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.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

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.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

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.

Single-cell transcriptomic-informed deconvolution of bulk data identifies immune checkpoint blockade resistance in urothelial cancer.

Interactions within the tumor microenvironment (TME) significantly influence tumor progression and treatment responses. While single-cell RNA sequencing (scRNA-seq) and spatial genomics facilitate TME exploration, many clinical cohorts are assessed at the bulk tissue level. Integrating scRNA-seq and bulk tissue RNA-seq data through computational deconvolution is essential for obtaining clinically relevant insights. Our method, ProM, enables the examination of major and minor cell types. Through evaluation against existing methods using paired single-cell and bulk RNA sequencing of human urothelial cancer (UC) samples, ProM demonstrates superiority. Application to UC cohorts treated with immune checkpoint inhibitors reveals pre-treatment cellular features associated with poor outcomes, such as elevated SPP1 expression in macrophage/monocytes (MM). Our deconvolution method and paired single-cell and bulk tissue RNA-seq dataset contribute novel insights into TME heterogeneity and resistance to immune checkpoint blockade.

Urine scRNAseq reveals new insights into the bladder tumor immune microenvironment.

Due to bladder tumors' contact with urine, urine-derived cells (UDCs) may serve as a surrogate for monitoring the tumor microenvironment (TME) in bladder cancer (BC). However, the composition of UDCs and the extent to which they mirror the tumor remain poorly characterized. We generated the first single-cell RNA-sequencing of BC patient UDCs with matched tumor and peripheral blood mononuclear cells (PBMC). BC urine was more cellular than healthy donor (HD) urine, containing multiple immune populations including myeloid cells, CD4+ and CD8+ T cells, natural killer (NK) cells, B cells, and dendritic cells (DCs) in addition to tumor and stromal cells. Immune UDCs were transcriptionally more similar to tumor than blood. UDCs encompassed cytotoxic and activated CD4+ T cells, exhausted and tissue-resident memory CD8+ T cells, macrophages, germinal-center-like B cells, tissue-resident and adaptive NK cells, and regulatory DCs found in tumor but lacking or absent in blood. Our findings suggest BC UDCs may be surrogates for the TME and serve as therapeutic biomarkers.

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, 134:3193-3200, 2024.

Molecular profiling of human substantia nigra identifies diverse neuron types associated with vulnerability in Parkinson's disease.

Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.

Single cell view of tumor microenvironment gradients in pleural mesothelioma.

Immunotherapies have shown great promise in pleural mesothelioma (PM), yet most patients still do not achieve significant clinical response, highlighting the importance of improving understanding of the tumor microenvironment (TME). Here, we utilized high-throughput, single-cell RNA-sequencing to de novo identify 54 expression programs and construct a comprehensive cellular catalogue of the PM TME. We found four cancer-intrinsic programs associated with poor disease outcome and a novel fetal-like, endothelial cell population that likely responds to VEGF signaling and promotes angiogenesis. Throughout cellular compartments, we observe substantial difference in the TME associated with a cancer-intrinsic sarcomatoid signature, including enrichment in fetal-like endothelial cells, CXCL9+ macrophages, cytotoxic, exhausted, and regulatory T cells, which we validated using imaging and bulk deconvolution analyses on independent cohorts. Finally, we show, both computationally and experimentally, that NKG2A-HLA-E interaction between NK and tumor cells represents an important new therapeutic axis in PM, especially for epithelioid cases.

HIV-1 activates oxidative phosphorylation in infected CD4 T cells in a human tonsil explant model.

Introduction: Human immunodeficiency virus type 1 (HIV-1) causes a chronic, incurable infection leading to immune activation and chronic inflammation in people with HIV-1 (PWH), even with virologic suppression on antiretroviral therapy (ART). The role of lymphoid structures as reservoirs for viral latency and immune activation has been implicated in chronic inflammation mechanisms. Still, the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue remain unexplored. Methods: In this study, we utilized human tonsil explants from healthy human donors and infected them with HIV-1 ex vivo. We performed single-cell RNA sequencing (scRNA-seq) to analyze the cell types represented in the tissue and to investigate the impact of infection on gene expression profiles and inflammatory signaling pathways. Results: Our analysis revealed that infected CD4+ T cells exhibited upregulation of genes associated with oxidative phosphorylation. Furthermore, macrophages exposed to the virus but uninfected showed increased expression of genes associated with the NLRP3 inflammasome pathway. Discussion: These findings provide valuable insights into the specific transcriptomic changes induced by HIV-1 infection in different cell types within lymphoid tissue. The activation of oxidative phosphorylation in infected CD4+ T cells and the proinflammatory response in macrophages may contribute to the chronic inflammation observed in PWH despite ART. Understanding these mechanisms is crucial for developing targeted therapeutic strategies to eradicate HIV-1 infection in PWH.

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.

Spinal cord injury regulates circular RNA expression in axons.

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

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.

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.

High-resolution transcriptomics informs glial pathology in human temporal lobe epilepsy.

The pathophysiology of epilepsy underlies a complex network dysfunction between neurons and glia, the molecular cell type-specific contributions of which remain poorly defined in the human disease. In this study, we validated a method that simultaneously isolates neuronal (NEUN +), astrocyte (PAX6 + NEUN-), and oligodendroglial progenitor (OPC) (OLIG2 + NEUN-) enriched nuclei populations from non-diseased, fresh-frozen human neocortex and then applied it to characterize the distinct transcriptomes of such populations isolated from electrode-mapped temporal lobe epilepsy (TLE) surgical samples. Nuclear RNA-seq confirmed cell type specificity and informed both common and distinct pathways associated with TLE in astrocytes, OPCs, and neurons. Compared to postmortem control, the transcriptome of epilepsy astrocytes showed downregulation of mature astrocyte functions and upregulation of development-related genes. To gain further insight into glial heterogeneity in TLE, we performed single cell transcriptomics (scRNA-seq) on four additional human TLE samples. Analysis of the integrated TLE dataset uncovered a prominent subpopulation of glia that express a hybrid signature of both reactive astrocyte and OPC markers, including many cells with a mixed GFAP + OLIG2 + phenotype. A further integrated analysis of this TLE scRNA-seq dataset and a previously published normal human temporal lobe scRNA-seq dataset confirmed the unique presence of hybrid glia only in TLE. Pseudotime analysis revealed cell transition trajectories stemming from this hybrid population towards both OPCs and reactive astrocytes. Immunofluorescence studies in human TLE samples confirmed the rare presence of GFAP + OLIG2 + glia, including some cells with proliferative activity, and functional analysis of cells isolated directly from these samples disclosed abnormal neurosphere formation in vitro. Overall, cell type-specific isolation of glia from surgical epilepsy samples combined with transcriptomic analyses uncovered abnormal glial subpopulations with de-differentiated phenotype, motivating further studies into the dysfunctional role of reactive glia in temporal lobe epilepsy.

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.

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.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

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.