Cancer immunotherapy is increasingly used in clinical practice, but its success rate is reduced by tumor escape from the immune system. This may be due to the genetic instability of tumor cells, which allows them to adapt to the immune response and leads to intratumoral immune heterogeneity. The study investigated spatial immune heterogeneity in the tumor microenvironment and its possible drivers in a mouse model of tumors induced by human papillomaviruses (HPV) following immunotherapy. Gene expression was determined by RNA sequencing and mutations by whole exome sequencing. A comparison of different tumor areas revealed heterogeneity in immune cell infiltration, gene expression, and mutation composition. While the mean numbers of mutations with every impact on gene expression or protein function were comparable in treated and control tumors, mutations with high or moderate impact were increased after immunotherapy. The genes mutated in treated tumors were significantly enriched in genes associated with ECM metabolism, degradation, and interactions, HPV infection and carcinogenesis, and immune processes such as antigen processing and presentation, Toll-like receptor signaling, and cytokine production. Gene expression analysis of DNA damage and repair factors revealed that immunotherapy upregulated Apobec1 and Apobec3 genes and downregulated genes related to homologous recombination and translesion synthesis. In conclusion, this study describes the intratumoral immune heterogeneity, that could lead to tumor immune escape, and suggests the potential mechanisms involved.

• MeSH
• imunoterapie * metody   MeSH
• infekce papilomavirem imunologie   virologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• mutace * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové mikroprostředí * imunologie   MeSH
• regulace genové exprese u nádorů MeSH
• sekvenování exomu MeSH
• únik nádoru z imunitní kontroly genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

PURPOSE: Patients with high-grade serous ovarian carcinoma (HGSOC) are virtually insensitive to immune checkpoint inhibitors (ICI) employed as standalone therapeutics, at least in part reflecting microenvironmental immunosuppression. Thus, conventional chemotherapeutics and targeted anticancer agents that not only mediate cytotoxic effects but also promote the recruitment of immune effector cells to the HGSOC microenvironment stand out as promising combinatorial partners for ICIs in this oncological indication. EXPERIMENTAL DESIGN: We harnessed a variety of transcriptomic, spatial, and functional assays to characterize the differential impact of neoadjuvant paclitaxel-carboplatin on the immunological configuration of paired primary and metastatic HGSOC biopsies as compared to neoadjuvant chemotherapy (NACT)-naïve HGSOC samples from five independent patient cohorts. RESULTS: We found NACT-driven endoplasmic reticulum stress and calreticulin exposure in metastatic HGSOC lesions culminates with the establishment of a dense immune infiltrate including follicular T cells (TFH cells), a prerequisite for mature tertiary lymphoid structure (TLS) formation. In this context, TLS maturation was associated with an increased intratumoral density of ICI-sensitive TCF1+PD1+ CD8+ T cells over their ICI-insensitive TIM-3+PD1+ counterparts. Consistent with this notion, chemotherapy coupled with a PD1-targeting ICI provided a significant survival benefit over either therapeutic approach in syngeneic models of HGSOC bearing high (but not low) tumor mutational burden. CONCLUSIONS: Altogether, our findings suggest that NACT promotes TLS formation and maturation in HGSOC lesions, de facto preserving an intratumoral ICI-sensitive T-cell phenotype. These observations emphasize the role of rational design, especially relative to the administration schedule, for clinical trials testing chemotherapy plus ICIs in patients with HGSOC. See related commentary by Bravo Melgar and Laoui, p. 10.

• MeSH
• CD8-pozitivní T-lymfocyty * imunologie   účinky léků   MeSH
• ektopické lymfoidní struktury * imunologie   patologie   MeSH
• hepatocytární jaderný faktor 1-alfa * genetika   metabolismus   MeSH
• inhibitory kontrolních bodů * terapeutické užití   farmakologie   MeSH
• karboplatina aplikace a dávkování   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• nádorové mikroprostředí * imunologie   účinky léků   MeSH
• nádory vaječníků * farmakoterapie   imunologie   patologie   MeSH
• neoadjuvantní terapie metody   MeSH
• paclitaxel aplikace a dávkování   terapeutické užití   farmakologie   MeSH
• protokoly protinádorové kombinované chemoterapie terapeutické užití   farmakologie   MeSH
• serózní cystadenokarcinom farmakoterapie   patologie   imunologie   MeSH
• stres endoplazmatického retikula účinky léků   imunologie   MeSH
• tumor infiltrující lymfocyty imunologie   účinky léků   metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Therefore, understanding the molecular regulatory mechanisms underlying the pathogenesis of DKD is imperative. In this study, we aimed to explore the molecular mechanisms of tubule region endothelial dysfunction in early DKD. Early-stage DKD model was established in 16-week-old female db/db mice for 16 weeks. Body weight, glucose level, and urine albumin-to-creatinine ratio (UACR) were measured. Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining were performed to evaluate pathological lesions. RNA sequencing data of the kidneys and integrated publicly available single-cell and spatial transcriptome datasets were used to investigate the mechanism of endothelial dysfunction. There was a significant increase in body weight (p = 0.001), glucose levels (p=0.0008), and UACR (p=0.006) in db/db mice compared with db/m mice. H&E and PAS staining showed that vacuolar lesions and protein casts of tubules were the major histopathological changes observed in early-stage DKD mice. The apoptotic pathway in endothelial cells was notably activated in DKD, and Thbs1 was identified as the central gene involved in this apoptotic process. Deconvolution of the cell composition in the RNA sequencing data showed a decrease in the proportion of endothelial cells in the DKD mice. Further analysis of the activity and regulatory network of transcription factors showed that Creb1 was activated in both mouse and human early-stage DKD, suggesting that Creb1 activation may be involved in early kidney injury. The endothelial cell apoptotic pathway is activated in DKD, and the proportion of endothelial cells was reduced in the DKD mice, which is significantly associated with Thbs1. Keywords: Diabetic kidney disease, Endothelial dysfunction, RNA sequencing,Thbs1, Creb1.

