ABSTRACT
BACKGROUND & AIMS: The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS: We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS: We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1+ macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligandâreceptor networks among malignant cells, SPP1+ macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1+ macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS: We identified that the TIB structure formed by the interaction of SPP1+ macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS: Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Mice , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Tumor Microenvironment , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy/methodsABSTRACT
BACKGROUND: The four major RNA adenosine modifications, i.e., m6A, m1A, alternative polyadenylation, and adenosine-to-inosine RNA editing, are mediated mostly by the "writer" enzymes and constitute critical mechanisms of epigenetic regulation in immune response and tumorigenesis. However, the cross-talk and potential roles of these "writers" in the tumor microenvironment (TME), drug sensitivity, and immunotherapy remain unknown. METHODS: We systematically characterized mRNA expression and genetic alterations of 26 RNA modification "writers" in colorectal cancer (CRC), and evaluated their expression pattern in 1697 CRC samples from 8 datasets. We used an unsupervised clustering method to assign the samples into two patterns of expression of RNA modification "writers". Subsequently, we constructed the RNA modification "writer" Score (WM_Score) model based on differentially expressed genes (DEGs) responsible for the RNA modification patterns to quantify the RNA modification-related subtypes of individual tumors. Furthermore, we performed association analysis for WM_Score and characteristics of TME, consensus molecular subtypes (CMSs), clinical features, transcriptional and post-transcriptional regulation, drug response, and the efficacy of immunotherapy. RESULTS: We demonstrated that multi-layer alterations of RNA modification "writer" are associated with patient survival and TME cell-infiltrating characteristics. We identified two distinct RNA modification patterns, characterized by a high and a low WM_Score. The WM_Score-high group was associated with worse patient overall survival and with the infiltration of inhibitory immune cells, such as M2 macrophages, EMT activation, and metastasis, while the WM_Score-low group was associated with a survival advantage, apoptosis, and cell cycle signaling pathways. WM_Score correlated highly with the regulation of transcription and post-transcriptional events contributing to the development of CRC. In response to anti-cancer drugs, WM_Score highly negatively correlated (drug sensitive) with drugs which targeted oncogenic related pathways, such as MAPK, EGFR, and mTOR signaling pathways, positively correlated (drug resistance) with drugs which targeted in apoptosis and cell cycle. Importantly, the WM_Score was associated with the therapeutic efficacy of PD-L1 blockade, suggesting that the development of potential drugs targeting these "writers" to aid the clinical benefits of immunotherapy. CONCLUSIONS: Our study is the first to provide a comprehensive analysis of four RNA modifications in CRC. We revealed the potential function of these writers in TME, transcriptional and post-transcriptional events, and identified their therapeutic liability in targeted therapy and immunotherapy. This work highlights the cross-talk and potential clinical utility of RNA modification "writers" in cancer therapy.
Subject(s)
Biomarkers, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Pharmacogenetics , RNA Processing, Post-Transcriptional , Tumor Microenvironment/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Combined Modality Therapy , Computational Biology/methods , Disease Management , Disease Susceptibility , Epithelial-Mesenchymal Transition , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Pharmacogenetics/methods , Prognosis , Proportional Hazards Models , RNA Processing, Post-Transcriptional/drug effects , Transcription, Genetic , Transcriptome , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
The tumor microenvironment (TME) represents a complex network in which tumor cells communicate not only with each other but also with stromal and immune cells. The intercellular interactions in the TME contribute to tumor initiation, progression, metastasis, and treatment outcome. Recent advances in spatial transcriptomics (ST) have revolutionized the molecular understanding of the TME at the spatial level. A comprehensive interactive analysis resource specifically designed for characterizing the spatial TME could facilitate further advances using ST. In this study, we collected 296 ST slides covering 19 cancer types and developed a computational pipeline to delineate the spatial structure along the malignant-boundary-nonmalignant axis. The pipeline identified differentially expressed genes and their functional enrichment, deconvoluted the cellular composition of the TME, reconstructed cell type-specific gene expression profiles at the sub-spot level, and performed cell-cell interaction analysis. Finally, the user-friendly database SpatialTME (http://www.spatialtme.yelab.site/) was constructed to provide search, visualization, and downloadable results. These detailed analyses are able to reveal the heterogeneous regulatory network of the spatial microenvironment and elucidate associations between spatial features and tumor development or response to therapy, offering a valuable resource to study the complex TME. SIGNIFICANCE: SpatialTME provides spatial structure, cellular composition, expression, function, and cell-cell interaction information to enable investigations into the tumor microenvironment at the spatial level to advance understanding of cancer development and treatment.
Subject(s)
Gene Expression Profiling , Tumor Microenvironment , Humans , Tumor Microenvironment/genetics , Gene Expression , Cell Transformation, Neoplastic , InternetABSTRACT
Stromal cells are physiologically essential components of the tumor microenvironment (TME) that mediates tumor development and therapeutic resistance. Development of a logical and unified system for stromal cell type identification and characterization of corresponding functional properties could help design antitumor strategies that target stromal cells. Here, we performed a pan-cancer analysis of 214,972 nonimmune stromal cells using single-cell RNA sequencing from 258 patients across 16 cancer types and analyzed spatial transcriptomics from 16 patients across seven cancer types, including six patients receiving anti-PD-1 treatment. This analysis uncovered distinct features of 39 stromal subsets across cancer types, including various functional modules, spatial locations, and clinical and therapeutic relevance. Tumor-associated PGF+ endothelial tip cells with elevated epithelial-mesenchymal transition features were enriched in immune-depleted TME and associated with poor prognosis. Fibrogenic and vascular pericytes (PC) derived from FABP4+ progenitors were two distinct tumor-associated PC subpopulations that strongly interacted with PGF+ tips, resulting in excess extracellular matrix (ECM) abundance and dysfunctional vasculature. Importantly, ECM-related cancer-associated fibroblasts enriched at the tumor boundary acted as a barrier to exclude immune cells, interacted with malignant cells to promote tumor progression, and regulated exhausted CD8+ T cells via immune checkpoint ligand-receptors (e.g., LGALS9/TIM-3) to promote immune escape. In addition, an interactive web-based tool (http://www.scpanstroma.yelab.site/) was developed for accessing, visualizing, and analyzing stromal data. Taken together, this study provides a systematic view of the highly heterogeneous stromal populations across cancer types and suggests future avenues for designing therapies to overcome the tumor-promoting functions of stromal cells. SIGNIFICANCE: Comprehensive characterization of tumor-associated nonimmune stromal cells provides a robust resource for dissecting tumor microenvironment complexity and guiding stroma-targeted therapy development across multiple human cancer types.
Subject(s)
Cancer-Associated Fibroblasts , Neoplasms , Humans , Tumor Microenvironment , Neoplasms/genetics , Neoplasms/therapy , Gene Expression Profiling , CD8-Positive T-LymphocytesABSTRACT
Although hypoxia is known to be associated with immune resistance, the adaptability to hypoxia by different cell populations in the tumor microenvironment and the underlying mechanisms remain elusive. This knowledge gap has hindered the development of therapeutic strategies to overcome tumor immune resistance induced by hypoxia. Here, bulk, single-cell, and spatial transcriptomics are integrated to characterize hypoxia associated with immune escape during carcinogenesis and reveal a hypoxia-based intercellular communication hub consisting of malignant cells, ALCAMhigh macrophages, and exhausted CD8+ T cells around the tumor boundary. A hypoxic microenvironment promotes binding of HIF-1α complex is demonstrated to the ALCAM promoter therefore increasing its expression in macrophages, and the ALCAMhigh macrophages co-localize with exhausted CD8+ T cells in the tumor spatial microenvironment and promote T cell exhaustion. Preclinically, HIF-1É inhibition reduces ALCAM expression in macrophages and exhausted CD8+ T cells and potentiates T cell antitumor function to enhance immunotherapy efficacy. This study reveals the systematic landscape of hypoxia at single-cell resolution and spatial architecture and highlights the effect of hypoxia on immunotherapy resistance through the ALCAMhigh macrophage-exhausted T cell axis, providing a novel immunotherapeutic strategy to overcome hypoxia-induced resistance in cancers.
