLS)‐positive melanoma cases both before and after immune checkpoint inhibitors (ICI) treatment, while CXCL13 levels were significantly elevated after ICI treatment. We successfully induced TLS by co‐administering CXCL13 and CCL21 in a mouse melanoma model, clarifying that this could enhance the therapeutic effect of ICI treatment.

AbstractFibroblast-like synoviocytes (FLSs) are important non-immune cells located mostly in the inner layer of the synovium. Indeed, these cells are specialized mesenchymal cells, implicated in collagen homeostasis of the articular joint and provide extracellular matrix (ECM) materials for cartilage and contribute to joint destruction via multiple mechanisms. RA FLS interactions with immune and non-immune cells lead to the development and organization of tertiary structures such as ectopic lymphoid-like structures (ELSs), tertiary lymphoid organs (TLOs), and secretion of proinflammatory cytokines. The interaction of RA FLS cells with immune and non-immune cells leads to stimulation and activation of effector immune cells. Pathological role of RA FLS cells has been reported for many years, while molecular and cellular mechanisms are not completely understood yet. In this review, we tried to summarize the latest findings about the role of FLS cells in ELS formation, joint destruction, interactions with immune and non-immune cells, as well as potential therapeutic options in rheumatoid arthritis (RA) treatment. Our study revealed data about interactions between RA FLS and immune/non-immune cells as well as the role of RA FLS cells in joint damage, ELS formation, and neoangiogenesis, which provide useful information for developing new approaches for RA treatment.