• MeSH
• apoptóza MeSH
• diabetické nefropatie * patologie   metabolismus   patofyziologie   genetika   MeSH
• endoteliální buňky metabolismus   patologie   MeSH
• ledvinové kanálky patologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• progrese nemoci * MeSH
• protein vázající cAMP responzivní element metabolismus   genetika   MeSH
• thrombospondin 1 metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Extramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor, even in the era of novel immunotherapies. Here, we applied spatial transcriptomics (RNA tomography for spatially resolved transcriptomics [tomo-seq] [n = 2] and 10x Visium [n = 12]) and single-cell RNA sequencing (n = 3) to a set of 14 EMD biopsies to dissect the 3-dimensional architecture of tumor cells and their microenvironment. Overall, infiltrating immune and stromal cells showed both intrapatient and interpatient variations, with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, which is consistent with genomic instability. We further identified the spatial expression differences between GPRC5D and TNFRSF17, 2 important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T cells. Notably, exhausted TIM3+/PD-1+ T cells diffusely colocalized with MM cells, whereas functional and activated CD8+ T cells showed a focal infiltration pattern along with M1 macrophages in tumor-free regions. This segregation of fit and exhausted T cells was resolved in the case of response to T-cell-engaging bispecific antibodies. MM and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.

• MeSH
• lidé MeSH
• mnohočetný myelom * genetika   patologie   imunologie   MeSH
• nádorové mikroprostředí * imunologie   genetika   MeSH
• stanovení celkové genové exprese MeSH
• T-lymfocyty imunologie   patologie   metabolismus   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

During pregnancy, two fetomaternal interfaces, the placenta-decidua basalis and the fetal membrane-decidua parietals, allow for fetal growth and maturation and fetal-maternal crosstalk, and protect the fetus from infectious and inflammatory signaling that could lead to adverse pregnancy outcomes. While the placenta has been studied extensively, the fetal membranes have been understudied, even though they play critical roles in pregnancy maintenance and the initiation of term or preterm parturition. Fetal membrane dysfunction has been associated with spontaneous preterm birth (PTB, < 37 weeks gestation) and preterm prelabor rupture of the membranes (PPROM), which is a disease of the fetal membranes. However, it is unknown how the individual layers of the fetal membrane decidual interface (the amnion epithelium [AEC], the amnion mesenchyme [AMC], the chorion [CTC], and the decidua [DEC]) contribute to these pregnancy outcomes. In this study, we used a single-cell transcriptomics approach to unravel the transcriptomics network at spatial levels to discern the contributions of each layer of the fetal membranes and the adjoining maternal decidua during the following conditions: scheduled caesarian section (term not in labor [TNIL]; n = 4), vaginal term in labor (TIL; n = 3), preterm labor with and without rupture of membranes (PPROM; n = 3; and PTB; n = 3). The data included 18,815 genes from 13 patients (including TIL, PTB, PPROM, and TNIL) expressed across the four layers. After quality control, there were 11,921 genes and 44 samples. The data were processed by two pipelines: one by hierarchical clustering the combined cases and the other to evaluate heterogeneity within the cases. Our visual analytical approach revealed spatially recognized differentially expressed genes that aligned with four gene clusters. Cluster 1 genes were present predominantly in DECs and Cluster 3 centered around CTC genes in all labor phenotypes. Cluster 2 genes were predominantly found in AECs in PPROM and PTB, while Cluster 4 contained AMC and CTC genes identified in term labor cases. We identified the top 10 differentially expressed genes and their connected pathways (kinase activation, NF-κB, inflammation, cytoskeletal remodeling, and hormone regulation) per cluster in each tissue layer. An in-depth understanding of the involvement of each system and cell layer may help provide targeted and tailored interventions to reduce the risk of PTB.

• MeSH
• amnion metabolismus   cytologie   MeSH
• chorion metabolismus   MeSH
• decidua * metabolismus   MeSH
• dospělí MeSH
• extraembryonální obaly * metabolismus   MeSH
• lidé MeSH
• porod v termínu genetika   MeSH
• předčasný odtok plodové vody genetika   metabolismus   MeSH
• předčasný porod * genetika   MeSH
• stanovení celkové genové exprese MeSH
• těhotenství MeSH
• transkriptom * MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The leishmaniases are globally important parasitic diseases for which no human vaccines are currently available. To facilitate vaccine development, we conducted an open-label observational study to establish a controlled human infection model (CHIM) of sand fly-transmitted cutaneous leishmaniasis (CL) caused by Leishmania major. Between 24 January and 12 August 2022, we exposed 14 participants to L. major-infected Phlebotomus duboscqi. The primary objective was to demonstrate effectiveness of lesion development (take rate) and safety (absence of CL lesion at 12 months). Secondary and exploratory objectives included rate of lesion development, parasite load and analysis of local immune responses by immunohistology and spatial transcriptomics. Lesion development was terminated by therapeutic biopsy (between days 14 and 42 after bite) in ten participants with clinically compatible lesions, one of which was not confirmed by parasite detection. We estimated an overall take rate for CL development of 64% (9/14). Two of ten participants had one and one of ten participants had two lesion recurrences 4-8 months after biopsy that were treated successfully with cryotherapy. No severe or serious adverse events were recorded, but as expected, scarring due to a combination of CL and the biopsy procedure was evident. All participants were lesion free at >12-month follow-up. We provide the first comprehensive map of immune cell distribution and cytokine/chemokine expression in human CL lesions, revealing discrete immune niches. This CHIM offers opportunities for vaccine candidate selection based on human efficacy data and for a greater understanding of immune-mediated pathology. ClinicalTrials.gov identifier: NCT04512742 .

• MeSH
• dospělí MeSH
• Leishmania major * imunologie   MeSH
• leishmanióza kožní * imunologie   parazitologie   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• parazitární zátěž MeSH
• Phlebotomus parazitologie   imunologie   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 (RAB31) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

• MeSH
• extracelulární vezikuly * metabolismus   MeSH
• glykolýza * MeSH
• jaterní cirhóza * metabolismus   patologie   genetika   MeSH
• jaterní hvězdicovité buňky * metabolismus   patologie   MeSH
• játra metabolismus   patologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Rab proteiny vázající GTP metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Spatial transcriptomics is revolutionizing modern biology, offering researchers an unprecedented ability to unravel intricate gene expression patterns within tissues. From pioneering techniques to newly commercialized platforms, the field of spatial transcriptomics has evolved rapidly, ushering in a new era of understanding across various disciplines, from developmental biology to disease research. This dynamic expansion is reflected in the rapidly growing number of technologies and data analysis techniques developed and introduced. However, the expanding landscape presents a considerable challenge for researchers, especially newcomers to the field, as staying informed about these advancements becomes increasingly complex. To address this challenge, we have prepared an updated review with a particular focus on technologies that have reached commercialization and are, therefore, accessible to a broad spectrum of potential new users. In this review, we present the fundamental principles of spatial transcriptomic methods, discuss the challenges in data analysis, provide insights into experimental considerations, offer information about available resources for spatial transcriptomics, and conclude with a guide for method selection and a forward-looking perspective. Our aim is to serve as a guiding resource for both experienced users and newcomers navigating the complex realm of spatial transcriptomics in this era of rapid development. We intend to equip researchers with the necessary knowledge to make informed decisions and contribute to the cutting-edge research that spatial transcriptomics offers.

• MeSH
• lidé MeSH
• stanovení celkové genové exprese * metody   MeSH
• transkriptom * MeSH
• výpočetní biologie metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

• MeSH
• alternativní sestřih genetika   MeSH
• endoteliální buňky metabolismus   MeSH
• expanze trinukleotidových repetic * genetika   MeSH
• Fuchsova endoteliální dystrofie * genetika   MeSH
• lidé MeSH
• rohovkový endotel metabolismus   patologie   MeSH
• transkripční faktor 4 * genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Our current understanding of whether B cell involvement in the tumor microenvironment benefits the patient or the tumor - in distinct cancers, subcohorts and individual patients - is quite limited. Both statements are probably true in most cases: certain clonal B cell populations contribute to the antitumor response, while others steer the immune response away from the desired mechanics. To step up to a new level of understanding and managing B cell behaviors in the tumor microenvironment, we need to rationally discern these roles, which are cumulatively defined by B cell clonal functional programs, specificities of their B cell receptors, specificities and isotypes of the antibodies they produce, and their spatial interactions within the tumor environment. Comprehensive analysis of these characteristics of clonal B cell populations is now becoming feasible with the development of a whole arsenal of advanced technical approaches, which include (1) methods of single-cell and spatial transcriptomics, genomics, and proteomics; (2) methods of massive identification of B cell specificities; (3) methods of deep error-free profiling of B cell receptor repertoires. Here we overview existing techniques, summarize their current application for B cells studies and propose promising future directions in advancing B cells exploration.

• MeSH
• B-lymfocyty MeSH
• lidé MeSH
• nádorové mikroprostředí MeSH
• nádory * MeSH
• podskupiny B-lymfocytů * MeSH
• receptory antigenů B-buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH