High-Resolution Profiling of Human Vocal Fold Cellular Landscapes With Single-Nuclei RNA Sequencing.

INTRODUCTION: The function of the vocal folds (VFs) is determined by the phenotype, abundance, and distribution of differentiated cells within specific microenvironments. Identifying this histologic framework is crucial in understanding laryngeal disease. A paucity of studies investigating VF cellular heterogeneity has been undertaken. Here, we examined the cellular landscape of human VFs by utilizing single-nuclei RNA-sequencing. METHODS: Normal true VF tissue was excised from five patients undergoing pitch elevation surgery. Tissue was snap frozen in liquid nitrogen and subjected to cellular digestion and nuclear extraction. Nuclei were processed for single-nucleus sequencing using the 10X Genomics Chromium platform. Sequencing reads were assembled using cellranger and analyzed with the scanpy package in python. RESULTS: RNA sequencing revealed 18 global cell clusters. While many were of epithelial origin, expected cell types, such as fibroblasts, immune cells, muscle cells, and endothelial cells were present. Subcluster analysis defined unique epithelial, immune, and fibroblast subpopulations. CONCLUSION: This study evaluated the cellular heterogeneity of normal human VFs by utilizing single-nuclei RNA-sequencing. With further confirmation through additional spatial sequencing and microscopic imaging, a novel cellular map of the VFs may provide insight into new cellular targets for VF disease. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

MacroH2A restricts inflammatory gene expression in melanoma cancer-associated fibroblasts by coordinating chromatin looping.

MacroH2A has established tumour suppressive functions in melanoma and other cancers, but an unappreciated role in the tumour microenvironment. Using an autochthonous, immunocompetent mouse model of melanoma, we demonstrate that mice devoid of macroH2A variants exhibit increased tumour burden compared with wild-type counterparts. MacroH2A-deficient tumours accumulate immunosuppressive monocytes and are depleted of functional cytotoxic T cells, characteristics consistent with a compromised anti-tumour response. Single cell and spatial transcriptomics identify increased dedifferentiation along the neural crest lineage of the tumour compartment and increased frequency and activation of cancer-associated fibroblasts following macroH2A loss. Mechanistically, macroH2A-deficient cancer-associated fibroblasts display increased myeloid chemoattractant activity as a consequence of hyperinducible expression of inflammatory genes, which is enforced by increased chromatin looping of their promoters to enhancers that gain H3K27ac. In summary, we reveal a tumour suppressive role for macroH2A variants through the regulation of chromatin architecture in the tumour stroma with potential implications for human melanoma.

An atlas of late prenatal human neurodevelopment resolved by single-nucleus transcriptomics.

Late prenatal development of the human neocortex encompasses a critical period of gliogenesis and cortical expansion. However, systematic single-cell analyses to resolve cellular diversity and gliogenic lineages of the third trimester are lacking. Here, we present a comprehensive single-nucleus RNA sequencing atlas of over 200,000 nuclei derived from the proliferative germinal matrix and laminating cortical plate of 15 prenatal, non-pathological postmortem samples from 17 to 41 gestational weeks, and 3 adult controls. This dataset captures prenatal gliogenesis with high temporal resolution and is provided as a resource for further interrogation. Our computational analysis resolves greater complexity of glial progenitors, including transient glial intermediate progenitor cell (gIPC) and nascent astrocyte populations in the third trimester of human gestation. We use lineage trajectory and RNA velocity inference to further characterize specific gIPC subpopulations preceding both oligodendrocyte (gIPC-O) and astrocyte (gIPC-A) lineage differentiation. We infer unique transcriptional drivers and biological pathways associated with each developmental state, validate gIPC-A and gIPC-O presence within the human germinal matrix and cortical plate in situ, and demonstrate gIPC states being recapitulated across adult and pediatric glioblastoma tumors.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.

Functional Effects of Cardiomyocyte Injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.

Multiparameter Single-Cell Characterization of Ovarian Intratumor Heterogeneity.

Cancer is a complex disease rooted in heterogeneity, which is the phenomenon of individual cells, tissues, or patients having distinct phenotypic and/or genetic characteristics. Observed divergent disease etiology is likely rooted, at least in part, in tumor heterogeneity and the classification of distinct and important subpopulations of cells within the tumor and its associated microenvironment has remained a technical challenge. Standard next-generation sequencing of bulk tumor tissue provides an overall average genetic profile of the sample, and masks contributions from individual cells and minor populations of cells, particularly in heterogeneous samples. Only with the advent of single-cell analysis and sequencing technologies has it become possible to characterize key contributions of cellular subpopulations in order to more comprehensively characterize disease. This chapter describes a method to generate linked phenotypic and genotypic data at single-cell resolution using a real-time single-cell resolved platform. Specifically, the example method provided here is used to link cellular growth kinetics and expression of a prognostic marker protein, CA-125, in cells derived from ovarian cancer patients with their single-cell genomic profiles, but the method is translatable to other cell types and phenotypes of interest.

Myeloid Cell-associated Resistance to PD-1/PD-L1 Blockade in Urothelial Cancer Revealed Through Bulk and Single-cell RNA Sequencing.

PURPOSE: To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer. EXPERIMENTAL DESIGN: We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures. RESULTS: We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade-resistant metastatic urothelial cancer. CONCLUSIONS: The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood.See related commentary by Drake, p. 4139.

Application of Single-Cell Sequencing to Immunotherapy.

Cancer is a highly complex and heterogeneous disease and immunotherapy has shown promise as a therapeutic approach. The increased resolution afforded by single-cell analysis offers the hope of finding and characterizing previously underappreciated populations of cells that could prove useful in understanding cancer progression and treatment. Urologic and prostate cancers are inherently heterogeneous diseases, and the potential for single-cell analysis to help understand and develop immunotherapeutic approaches to treat these diseases is very exciting. In this review, we view cancer immunotherapy through a single-cell lens and discuss the state-of-the-art technologies that enable advances in this field.

Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells.

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

Plectin-containing, centrally localized focal adhesions exert traction forces in primary lung epithelial cells.

Receptor clustering upon cell attachment to the substrate induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, the extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micropatterned substrates: one ring of FAs located at the cell periphery (pFAs), and another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is normally absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent, with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties.