CAR-T cell therapy is effective in hematologic malignancies but remains challenging in solid tumors owing to antigen heterogeneity and tumor microenvironment-induced exhaustion. Here, gene editing of the nuclear receptor NR2F6 restores CAR-T cell functionality, sustaining a TCF1⁺ progenitor-exhausted phenotype, enhancing metabolic fitness, and preserving cytotoxic potency under chronic antigen exposure. In immunocompetent models, Nr2f6-deficient CAR-T cells suppress solid tumor growth and induce robust, polyclonal host antitumor responses that persist after CAR-T clearance, as demonstrated by tumor re-challenge protection. Although infused CAR-T cells disappear within 2 weeks, durable tumor control coincides with epitope spreading and secondary immune responses, likely via dendritic cell reactivation. Protection against antigen-negative tumors and transferable immunity reveal a dual mode of direct cytotoxicity followed by durable immune reprogramming. This broadened host immunity may offset immune escape driven by antigen heterogeneity or loss, establishing NR2F6 inhibition as a promising CAR-T engineering strategy for durable, antigen-agnostic solid-tumor immunotherapy.

Abstract Classic hairy cell leukemia (HCL) is a rare indolent B-cell lymphoproliferative disorder characterized by the driver mutation BRAF V600E. Standard treatment with purine analogs (eg, cladribine) induces long-term remissions, but up to 25% of patients relapse early. Even when targeting BRAF V600E, residual HCL cells frequently persist in the bone marrow (BM). To identify additional biological alterations contributing to HCL persistence, we performed single-cell RNA sequencing of sorted primary HCL cells from long-term vs short-term cladribine responders (ST-R; >10 vs ≤3 years of progression-free survival) at diagnosis and from ST-R at diagnosis vs relapse. We identified a distinct HCL subcluster characterized by increased DUSP1, FOS, and JUND expression that was detected in all patients and persisted or even expanded at relapse. Cancer pathway analysis suggested enhanced tumor microenvironment dependence, as reflected by suppression of the p38 mitogen-activated protein kinase pathway. In the absence of a suitable BRAF V600E-mutated HCL cell line, we validated HAIR-M cells (BRAF D594E) as a bona fide experimental HCL model. The activating BRAF D594E mutation mimics V600E-induced downstream signaling that can be effectively targeted by BRAF inhibitors (BRAFi). HAIR-M coculture with BM stromal cells (BMSC) strongly induced DUSP1 and was accompanied by protection from BRAFi-induced HAIR-M apoptosis. The functional importance of DUSP1 was corroborated by showing that BMSC-induced protection from cell death could be overcome by DUSP1 inhibition. Our results may set the stage for future clinical testing of DUSP1 inhibition to eliminate minimal residual disease and prevent relapse in HCL.

The immune composition of the tumor microenvironment has a major impact on the therapy response in patients with colorectal cancer. Here, we built an atlas with 4.27 million single cells from 1,670 patient samples and complemented it with single-cell profiles from 266 patients, including cells with low mRNA content, spatial transcriptomics from 3.7 million cells, and protein profiles from 0.7 million cells. The analysis of the atlas allows tumor classification into immune desert, B cell enriched, T cell enriched, and myeloid cell enriched immune phenotypes. Within the myeloid compartment, we identify consensus myeloid gene expression programs with four immunomodulatory programs, and uncover a subpopulation of neutrophils with antigen-presenting properties. Moreover, functional experiments using patient-derived organoids show KRAS-dependent pro-tumorigenic polarization of neutrophils. Further, spatial multimodal single-cell profiling reveals niches with IL-1 signaling-based neutrophil-fibroblast interaction. Finally, using an orthotopic mouse model, we show that cancer-derived signals modify neutrophil production in the bone marrow.

Aims:Cardiac shockwave therapy (SWT) improves left ventricular (LV) function in patients with ischaemic cardiomyopathy. Shockwave therapy activates Toll-like receptor 3 (TLR3), a receptor-inducing chromatin remodelling and nuclear reprogramming of cardiac cells. We hypothesized that mechanical activation of TLR3 facilitates reprogramming of fibroblasts towards endothelial cells restoring myocardial perfusion and function. Methods and results:Human cardiac fibroblasts were treated by mechanical stimulation via SWT or TLR3 agonist Poly(I:C) in the presence of endothelial induction medium. A lineage tracing experiment was performed in a transgenic mouse model of Fsp1-Cre/LacZ mice after coronary occlusion. Left ventricular function and scarring were assessed. Single-cell sequencing including RNA trajectory analysis was performed. Chromatin remodelling and epigenetic plasticity were evaluated via western blot and Assay for Transposase-Accessible Chromatin sequencing. Mechanical stimulation of human fibroblasts with SWT activated TLR3 signalling and enhanced the expression of endothelial genes in a TLR3-dependent fashion. The induced endothelial cells (ECs) resembled genuine ECs in that they produced endothelial nitric oxide and formed tube-like structures in Matrigel. In a lineage tracing experiment in Fsp1-Cre/LacZ mice, shockwave treatment increased LacZ/CD31-positive cells (indicating transdifferentiation) after coronary occlusion. Furthermore, SWT reduced myocardial scar size and improved LV function. Single-cell RNA-seq and RNA trajectory analyses revealed that SWT induced an endothelial fibroblast cluster and mechanical stimulation induced significant changes in chromatin organization, with chromatin being more accessible after both treatments in 1705 genomic regions. Conclusion:Shockwave therapy enhances DNA accessibility via TLR3 activation and facilitates the transdifferentiation of fibroblasts towards endothelial cells in ischaemic myocardium.

Colorectal cancer liver metastasis (CRLM) is associated with poor survival, primarily due to acquired therapy resistance. While novel therapies arise, translation is limited by the lack of tumor models accurately representing dynamic microenvironmental interplay. Here, we show that ex vivo normothermic machine perfusion (NMP) offers a novel preclinical framework to study the intratumoral dynamics of CRLM biology. Six resected metastatic human livers were preserved for two days and subjected to multi-omic profiling of serially sampled adjacent liver and metastatic tissue using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST). Tissue integrity was assessed and cross-validated by immunofluorescence (IF), high-resolution respirometry (HRR) and flow-cytometry. NMP was successfuly applied to metastatic livers with minimal surgical adaptations, preserving both intrinsic hepatic properties and tissue viability over an extended duration. Single-cell and spatial mapping confirmed preservation of CRLM phenotypic properties and demonstrated high clinical translatability by applicability of the intrinsic epithelial consensus molecular subtypes to metastasis. Spatially deconvoluted pathway activities reflected functional tissue-microenvironments. Transcriptomic profiles – including those of tumor-associated myeloid cells – were preserved during NMP. Finally, we demonstrate tumor-associated myeloid cell persistence as a driver of disease progression and poor survival in colorectal cancer. Our findings represent the basis for future innovative applications adopting NMP in the context of CRLM, providing a new preclinical tumor model avenue.

Background:Dynamic remodelling of the tumour microenvironment (TME) plays a central role in prostate cancer (PCa) progression, immune evasion and therapy resistance. However, the co-existence of both tumour-promoting and tumour-restraining stromal elements necessitates extensive characterisation of the TME for effective targeting. Fibromuscular cell heterogeneity in PCa remains poorly characterised, in part due to challenges in isolating cells embedded within the desmoplastic stroma. This study therefore aimed to better characterise fibroblast and smooth muscle cell (SMC) populations as the major tissue-resident stromal cell subtypes within the PCa TME. Methods:A PCa single-cell RNA sequencing (scRNA-seq) dataset was re-analysed to define fibromuscular subtypes. Due to low fibroblast yields, an optimised tissue dissociation protocol was developed and benchmarked against two commercial kits via flow cytometry, immunostaining of clinical specimens and ex vivo culture. Dimensionality reduction and clustering were applied to the CD31- stromal fraction using a multiparameter surface marker panel. Annotation of the resulting clusters based on their surface marker profile was supported by integrating scRNA-seq and immuno-histological findings. Results:The optimised protocol yielded over twice the viable cells/mg tissue compared to two commercial kits, preserved surface marker integrity, enhanced successful cultivation of mesenchymal cells and recovered diverse stromal subpopulations from benign and malignant samples. Dimensionality reduction and clustering of flow cytometry counts identified 11 distinct CD31- stromal populations. Integration with transcriptomic data and immunofluorescence of clinical specimens identified spatially- and prognostically-distinct fibroblast subtypes, including inflammatory and myofibroblastic cancer-associated fibroblasts, pericytes linked to poor prognosis and a novel SMC subset associated with stromal activation. Conclusion:This study presents a robust workflow for improved isolation and characterisation of fibromuscular stromal cells in PCa. The multimodal approach enabled refined characterisation of phenotypically distinct and clinically-relevant stromal subpopulations within their spatial context providing a foundation for future TME-targeted therapies.

BackgroundHead and neck squamous cell carcinomas (HNSCC) are highly heterogeneous tumors. In the harsh tumor microenvironment (TME), metabolic reprogramming and mitochondrial dysfunction may lead to immunosuppressive phenotypes. Aerobic glycolysis is needed for the activation of cytotoxic T-cells and the absence of glucose may hamper the full effector functions of cytotoxic T-cells. To test the effect of mitochondrial dysfunction on cytotoxic T cell function, slice cultures (SC) of HNSCC cancer were cultivated under different metabolic conditions.MethodsTumor samples from 21 patients with HNSCC were collected, from which, SC were established and cultivated under six different conditions. These conditions included high glucose, T cell stimulation, and temporarily induced mitochondrial dysfunction (MitoDys) using FCCP and oligomycin A with or without additional T cell stimulation, high glucose and finally, a control medium. Over three days of cultivation, sequential T cell stimulation and MitoDys treatments were performed. Supernatant was collected, and SC were fixed and embedded. Granzyme B was measured in the supernatant and in the SC via immunohistochemistry (IHC). Staining of PD1, CD8/Ki67, and cleaved­caspase­3 (CC3) were performed in SC.ResultsHematoxylin eosin stains showed that overall SC quality remained stable over 3 days of cultivation. T cell stimulation, both alone and combined with MitoDys, led to significantly increased granzyme levels in SC and in supernatant. Apoptosis following T cell stimulation was observed in tumor and stroma. Mitochondrial dysfunction alone increased apoptosis in tumor cell aggregates. High glucose concentration alone had no impact on T cell activity and apoptosis. Apoptosis rates were significantly lower under conditions with high glucose and MitoDys (p=0.03).ConclusionStimulation of tumor-infiltrating lymphocytes in SC was feasible, which led to increased apoptosis in tumor cells. Induced mitochondrial dysfunction did not play a significant role in the activation and function of TILs in SC of HNSCC. Moreover, high glucose concentration did not promote cytotoxic T cell activity in HNSCC SC.

Colorectal cancer (CRC) features molecular heterogeneity and differing immune cell infiltration patterns, affecting how patients respond to therapy. However, a comprehensive understanding of immune cell phenotypes across patient subgroups is missing. Here, we dissect the CRC tumor microenvironment by integrating 4,27 million single cells from 1670 samples and 650 patients across 49 studies (77 datasets) representing 7 billion expression values. These samples encompass the full spectrum of disease progression, from normal colon to polyps, primary tumors, and metastases, covering both early and advanced stages of CRC. We present a high-resolution view of CRC with 62 major cell types or states, revealing different cell-type composition patterns in CRC subtypes. The atlas allows for refined tumor classification and patient stratification into four immune phenotypes: immune desert, myeloid, B cell, and T cell subtypes. Using bulk samples with genomic and clinical information, we identify cellular components associated with tumor histology and genotypes. These findings could have important implications for developing improved cancer immunotherapy approaches in CRC. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Cancer immune evasion is critical in non-small cell lung cancer (NSCLC) and has been targeted by immunotherapy. High soluble (s)PD-L1 is associated with reduced survival and treatment failure in advanced stages. Here we evaluated the effects of sPD-L1 on T cells, relapse free survival, and overall survival in early stage NSCLC. METHODS:In vitro T cell stimulation was performed in the presence of sPD-L1 to evaluate its immunomodulatory activity. Data from The Cancer Genome Atlas (TCGA) were investigated for PD-L1 splice variants and enzymes involved in proteolytic cleavage (i.e. ADAM10). Plasma from 74 NSCLC (stage IA-IIIB), as well as an additional 73 (control cohort) patients was collected prior to curative surgery. Thereafter sPD-L1 levels from an immunosorbent assay were correlated with patient outcome. RESULTS:In vitro sPD-L1 inhibited IFN-γ production and proliferation of T cells and induced a terminal effector CD4 T cell subtype expressing CD27. Data from the TCGA demonstrated that elevated mRNA levels of ADAM10 is a negative predictor of outcome in NSCLC patients. To investigate the clinical relevance of these in vitro and TCGA findings, we quantified sPD-L1 in the plasma of early-stage NSCLC patients. In the first cohort we found significantly higher sPD-L1 levels in relapsing NSCLC patients, with a multivariate analysis revealing high sPD-L1 (>1000 pg/mL) as an independent predictor of survival. However, these findings could not be validated in two independent control cohorts. DISCUSSION:Although in vitro and TCGA data support the suppressive effect of sPD-L1 we were unable to translate this in our clinical setting. These results may be due to the small patient number and their heterogeneity as well as the lack of a standardized sPD-L1 ELISA. Our inconclusive results regarding the value of sPD-L1 in early stage NSCLC warrant assay validation and further investigation in larger (neo-)adjuvant trials.

The present structure-activity relationship study investigates the development of novel chemosensitizers targeting therapy-resistant cancer stem cells (CSCs). We used 4 '-((2-propyl-1H-benzo[d]imidazole-1-yl)methyl)-[1,1 '-biphenyl]-2-carboxylic acid, derived from the angiotensin II type 1 receptor blocker telmisartan, as a lead structure, demonstrating that the biphenyl moiety is essential for chemosensitizing activity. Introducing a methyl carboxylate or carboxamide instead of the COOH-group significantly enhanced this effect, leading to the development of highly potent compounds. These novel, noncytotoxic chemosensitizers effectively target CSCs and overcome drug resistance by interfering with CSC persistence mechanisms-hyperactivated STAT5 signaling and increased drug transporter activity-with demonstrated efficacy in leukemia, ovarian, and prostate cancers. The carboxamide of telmisartan (telmi-amide, 7c) significantly reduced tumor growth in an imatinib-resistant leukemia xenograft model, both as monotherapy and combined with imatinib, showing promising oral bioavailability and tolerability. In summary, telmisartan derivatives act as effective chemosensitizers and offer an innovative strategy for targeting CSCs in various malignant diseases.

The successful recovery of immune cells, particularly those with low mRNA content, by single-cell RNA sequencing (scRNA-seq) remains a significant challenge. Tissue dissociation and selection of the appropriate scRNA-seq technology are crucial. Our protocol efficiently recovers low-mRNA content immune cells using the BD Rhapsody scRNA-seq platform. It includes optimized tissue dissociation for prostate, lung, and liver tissues, cell labeling with Sample Tag antibodies, microwell-based single-cell capture, cDNA synthesis, library preparation, and data pre-processing with basic quality control analysis.For complete details on the use and execution of this protocol, please refer to Salcher et al.1

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy.Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low-mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs, and various nonlymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single-cell suspensions from human nonlymphoid tissues including lung, skin, gingiva, intestine as well as from tumors and tumor-draining lymph nodes with a subsequent analysis of dendritic cells by flow cytometry. Further, prepared single-cell suspensions can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, etc. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

ABSTRACT The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy. Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low-mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results. SYNOPSIS Comparison of scRNA-seq protocols uncovers disparities in RNA-to-library conversion Microwell-based scRNA-seq technology excels in capturing low-mRNA content cells Biased transcriptomes due to gene specific RNA detection efficacies by both platforms The study guides in informed scRNA-seq platform selection and data interpretation

BACKGROUND:Elevated TCRαβ+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE:We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS:Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS:Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION:A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Introduction: Standard treatment with purine analogs such as cladribine (2-CdA) achieves excellent remissions in classic hairy cell leukemia (HCL), but up to 25% of patients relapse early and there is a non-diminishing risk of late relapse in complete responders. Even when targeting the recurrent genetic driver mutation BRAF V600E, HCL cells frequently persist in the bone marrow suggesting cooperating biologic alterations facilitating disease survival and persistence. Methods: Single-cell RNA sequencing (scRNA-Seq) was performed in primary HCL cells derived from three long-term (>10 years progression-free survival, PFS) and three short-term responders after 2-CdA treatment (SR, <3 years PFS) and from up to three time points (HCL diagnosis, first, and second relapse). Functional studies were performed using the BRAF D594E-mutated HAIR-M cell line after validating BRAF D594E as an activating mutation with similar downstream signaling and inhibitory potential with BRAF inhibitors as compared to BRAF V600E in primary HCL cells. Results: scRNA-Seq revealed distinct HCL cell clusters characterized by marked overexpression of DUSP1, FOS and JUND in all six patients that were outgrowing at first and second relapse in all three SR. In these DUSP1hi/FOShi/JUNDhi HCL cells, PROGENy cancer pathway analysis demonstrated induced activation of MAPK pathways and gene ontology analysis found negative regulation of the pro-apoptotic p38-MAPK-cascade as potential underlying biologic alteration. BRAF D594E-mutated HAIR-M cells were selected as substitute for primary HCL cells in cell culture due to comparable changes upon exposure to BRAF inhibitors in BRAF kinase activity conformation as assessed by the modular kinase conformation biosensor platform (KinCon) and similar reduction in ERK phosphorylation levels in HEK293T cells transiently overexpressing either flag-tagged BRAF D594E or BRAF V600E. In HAIR-M, we confirm stable expression levels of DUSP1, FOS and JUND, which were highly increased when co-cultured with HS5 stromal cells. Whereas increased expression of FOS and JUND was reversible upon exposure to BRAF inhibitors, expression of DUSP1, a direct target of BRAF-MEK-ERK signaling, and phosphorylation of p38 remained unaffected. As DUSP1 preferentially dephosphorylates/inactivates p38, we studied exposure of the DUSP1-inhibitor BCI in co-culture and found re-phosphorylated p38. Conclusion: DUSP1hi/FOShi/JUNDhi HCL cells may represent a distinct subset of HCL cells highly dependent on environmental cues with an inherently more resistant phenotype to treatment with 2-CdA or BRAF-inhibitors. Inhibition of DUSP1 may represent a novel therapeutic approach by effectively truncating HCL cells from extracellular pro-survival stimuli. Finally, our results suggest that BRAF D594E-mutated HAIR-M cells may serve as a representative disease model for functional studies. Keywords: bioinformatics, computational and systems biology, indolent non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

LSC analysis at 3 months time point by FISH

IntroductionNaïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells.MethodsHere we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions. ResultsThe quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate. DiscussionOverall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells.