Cell Type-specific Differences Research Articles

Adaptive digital tissue deconvolution.

The inference of cellular compositions from bulk and spatial transcriptomics data increasingly complements data analyses. Multiple computational approaches were suggested and recently, machine learning techniques were developed to systematically improve estimates. Such approaches allow to infer additional, less abundant cell types. However, they rely on training data which do not capture the full biological diversity encountered in transcriptomics analyses; data can contain cellular contributions not seen in the training data and as such, analyses can be biased or blurred. Thus, computational approaches have to deal with unknown, hidden contributions. Moreover, most methods are based on cellular archetypes which serve as a reference; e.g. a generic T-cell profile is used to infer the proportion of T-cells. It is well known that cells adapt their molecular phenotype to the environment and that pre-specified cell archetypes can distort the inference of cellular compositions. We propose Adaptive Digital Tissue Deconvolution (ADTD) to estimate cellular proportions of pre-selected cell types together with possibly unknown and hidden background contributions. Moreover, ADTD adapts prototypic reference profiles to the molecular environment of the cells, which further resolves cell-type specific gene regulation from bulk transcriptomics data. We verify this in simulation studies and demonstrate that ADTD improves existing approaches in estimating cellular compositions. In an application to bulk transcriptomics data from breast cancer patients, we demonstrate that ADTD provides insights into cell-type specific molecular differences between breast cancer subtypes. A python implementation of ADTD and a tutorial are available at Gitlab and zenodo (doi:10.5281/zenodo.7548362).

Calcium plays an essential role in early-stage dendrite injury detection and regeneration

Dendrites are injured in a variety of clinical conditions such as traumatic brain and spinal cord injuries and stroke. How neurons detect injury directly to their dendrites to initiate a pro-regenerative response has not yet been thoroughly investigated. Calcium plays a critical role in the early stages of axonal injury detection and is also indispensable for regeneration of the severed axon. Here, we report cell and neurite type-specific differences in laser injury-induced elevations of intracellular calcium levels. Using a human KCNJ2 transgene, we demonstrate that hyperpolarizing neurons only at the time of injury dampens dendrite regeneration, suggesting that inhibition of injury-induced membrane depolarization (and thus early calcium influx) plays a role in detecting and responding to dendrite injury. In exploring potential downstream calcium-regulated effectors, we identify L-type voltage-gated calcium channels, inositol triphosphate signaling, and protein kinase D activity as drivers of dendrite regeneration. In conclusion, we demonstrate that dendrite injury-induced calcium elevations play a key role in the regenerative response of dendrites and begin to delineate the molecular mechanisms governing dendrite repair.

Germ fate determinants protect germ precursor cell division by reducing septin and anillin levels at the cell division plane.

Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formincyk-1(ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septinUNC-59 and anillinANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.

Linking Altered Nephrogenesis with Hypertension using Single Nuclei RNA Sequencing of Fetal Kidney at E15.5, E16.5 and E17.5 in a Novel Nephron Deficient Rat Model

This study sought to elucidate molecular mechanisms linking altered nephrogenesis in the HSRA rat, a novel model of nephron deficiency, to the development of hypertension and chronic kidney disease (CKD) later in life. The HSRA model gives rise to offspring born with a single kidney (-S) 50-75% of the time. The single kidneys from HSRA-S animals exhibit reduced nephron endowment compared to either kidney from two-kidney offspring (-C). We hypothesize that early molecular factors during development contribute to the observed reduction in nephron number in -S animals. Timed breeding and embryonic kidney isolation from -S and -C animals were performed at gestational days (GD) 15.5, 16.5, or 17.5. Single nuclei RNA sequencing (snRNA-seq) was conducted using n=3 -S kidneys and n=3 -C kidneys for each timepoint. The library was prepared using a 10x Chromium Single Cell 3’ kit, followed by sequencing on an Illumina NextSeq2000. The average number of cells captured per sample was 16,997, yielding an average of 25,335 reads/cell, with a median of 1,512 genes/cell identified. Bioinformatic analysis using Cell Ranger and Seurat (v4) revealed various renal progenitor cell types including metanephric mesenchymal (MM) and ureteric bud cells, alongside tubular, stromal, and glomerular endothelial progenitors. Podocytes emerged by GD17.5, originating from the MM cell population, as evidenced by their extension from this cluster. Global differential expression analysis revealed minimal transcriptional differences between -S and -C fetal kidneys at GD 16.5 and 17.5. However, a down-regulation of ribosomal genes was observed on GD15.5 in the -S group, suggesting premature cellular differentiation contributing to the diminished nephron formation. Markedly, cell type-specific gene expression differences were observed at GD 16.5 and 17.5, highlighting the superior resolution that snRNAseq provides compared to bulk RNA sequencing methods. We are currently performing trajectory analysis using Monocle3 to investigate cellular fate pathways and uncover genes involved during nephron progenitor cell differentiation. These preliminary findings shed light on the cellular and molecular landscape during critical developmental stages in the kidney of the HSRA rat that could link developmental factors affecting nephron number to hypertension and CKD pathologies later in life. This research was generously supported by the Graduate School of Health Sciences at UMMC. MRG is supported by NIH R01HL137673. The UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS, including Molecular Center of Health and Disease-COBRE (P20GM144041-Garrett), Mississippi INBRE (P20GM103476-Flynt), and Cardiorenal and Metabolic Diseases-COBRE (P20GM104357-Hall). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

Cell-type specific inhibitory plasticity in subicular pyramidal cells.

The balance between excitation and inhibition is essential to the proper function of cortical circuits. To maintain this balance during dynamic network activity, modulation of the strength of inhibitory synapses is a central requirement. In this study, we aimed to characterize perisomatic inhibition and its plasticity onto pyramidal cells (PCs) in the subiculum, the main output region of the hippocampus. We performed whole-cell patch-clamp recordings from the two main functional PC types, burst (BS) and regular spiking (RS) neurons in acute rat hippocampal slices and applied two different extracellular high-frequency stimulation paradigms: non-associative (presynaptic stimulation only) and associative stimulation (concurrent pre-and postsynaptic stimulation) to induce plasticity. Our results revealed cell type-specific differences in the expression of inhibitory plasticity depending on the induction paradigm: While associative stimulation caused robust inhibitory plasticity in both cell types, non-associative stimulation produced long-term potentiation in RS, but not in BS PCs. Analysis of paired-pulse ratio, variance of IPSPs, and postsynaptic Ca2+ buffering indicated a dominant postsynaptic calcium-dependent signaling and expression of inhibitory plasticity in both PC types. This divergence in inhibitory plasticity complements a stronger inhibition and a higher intrinsic excitability in RS as compared to BS neurons, suggesting differential involvement of the two PC types during network activation and information processing in the subiculum.

Single-nucleus RNA velocity reveals critical synaptic and cell-cycle dysregulations in neuropathologically confirmed Alzheimer’s disease

Typical differential single-nucleus gene expression (snRNA-seq) analyses in Alzheimer’s disease (AD) provide fixed snapshots of cellular alterations, making the accurate detection of temporal cell changes challenging. To characterize the dynamic cellular and transcriptomic differences in AD neuropathology, we apply the novel concept of RNA velocity to the study of single-nucleus RNA from the cortex of 60 subjects with varied levels of AD pathology. RNA velocity captures the rate of change of gene expression by comparing intronic and exonic sequence counts. We performed differential analyses to find the significant genes driving both cell type-specific RNA velocity and expression differences in AD, extensively compared these two transcriptomic metrics, and clarified their associations with multiple neuropathologic traits. The results were cross-validated in an independent dataset. Comparison of AD pathology-associated RNA velocity with parallel gene expression differences reveals sets of genes and molecular pathways that underlie the dynamic and static regimes of cell type-specific dysregulations underlying the disease. Differential RNA velocity and its linked progressive neuropathology point to significant dysregulations in synaptic organization and cell development across cell types. Notably, most of the genes underlying this synaptic dysregulation showed increased RNA velocity in AD subjects compared to controls. Accelerated cell changes were also observed in the AD subjects, suggesting that the precocious depletion of precursor cell pools might be associated with neurodegeneration. Overall, this study uncovers active molecular drivers of the spatiotemporal alterations in AD and offers novel insights towards gene- and cell-centric therapeutic strategies accounting for dynamic cell perturbations and synaptic disruptions.

Abstract 1541: Heterogeneity of the hormone receptor-positive breast tumor immune microenvironment according to patient’s body mass index

Abstract Background: Obesity is a risk factor for postmenopausal hormone receptor-positive (HR+) breast cancer (BC) and is associated with poorer outcomes. “Obesity-paradox”, a phenomenon where obesity (BMI ≥ 30) is associated with better outcomes in patients treated with immune checkpoint inhibitors (ICI), has been observed in several cancer types. It is however under-explored in BC, especially HR+ BC. Emerging data from recent trials (KEYNOTE-756 and CheckMate 7FL) have shown the potential benefits of ICI for treating early HR+ BC. This prompted the need for better insights into the biological link between obesity and HR+ BC, particularly its tumor immune microenvironment (TIME). Patients & Methods: A monocentric cohort of 33 patients (11 lean, 11 overweight, 11 obese) with early HR+ BC was selected for the study, whose tumor tissues were collected and subjected to snRNA-seq. Unsupervised clustering and cluster annotation were performed on the processed data leading to identification of cell types and subtypes. We then performed differential gene expression (DGEA) and gene set enrichment analyses (GSEA) according to BMI category for each of the immune cell types. Results: We identified a total of 76,773 immune cells belonging to six major cell types, namely B/plasma cell, dendritic cell (DC), mast cell, monocyte/macrophage (mono/macro), T cell, and natural killer (NK) cell. Their proportions varied between patients, however immune cellular composition was not associated with BMI. DGEA and GSEA revealed highly cell type-specific obesity-associated differences in the expression profile of immune cells. Notably, an enrichment of B cell-mediated immune activities, substantiated by the upregulation of Ig genes, was observed in B cells and plasma cells. This was however coupled with an overexpression of the checkpoint BTLA in B cells. CD8+ and CD4+ T cells showed a lower level of activation (higher LEF1, TCF7, lower ANXA1, IL4R), and NK cells a decreased cytokine production (CD96, CD226) and conflicting differential expression of cytotoxic molecules (lower NKG7, higher granzymes). Checkpoint genes such as PDCD1, CTLA4, TIGIT, LAG3, HAVCR2 did not show significant changes in their expression in T/NK cells. Mast cells displayed a downregulation of antigen presenting genes (CD74, HLA-A/B/E). DC and mono/macro populations also exhibited both up- and down-regulation of genes involved in different immune pathways. Conclusion: Our current data suggested that obesity was associated with multi-directional changes in the immune activities, possibly implying an unresolved inflammation in the HR+ BC tissue. Further investigation incorporating other cell types and their interaction with immune cells in both tumor and matched normal mammary tissues is warranted and on-going, which could provide more understanding of HR+ breast TIME and lead to speculation on therapeutic implications. Citation Format: Ha-Linh Nguyen, Tatjana Geukens, Maxim De Schepper, Karen Van Baelen, Marion Maetens, Gitte Zels, Amena Mahdami, Edoardo Isnaldi, Sigrid Hatse, Joke Verbeke, Christophe Matthys, Bernard Thienpont, Evy Vanderheyden, Thomas Van Brussel, Rogier Schepers, Gino Philips, Bram Boeckx, Diether Lambrechts, Giuseppe Floris, François Richard, Christine Desmedt. Heterogeneity of the hormone receptor-positive breast tumor immune microenvironment according to patient’s body mass index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1541.

Argon plasma-modified bacterial nanocellulose: Cell-specific differences in the interaction with fibroblasts and endothelial cells

Unique properties of bacterial nanocelullose (BNC), such as high purity, water retention, nanofibrillar structure and non-cytotoxicity, allow its application in regenerative medicine, particularly for active wound healing and for skin tissue engineering. However, its biological activity is not fully sufficient to be considered a good cell scaffold. This can be enhanced by physicochemical modifications, particularly plasma treatment. In this work, air-dried (AD) or lyophilized (L) BNC samples were modified in a direct current Ar+ plasma discharge (PM). BNC_AD samples showed higher surface roughness, tensile strength and Young's modulus but a lower swelling ratio than BNC_L samples. The swelling ratio of BNC_L samples further increased after PM. The samples were subjected to six-day in vitro cell culture tests with normal human dermal fibroblasts (NHDFs) or human umbilical vein endothelial cells (HUVECs), i.e. cell types important for skin defect healing. NHDFs adhered in higher numbers, by a larger cell spreading area and reached higher cell population densities on PM samples than on unmodified samples. However, in HUVECs, this effect of PM on cell growth was less pronounced or even opposite, especially on mechanically weaker and more swellable BNC_L. At the same time, both cell types preferred mechanically stronger BNC_AD for their adhesion and growth, which was more apparent in HUVECs. The expression of mRNA for markers of cell adhesion and phenotypic maturation in NHDFs (talin, vinculin, CD90) was generally similar on BNC_PM and on tissue culture polystyrene. In HUVECs, the expression of vinculin and PECAM-1 on all BNC samples was lower than on polystyrene, but the expression of KDR (VEGF receptor 2) was higher, especially on BNC_PM. These results indicate cell type-specific differences in the response to various BNC modifications, which must be properly balanced to accommodate various cell types needed for wound healing and skin tissue engineering.

Single-Cell Transcriptomic Profiling Identifies Molecular Phenotypes of Newborn Human Lung Cells.

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

GSK3α/β Restrain IFN-γ-Inducible Costimulatory Molecule Expression in Alveolar Macrophages, Limiting CD4+ T Cell Activation.

Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFN-γ is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFN-γ-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages. Our findings reveal that IFN-γ robustly activates both macrophage types; however, the profile of activated IFN-γ-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFN-γ. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3α/β alters the IFN-γ response. GSK3α/β controlled distinct IFN-γ responses, and in AM-like cells, we found that GSK3α/β restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFN-γ is restricted in a GSK3α/β-dependent manner and that IFN-γ responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

Astrocytes Drive Divergent Metabolic Gene Expression in Humans and Chimpanzees.

The human brain utilizes ∼20% of all of the body's metabolic resources, while chimpanzee brains use <10%. Although previous work shows significant differences in metabolic gene expression between the brains of primates, we have yet to fully resolve the contribution of distinct brain cell types. To investigate cell type-specific interspecies differences in brain gene expression, we conducted RNA-seq on neural progenitor cells, neurons, and astrocytes generated from induced pluripotent stem cells from humans and chimpanzees. Interspecies differential expression analyses revealed that twice as many genes exhibit differential expression in astrocytes (12.2% of all genes expressed) than neurons (5.8%). Pathway enrichment analyses determined that astrocytes, rather than neurons, diverged in expression of glucose and lactate transmembrane transport, as well as pyruvate processing and oxidative phosphorylation. These findings suggest that astrocytes may have contributed significantly to the evolution of greater brain glucose metabolism with proximity to humans.

K+ and pH homeostasis in plant cells is controlled by a synchronized K+ /H+ antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca2+ and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations. K+ and anion fluxes at both, PM and VM, affected H+ and Ca2+ , as changes in extracellular KCl or KNO3 concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca2+ ]cyt and the membrane potential. At both membranes, the K+ transporter networks mediated an antiport of K+ and H+ . By contrast, net transport of anions was accompanied by parallel H+ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K+ /H+ transport but cell type-specific differences in [H+ ]cyt and pH-dependent [Ca2+ ]cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H+ /K+ homeostasis and points to transceptor-like Ca2+ /H+ -based ion signaling in plant cells.

A comparative study of in vitro air–liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

BackgroundThe airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air–liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized.MethodsBCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens.ResultsWe confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus.ConclusionsTaken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

Yersinia deploys type III-secreted effectors to evade caspase-4 inflammasome activation in human cells.

Yersinia are responsible for significant disease burden in humans, ranging from recurrent disease outbreaks (yersiniosis) to pandemics (Yersinia pestis plague). Together with rising antibiotic resistance rates, there is a critical need to better understand Yersinia pathogenesis and host immune mechanisms, as this information will aid in developing improved immunomodulatory therapeutics. Inflammasome responses in human cells are less studied relative to murine models of infection, though recent studies have uncovered key differences in inflammasome responses between mice and humans. Here, we dissect human intestinal epithelial cell and macrophage inflammasome responses to Yersinia pseudotuberculosis. Our findings provide insight into species- and cell type-specific differences in inflammasome responses to Yersinia.

Single-cell profiling of murine bladder cancer identifies sex-specific transcriptional signatures with prognostic relevance

Bladder cancer (BLCA) is more common in men but more aggressive in women. Sex-based differences in cancer biology are commonly studied using a murine model with BLCA generated by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). While tumors in the BBN model have been profiled, these profiles provide limited information on the tumor microenvironment. Here, we applied single-cell RNA sequencing to characterize cell-type specific transcriptional differences between male and female BBN-induced tumors. We found proportional and gene expression differences in epithelial and non-epithelial subpopulations between male and female tumors. Expression of several genes predicted sex-specific survival in several human BLCA datasets. We identified novel and clinically relevant sex-specific transcriptional signatures including immune cells in the tumor microenvironment and it validated the relevance of the BBN model for studying sex differences in human BLCA. This work highlights the importance of considering sex as a biological variable in the development of new and accurate cancer markers.

Diverse intratumoral heterogeneity and immune microenvironment of two HPV-related cervical cancer types revealed by single-cell RNA sequencing.

Cervical squamous cell carcinoma (SCC) and adenocarcinoma (AD) are the main histological types of human papillomavirus-related cervical cancer. However, there are few reports on cell type-specific molecular differences between SCC and AD. Here, we used unbiased droplet-based single-cell RNA sequencing to elucidate the cellular differences between SCC and AD in tumor heterogeneity, and tumor microenvironment (TME). A total of 61 723 cells from three SCC and three AD patients, were collected and divided into nine cell types. Epithelial cells exhibited high intra- and interpatient heterogeneity and functional diversity. Signaling pathways, such as epithelial-to-mesenchymal-transition (EMT), hypoxia and inflammatory response were upregulated in SCC, while cell cycle-related signaling pathways were highly enriched in AD. SCC was associated with high infiltration of cytotoxicity CD8 T, effector memory CD8 T, proliferativenatural killer (NK), and CD160+ NK cells as well as tumor-associated macrophages (TAMs) with high major histocompatibility complex-II genes. AD exhibited a high proportion of naive CD8 T, naive CD4 T, Treg CD4, central memory CD8, and TAMs with immunomodulatory functions. Additionally, we also observed that the majority of cancer-associated fibroblasts (CAFs) were from AD, and participated in inflammation regulation, while SCC-derived CAFs exhibited similar functions to tumor cells, such as EMT and hypoxia. This study revealed the widespread reprogramming of multiple cell populations in SCC and AD, dissected the cellular heterogeneity and characteristics in TME, and proposed potential therapeutic strategies for CC, such as targeted therapy and immunotherapy.

Proximity Sequencing Enables Measurement of Protein Complexes in Single Cells.

Advances in single-cell sequencing technology have transformed our understanding of cellular heterogeneity and identity, providing insight on topics ranging from the diversity of microbial ecosystems to tumor biological behaviors. However, accurate measurement of protein complexes in single cells remains challenging as relatively few proteins can form large numbers of complexes that must be distinguished accurately. The inability to accurately differentiate protein complexes hinders in-depth study of critical biological functions such as immune signaling and cellular differentiation. In a recent article in Nature Methods, Vistain et al. (1) present a solution to this problem by combining proximity ligation assay (PLA) with single-cell RNA sequencing (scRNA-seq). This method, which they call proximity sequencing (Prox-seq), can measure hundreds of protein complexes and mRNAs in single cells simultaneously. Prox-seq introduces pairs of DNA-conjugated antibodies to single-cell sequencing methods. When proteins enter a complex, the DNA oligomers on the antibodies come into proximity and are ligated. These ligated DNA oligomers (PLA products) are then sequenced to retrieve protein complex information. Prox-seq also generates transcriptome data through mRNA sequencing, allowing multimodal analyses of single cells. To validate this assay, the authors first demonstrated Prox-seq’s ability to differentiate T and B cells in a mixed population using probes designed for 11 cell-specific protein targets. Prox-seq measurements showed that T and B cells could be accurately differentiated using mRNAs, proteins, or total PLA products. An orthogonal method of flow cytometry was used to confirm that the enriched PLA products in each cell type were cell-specific proteins. This confirmed that PLA products can be measured using scRNA-seq, and PLA data displays cell type-specific differences in protein expression. Quantification of protein expression analysis in the same cell types with a different panel of 13 Prox-seq probes showed high correlation with flow cytometry which confirmed accurate protein characterization and quantification by Prox-seq in single cells. The authors also tested Prox-seq’s ability to measure large numbers of protein complexes in 8700 human peripheral blood mononuclear cells with a panel of probe pairs targeting 38 immune cell markers, and up to 741 unique protein complexes. The data showed 37 protein complexes and measurements identifying the expected cell types. In addition, they identified a novel CD8 and CD9 interaction in naïve CD8 T cells. Lastly, authors demonstrated that the assay can identify variability of receptor components in individual human macrophages after activation of an innate immunity signaling pathway by measuring PLA products detected within a panel designed for 15 pathway-specific protein targets.

Effects of acute low-moderate dose ionizing radiation to human brain organoids.

Human exposure to low-to-moderate dose ionizing radiation (LMD-IR) is increasing via environmental, medical, occupational sources. Acute exposure to LMD-IR can cause subclinical damage to cells, resulting in altered gene expression and cellular function within the human brain. It has been difficult to identify diagnostic and predictive biomarkers of exposure using traditional research models due to factors including lack of 3D structure in monolayer cell cultures, limited ability of animal models to accurately predict human responses, and technical limitations of studying functional human brain tissue. To address this gap, we generated brain/cerebral organoids from human induced pluripotent stem cells to study the radiosensitivity of human brain cells, including neurons, astrocytes, and oligodendrocytes. While organoids have become popular models for studying brain physiology and pathology, there is little evidence to confirm that exposing brain organoids to LMD-IR will recapitulate previous in vitro and in vivo observations. We hypothesized that exposing brain organoids to proton radiation would (1) cause a time- and dose-dependent increase in DNA damage, (2) induce cell type-specific differences in radiosensitivity, and (3) increase expression of oxidative stress and DNA damage response genes. Organoids were exposed to 0.5 or 2 Gy of 250 MeV protons and samples were collected at 30 minute, 24 hour, and 48 hour timepoints. Using immunofluorescence and RNA sequencing, we found time- and dose-dependent increases in DNA damage in irradiated organoids; no changes in cell populations for neurons, oligodendrocytes, and astrocytes by 24 hours; decreased expression of genes related to oligodendrocyte lineage, astrocyte lineage, mitochondrial function, and cell cycle progression by 48 hours; increased expression of genes related to neuron lineage, oxidative stress, and DNA damage checkpoint regulation by 48 hours. Our findings demonstrate the possibility of using organoids to characterize cell-specific radiosensitivity and early radiation-induced gene expression changes within the human brain, providing new avenues for further study of the mechanisms underlying acute neural cell responses to IR exposure at low-to-moderate doses.

Tissue specificity of DNA damage and repair.

DNA is a remarkable biochemical macromolecule tasked with storing the genetic information that instructs life on planet Earth. However, its inherent chemical instability within the cellular milieu is incompatible with the accurate transmission of genetic information to subsequent generations. Therefore, biochemical pathways that continuously survey and repair DNA are essential to sustain life, and the fundamental mechanisms by which different DNA lesions are repaired have remained well conserved throughout evolution. Nonetheless, the emergence of multicellular organisms led to profound differences in cellular context and physiology, leading to large variations in the predominant sources of DNA damage between different cell types, and in the relative contribution of different DNA repair pathways towards genome maintenance in different tissues. While we continue to make large strides into understanding how individual DNA repair mechanisms operate on a molecular level, much less attention is given to these cell type-specific differences. This short review aims to provide a broad overview of DNA damage and repair mechanism to non-specialists, and to highlight some fundamental open questions in tissue and cell-type-specificity of these processes, which may have profound implications for our understanding of important pathophysiological processes such as cancer, neurodegeneration and aging.