ABSTRACT
Intestinal fibrosis, a severe complication of Crohn's disease (CD), is characterized by excessive extracellular matrix (ECM) deposition and induces intestinal strictures, but there are no effective anti-fibrosis drugs available for clinical application. We performed single-cell RNA sequencing (scRNA-seq) of fibrotic and non-fibrotic ileal tissues from CD patients with intestinal obstruction. Analysis revealed mesenchymal stromal cells (MSCs) as the major producers of ECM and the increased infiltration of its subset FAP+ fibroblasts in fibrotic sites, which was confirmed by immunofluorescence and flow cytometry. Single cell transcriptomic profiling of chronic Dextran Sulfate Sodium Salt (DSS) murine colitis model revealed Cd81+Pi16- fibroblasts exhibited transcriptomic and functional similarities to human FAP+ fibroblasts. Consistently, FAP+ fibroblasts were identified as the key subtype with the highest level of ECM production in fibrotic intestines. Furthermore, specific knockout or pharmacological inhibition of TWIST1, which was highly expressed by FAP+ fibroblasts, could significantly ameliorate fibrosis in mice. In addition, TWIST1 expression was induced by CXCL9+ macrophages enriched in fibrotic tissues via IL-1ß and TGF-ß signal. These findings suggest the inhibition of TWIST1 as a promising strategy for CD fibrosis treatment.
ABSTRACT
Although advances in spatial transcriptomics (ST) enlarge to unveil spatial landscape of tissues, it remains challenging to delineate pathology-relevant and cellular localizations, and interactions exclusive to a spatial niche (e.g., tumor boundary). Here, we develop Cottrazm, integrating ST with hematoxylin and eosin histological image, and single-cell transcriptomics to delineate the tumor boundary connecting malignant and non-malignant cell spots in tumor tissues, deconvolute cell-type composition at spatial location, and reconstruct cell type-specific gene expression profiles at sub-spot level. We validate the performance of Cottrazm along the malignant-boundary-nonmalignant spatial axis. We identify specific macrophage and fibroblast subtypes localized around tumor boundary that interacted with tumor cells to generate a structural boundary, which limits T cell infiltration and promotes immune exclusion in tumor microenvironment. In this work, Cottrazm provides an integrated tool framework to dissect the tumor spatial microenvironment and facilitates the discovery of functional biological insights, thereby identifying therapeutic targets in oncologic ST datasets.
Subject(s)
Fibroblasts , Tumor Microenvironment , Eosine Yellowish-(YS) , Gene Expression Profiling , HematoxylinABSTRACT
Colorectal cancer (CRC) is among the most common malignancies with limited treatments other than surgery. The tumor microenvironment (TME) profiling enables the discovery of potential therapeutic targets. Here, we profile 54,103 cells from tumor and adjacent tissues to characterize cellular composition and elucidate the potential origin and regulation of tumor-enriched cell types in CRC. We demonstrate that the tumor-specific FAP+ fibroblasts and SPP1+ macrophages were positively correlated in 14 independent CRC cohorts containing 2550 samples and validate their close localization by immuno-fluorescent staining and spatial transcriptomics. This interaction might be regulated by chemerin, TGF-ß, and interleukin-1, which would stimulate the formation of immune-excluded desmoplasic structure and limit the T cell infiltration. Furthermore, we find patients with high FAP or SPP1 expression achieved less therapeutic benefit from an anti-PD-L1 therapy cohort. Our results provide a potential therapeutic strategy by disrupting FAP+ fibroblasts and SPP1+ macrophages interaction to improve immunotherapy.