Fig 5 - available from: Nature Communications
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Response signatures outperform pathway methods for patient survival. a Pan-cancer associations between pathway scores and patient survival. Pathways on the horizontal axis and different methods on the vertical axis. Associations of survival increase (green) and decrease. Significance labels as indicated. Shades correspond to effect size, p values as indicated. b Volcano plot of cancers-specific associations between patient survival and inferred pathway score using PROGENy. Effect size on the horizontal axis. Below zero indicates increased survival (green), above decreased survival (red). FDRadjusted p values on the vertical axis. Size of the dots corresponds to number of patients in each cohort. c Kaplan-Meier curves of individual associations for kidney (KIRC), low-grade glioma (LGG), and adrenocortical carcinoma (ACC). Pathway scores are split in top and bottom quartiles and center half. Lines show the fraction of patients (vertical axis) that are alive at a given time (horizontal axis) within one year. P values for discretized scores
Source publication
Aberrant cell signaling can cause cancer and other diseases and is a focal point of drug research. A common approach is to infer signaling activity of pathways from gene expression. However, mapping gene expression to pathway components disregards the effect of post-translational modifications, and downstream signatures represent very specific expe...
Contexts in source publication
Context 1
... a leave-one-out strategy of model building and perturbation scoring, our inferred pathway activation is strongly (p < 10 -10 , except p < 10 -5 for Trail) associated with the pathway that was experimentally perturbed. The associations of a pathway signature with other pathways are weaker (p > 10 -5 ), except for EGFR with MAPK/PI3K and TNFa with NFkB/MAPK ( Fig. 2a and Supplementary Fig. 5, left), where there is biologically known cross-activation 27 . Relative activation patterns are consistent across input experiments ( Supplementary Fig. 5, right). ...
Context 2
... associations of a pathway signature with other pathways are weaker (p > 10 -5 ), except for EGFR with MAPK/PI3K and TNFa with NFkB/MAPK ( Fig. 2a and Supplementary Fig. 5, left), where there is biologically known cross-activation 27 . Relative activation patterns are consistent across input experiments ( Supplementary Fig. 5, right). ...
Context 3
... all cancer types, PROGENy found a strong association between the activation of EGFR, MAPK, PI3K, and Hypoxia pathways and decreased survival, similar to other signature methods (Fig. 5a). Gene Ontology found much weaker associations for expression of those pathways, and the other pathway mapping methods missed them almost entirely. PROGENy is the only method to find an increase in survival associated with the activation of the Trail/apoptosis pathway, while other methods show either a decrease or no effect, or we did ...
Context 4
... individual cancer types, PROGENy finds a similar separation between oncogenic and tumor-suppressor pathways (Fig. 5b), showing that it can capture both general and specific patterns in gene expression changes. Importantly, pathway mapping methods do not provide this separation and our associations are significant for more cancer types and more specific to individual pathways ( Supplementary Fig. 11). In addition, we find cancer-specific associations ...
Context 5
... and Low-Grade Glioma (LGG) show decreased survival with TNFa and JAK- STAT pathways, respectively, where specific activating mutations are much less known than for EGFR/MAPK. For these three associations, the top and bottom quartiles of PROGENy pathway activity were able to stratify patients in groups with over 25% difference in one year survival (Fig. 5c). These associations are stable when resampling patients (Supplementary Table 7). In summary, we can observe that signature-based methods generally outperform pathway mapping for survival associations, but the difference between PROGENy and the signatures of SPEED 5,26 and Gatza et al. 18 is less pronounced than for driver mutations or ...
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Citations
... Further analysis covered a wide range of cellular phenotypes and focused on calculating the gene signature scores of T cells [15], NK cells [17], B cells [18], macrophages [19], dendritic cells [19], and fibroblasts [20]. These scores were derived via the AddModuleScore function in the seurat framework, which incorporates gene sets (Table S2-7) curated from previous studies [15,[17][18][19][20]. Pathway-responsive genes for activity inference (Progeny) [21] analysis were also utilized to compare and assess the activation levels of specific tumour signaling pathways in epithelial cells between PL and LM. ...
Background
Colorectal neuroendocrine tumors with liver metastases (CRNELM) are associated with a poorer prognosis compared to their nonmetastatic counterparts. A comprehensive understanding of the tumor microenvironment (TME) heterogeneity between primary lesions (PL) and liver metastases (LM) could provide crucial insights for enhancing clinical management strategies for these patients.
Methods
We utilized single-cell RNA sequencing to analyze fresh tissue samples from CRNELM patients, aiming to elucidate the variations in TME between PL and LM. Complementary multidimensional validation was achieved through spatial transcriptomics, bulk RNA sequencing, and multiplex immunohistochemistry/immunofluorescence.
Results
Our single-cell RNA sequencing analysis revealed that LM harboured a higher proportion of CD8 + T cells, CD4 + T cells, NK cells, NKT cells, and B cells exhibiting a stress-like phenotype compared to PL. RGS5 + pericytes may play a role in the stress-like phenotype observed in immune cells within LM. MCs in PL (PL_MCs) and LM (LM_MCs) exhibit distinct activation of tumor-associated signaling pathways. Notably, COLEC11 + matrix cancer-associated fibroblasts (COLEC11_mCAFs) were found to be significantly associated with LM_MCs. Cell communication analysis unveiled potential targetable receptor-ligand interactions between COLEC11_mCAFs and LM_MCs. Multidimensional validation confirmed the prominence of the characteristic stress-like phenotypes, including HSPA6_CD8_Tstr, HSPA6_NK, and COLEC11_mCAFs in LM. Moreover, a higher abundance of COLEC11_mCAFs correlated with poorer survival rates in the neuroendocrine tumor patient cohort.
Conclusion
Overall, our study provides the first single-cell analysis of the cellular and molecular differences between PL and LM in CRNELM patients. We identified distinct cell subsets and receptor-ligand interactions that may drive TME discrepancies and support metastatic tumor growth. These insights highlight potential therapeutic targets and inform strategies for better managing CRNELM patients.
... These results revealed that the JAK/STAT3 pathway highly activated basal bladder cancer. The previous study provided a PROGENy algorithm showing a common core of pathwayresponsive genes by leveraging a large-scale compendium of available perturbation experiments (20). The weight > 0 of the gene indicates that the activation of the pathways accompanies gene expression. ...
As an antibody-drug conjugate (ADC), disitamab vedotin (RC48) is a promising treatment targeting ERBB2 for locally advanced and metastatic bladder cancer (BLCA). However, the subtype heterogeneity of muscle-invasive bladder cancer (MIBC) often leads to different therapeutic outcomes. In our study, we aim to explore sensitivity differences and mechanisms of different molecular subtypes of MIBC to RC48 treatment and develop a strategy for combination therapy against cancer. Using large-scale mRNA expression profile datasets, Western blotting, and immunohistochemistry, we first found that ERBB2 is upregulated in the luminal type but downregulated in basal bladder cancer. In addition, luminal cells showed higher sensitivity to RC48 than basal cells. Basal cells with ERBB2 overexpression demonstrated increased sensitivity to RC48 in vitro and in vivo, indicating that ERBB2 expression mediates RC48’s therapeutic efficacy against cancer. In basal or RC48-exposed luminal cells, the JAK/STAT3 pathway was highly enriched, suggesting that downregulation or pharmacological inhibition of ERBB2 leads to compensatory activation of this pathway. Silencing STAT3 increased the inhibitory efficacy of RC48. In addition, artesunate (ART, a STAT3 inhibitor) showed a synergistic effect with RC48 against basal bladder cancer both in vitro and in vivo. In summary, these findings provide a theoretical foundation for subsequent clinical trials combining RC48 and ART in MIBC based on molecular subtypes.
... Enriched pathways with P value and adjust p value < 0.05 were examined. PROGENy: Pathway RespOnsive GENes for activity inference was used to infer activities of 14 pathways [21]. PROGENy pathway activity scores were calculated for the Seurat object running 'progeny' command (organism="Human", top = 500, perm = 1, return_assay = TRUE). ...
Cancer-associated Fibroblasts (CAFs) have emerged as critical regulators of anti-tumour immunity, with both beneficial and detrimental properties that remain poorly characterised. To investigate this, we performed single-cell and spatial transcriptomic analysis, comparing head & neck squamous cell carcinoma (HNSCC) subgroups, which although heterogenous, can be considered broadly immune-hot and immune-cold (human papillomavirus [HPV]+ve and HPV-ve tumours respectively). This identified six fibroblast subpopulations, including two with immunomodulatory gene expression profiles (IL-11 + inflammatory [i]CAF and CCL19 + fibroblastic reticular cell [FRC]-like). IL-11 + iCAF were spatially associated with inflammatory monocytes and regulated in vitro through synergistic activation of canonical NF-κB signalling by IL-1β and TNF-α. FRC-like were enriched in immune-hot HPV+ve tumours, associated with CD4 + T-cells and B-cells in tertiary lymphoid structures and regulated through non-canonical NF-κB signalling via lymphotoxin. Pan-cancer analysis revealed several ‘iCAF’ subgroups present in both normal and cancer tissues; IL11 + iCAF were found in cancers from the gastrointestinal (GI) tract and transcriptomically distinct from iCAFs previously described in pancreatic and breast cancers with greater inflammatory properties; FRC-like fibroblasts were present at low frequencies in all tumour types, and were associated with significantly better survival in patients receiving checkpoint immunotherapy. This work clarifies and expands current literature on immunomodulatory CAFs, highlighting links with important immunological niches.
... However, to infer causal connections within the core network pathways regulating both OSS and STAT states and EC transitions, it is imperative to ascertain the responses in pathway activities, including the kinases and BRD proteins that were affected by drug perturbations. The existing methods for identifying enzyme transcriptional signatures and estimating enzyme activities are founded on database knowledge and cover only a few well-studied pathways (21,36). Therefore, we developed a data-driven identification of enzyme activity signatures. ...
Fluid shear stress (FSS) from blood flow sensed by vascular endothelial cells (ECs) determines vessel behavior, but regulatory mechanisms are only partially understood. We used cell state transition assessment and regulation (cSTAR), a powerful computational method, to elucidate EC transcriptomic states under low shear stress (LSS), physiological shear stress (PSS), high shear stress (HSS), and oscillatory shear stress (OSS) that induce vessel inward remodeling, stabilization, outward remodeling, or disease susceptibility, respectively. Combined with a publicly available database on EC transcriptomic responses to drug treatments, this approach inferred a regulatory network controlling EC states and made several notable predictions. Particularly, inhibiting cell cycle-dependent kinase (CDK) 2 was predicted to initiate inward remodeling and promote atherogenesis. In vitro, PSS activated CDK2 and induced late G 1 cell cycle arrest. In mice, EC deletion of CDK2 triggered inward artery remodeling, pulmonary and systemic hypertension, and accelerated atherosclerosis. These results validate use of cSTAR and identify key determinants of normal and pathological artery remodeling.
... The SFRP2+ RetD had the highest ECM score and collagen score of all fibroblast subpopulations (Figures 4A), while the PI16+ FB had the highest glycoproteins and proteoglycans scores ( Figure S8A), pointing to specific roles in ECM production for the different subsets. The TGFβ pathway activity inferred by using PROGENy signatures (20) was most enriched in SFRP2+ RetD FB ( Figure 4B). Comparison of fibroblast populations in SSc and normal skin revealed higher ECM and collagens scores and of TGFβ activity for several fibroblast populations in SSc skin ( Figure S8B). ...
Spatially non-resolved transcriptomic data identified functionally distinct populations of fibroblasts in health and disease. However, in-depth transcriptional profiling in situ at single-cell resolution has not been possible so far. Here, we studied fibroblast populations in the skin of SSc patients and healthy individuals using cyclic in situ hybridization (cISH) as a novel approach for spatially-resolved transcriptional phenotyping with subcellular resolution. cISH deconvoluted the heterogeneity of 20,979 cells including 3,764 fibroblasts (FB). BANKSY-based spatially-informed clustering identified nine FB subpopulations, with SFRP2+ RetD FB and CCL19+ nonPV FB as novel subpopulations that reside in specific cellular niches and display unique gene expression profiles. SFRP2+ RetD FB and CCL19+ nonPV FB as well as COL8A1+ FB, display altered frequencies in SSc skin and play specific, disease-promoting roles for extracellular matrix release and leukocyte recruitment as revealed by their transcriptional profile, their cellular interactions and ligand-receptor analyses. The frequencies of COL8A1+ FB and their interactions with monocytic cells and B cells are associated with progression of skin fibrosis in SSc. In summary, our spatially-resolved transcriptomic approach identified novel fibroblast subpopulations deregulated in SSc skin with specific pathogenic roles, some of which may potentially serve as biomarkers for progression of skin fibrosis.
... Viral read identification was based on the GATK Pathseq software kit 2.0 [27] with quantitative assessment expressed in VRM (viral read per million human reads). Viral status and type verification were performed using the VIRTUS pipeline [28]. ...
Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV-positive HNSCC) has distinct biological characteristics from HPV-negative HNSCC. Using an AI-based analytical platform on meta cohorts, we profiled expression patterns of viral transcripts and HPV viral genome integration, and classified the tumor microenvironment (TME). Unsupervised clustering analysis revealed five distinct and novel TME subtypes across patients (immune-enriched, highly immune and B-cell enriched, fibrotic, immune-desert, and immune-enriched luminal). These TME subtypes were highly correlated with patient prognosis. In order to understand specific factors associated with prognosis, we used the unsupervised clustering of an HPV-positive HNSCC cohort from The Cancer Genome Atlas (TCGA) (n = 53) based on HPV transcript expression, and identified four HPV-related subtypes (E2/E5, E6/E7, E1/E4 and L1/L2). Utilizing both viral transcript and TME subtypes, we found that the E2/E5 HPV subtype was associated with an immune-enriched TME and had a higher overall survival rate compared to other subtypes. The E2/E5 subtype was also enriched for samples without HPV-genome integration, suggesting that the episomal HPV status and E2/E5 expression pattern may be associated with an inflamed microenvironment and improved prognosis. In contrast, E6/E7 subtype samples were associated with the fibrotic and immune-desert TME subtypes, with lower values of T-cell and B-cell gene expression signatures and lower overall survival. Both E1/E4 and L1/L2 subtypes were associated with the immune-enriched luminal subtype. Our results suggest that HPV-transcript expression patterns may drive the modulation of the TME, and thereby impact prognosis.
... Additionally, tools originally developed for bulk RNA data have been adapted for single-cell analysis. DoRothEA [9] and PROGENy [26] designed for inferring transcription factor (TF)-target activities, are prime examples. Interestingly, Holland et al. [12] found that these bulk RNA-seq methods perform optimally even on simulated scRNAseq data, sometimes outperforming tools specifically designed for scRNA-seq analysis. ...
... While gene set tests evaluate pathway enrichment between conditions (e.g., healthy donors versus non-healthy patients), an alternative approach involves scoring pathway or gene signature activity in individual cells. In our initial benchmark tests, we compared several widely used tools for inferring gene set activity, including pathway activity in individual cells: AUCell, pathway over dispersion analysis using GSEA (Subramanian et al., 2005), PROGENy [26], Seurat [25] and PathSingle. ...
Pathway analysis is a powerful approach for elucidating insights from gene expression data and associating such changes with cellular phenotypes. The overarching objective of pathway research is to identify critical molecular drivers within a cellular context and uncover novel signaling networks from groups of relevant biomolecules. In this work, we present PathSingle, a Python-based pathway analysis tool tailored for single-cell data analysis. PathSingle employs a unique graph-based algorithm to enable the classification of diverse cellular states, such as T cell subtypes. Designed to be open-source, extensible, and computationally efficient, PathSingle is available at https://github.com/zurkin1/PathSingle under the MIT license. This tool provides researchers with a versatile framework for uncovering biologically meaningful insights from high-dimensional single-cell transcriptomics data, facilitating a deeper understanding of cellular regulation and function.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13040-024-00416-7.
... Ratios of spliced and unspliced mRNAs reveal trajectory toward trNK cluster. (I) Color-coded TGF-β pathway activity quantified with PROGENy (Schubert et al., 2018). (J) Colorcoded expression of indicated genes. ...
... Following normal processing, two clusters which likely represented doublets and stressed cells were excluded. TGF-β imprinting was measured using AddModuleScore with a list of TGF-β response genes (Plasari et al., 2009) and TGF-β pathway activity using PROGENy (Schubert et al., 2018). Single-cell regulatory network inference and clustering (SCENIC) on P20 group 1 ILCs was performed using the pySCENIC workflow with standard parameters (Aibar et al., 2017). ...
Group 1 innate lymphoid cells (ILCs) encompass NK cells and ILC1s, which have non-redundant roles in host protection against pathogens and cancer. Despite their circulating nature, NK cells can establish residency in selected tissues during ontogeny, forming a distinct functional subset. The mechanisms that initiate, maintain, and regulate the conversion of NK cells into tissue-resident NK (trNK) cells are currently not well understood. Here, we identify autocrine transforming growth factor-β (TGF-β) as a cell-autonomous driver for NK cell tissue residency across multiple glandular tissues during development. Cell-intrinsic production of TGF-β was continuously required for the maintenance of trNK cells and synergized with Hobit to enhance cytotoxic function. Whereas autocrine TGF-β was redundant in tumors, our study revealed that NK cell–derived TGF-β allowed the expansion of cytotoxic trNK cells during local infection with murine cytomegalovirus (MCMV) and contributed to viral control in the salivary gland. Collectively, our findings reveal tissue-specific regulation of trNK cell differentiation and function by autocrine TGF-β1, which is relevant for antiviral immunity.
... Tumour-associated signalling pathway activity of tumour cells was defined by PROGENy [58] v1. 16.0 based on the scRNA-Seq dataset. ...
T‐cell acute lymphoblastic leukaemia (T‐ALL) is a heterogeneous malignant disease with high relapse and mortality rates. To characterise the multiomics features of T‐ALL, we conducted integrative analyses using single‐cell RNA, TCR and chromatin accessibility sequencing on pre‐ and post‐treatment peripheral blood and bone marrow samples of the same patients. We found that there is transcriptional rewiring of gene regulatory networks in T‐ALL cells. Some transcription factors, such as TCF3 and KLF3, showed differences in activity and expression levels between T‐ALL and normal T cells and were associated with the prognosis of T‐ALL patients. Furthermore, we identified multiple malignant TCR clonotypes among the T‐ALL cells, where the clonotypes consisted of distinct combinations of the same TCR α and β chain per patient. The T‐ALL cells displayed clonotype‐specific immature thymocyte cellular characteristics and response to chemotherapy. Remarkably, T‐ALL cells with an orphan TCRβ chain displayed the strongest stemness and resistance to chemotherapy. Our study provided transcriptome and epigenome characterisation of T‐ALL cells categorised by TCR clonotypes, which may be helpful for the development of novel predictive markers to evaluate treatment effectiveness for T‐ALL.
... Unsupervised re-clustering of the 2449 cells bearing tumor-reactive TCRs based on differentially expressed genes and developmental trajectories revealed three pathways consisting of eight subclusters of tumor-reactive CD8 TILs (Fig. 5, A and B, and data file S6). Analysis of the signaling pathway activity of subclusters using decoupleR (36) and PROGENy (37) revealed that the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways were active in effector_3, effector_4, proliferating_1, and proliferating_2 ( Fig. 5C). In addition, analysis of transcription factor activity using the CollecTRI database (38) revealed the increased activity of transcription factor 7 (TCF7) in effector_3, effector_4, and proliferating_2 (Fig. 5D). ...
... (C) heatmap of the normalized signaling pathway activity for tumor-reactive cD8 Tils. Pathway activity was inferred by ProGeny (37) and decoupler (36). (D) heatmap of normalized transcription factor activity for tumor-reactive cD8 Tils inferred from collecTri (38) and decoupler (36). ...
Acral lentiginous melanoma (ALM) is the most common melanoma subtype in non-Caucasians. Despite advances in cancer immunotherapy, current immune checkpoint inhibitors remain unsatisfactory for ALM. Hence, we conducted comprehensive immune profiling using single-cell phenotyping with reactivity screening of the T cell receptors of tumor-infiltrating T lymphocytes (TILs) in ALM. Compared with cutaneous melanoma, ALM showed a lower frequency of tumor-reactive CD8 clusters and an enrichment of regulatory T cells with direct tumor recognition ability, suggesting a suppressive immune microenvironment in ALM. Tumor-reactive CD8 TILs showed heterogeneous expression of coinhibitory molecules, including KLRC1 (NKG2A), in subpopulations with therapeutic implications. Overall, our study provides a foundation for enhancing the efficacy of immunotherapy in ALM.
