Distinct Cell Subpopulations Research Articles

An atlas of immune cell transcriptomes in human immunodeficiency virus-infected immunological non-responders identified marker genes that control viral replication.

Previous studies have examined the bulk transcriptome of peripheral blood immune cells in acquired immunodeficiency syndrome patients experiencing immunological non-responsiveness. This study aimed to investigate the characteristics of specific immune cell subtypes in acquired immunodeficiency syndrome patients who exhibit immunological non-responsiveness. A single-cell transcriptome sequencing of peripheral blood mononuclear cells obtained from both immunological responders (IRs) (CD4 + T-cell count >500) and immunological non-responders (INRs) (CD4 + T-cell count <300) was conducted. The transcriptomic profiles were used to identify distinct cell subpopulations, marker genes, and differentially expressed genes aiming to uncover potential genetic factors associated with immunological non-responsiveness. Among the cellular subpopulations analyzed, the ratios of monocytes, CD16 + monocytes, and exhausted B cells demonstrated the most substantial differences between INRs and IRs, with fold changes of 39.79, 11.08, and 2.71, respectively. In contrast, the CD4 + T cell ratio was significantly decreased (0.39-fold change) in INRs compared with that in IRs. Similarly, the ratios of natural killer cells and terminal effector CD8 + T cells were also lower (0.37-fold and 0.27-fold, respectively) in the INRs group. In addition to several well-characterized immune cell-specific markers, we identified a set of 181 marker genes that were enriched in biological pathways associated with human immunodeficiency virus (HIV) replication. Notably, ISG15 , IFITM3 , PLSCR1 , HLA-DQB1 , CCL3L1 , and DDX5 , which have been demonstrated to influence HIV replication through their interaction with viral proteins, emerged as significant monocyte marker genes. Furthermore, the differentially expressed genes in natural killer cells were also enriched in biological pathways associated with HIV replication. We generated an atlas of immune cell transcriptomes in HIV-infected IRs and INRs. Host genes associated with HIV replication were identified as markers of, and were found to be differentially expressed in, different types of immune cells.

Molecular insights using spatial transcriptomics of the distal lung in congenital diaphragmatic hernia.

Abnormal pulmonary vascular development and function in congenital diaphragmatic hernia (CDH) is a significant factor leading to pulmonary hypertension. The lung is a very heterogenous organ and has marked cellular diversity that is differentially responsive to injury and therapeutic agents. Spatial transcriptomics provides the unmatched capability of discerning the differences in the transcriptional signature of these distinct cell subpopulations in the lung with regional specificity. We hypothesized that the distal lung parenchyma (selected as a region of interest) would show a distinct transcriptomic profile in the CDH lung compared with control (normal lung). We subjected lung sections obtained from male and female CDH and control neonates to spatial transcriptomics using the Nanostring GeoMx platform. Spatial transcriptomic analysis of the human CDH and control lung revealed key differences in the gene expression signature. Increased expression of alveolar epithelial-related genes (SFTPA1 and SFTPC) and angiogenesis-related genes (EPAS1 and FHL1) was seen in control lungs compared with CDH lungs. Response to vitamin A was enriched in the control lungs as opposed to abnormality of the coagulation cascade and TNF-alpha signaling via NF-kappa B in the CDH lung parenchyma. In male patients with CDH, higher expression of COL1A1 (ECM remodeling) and CD163 was seen. Increased type 2 alveolar epithelial cells (AT-2) and arterial and lung capillary endothelial cells were seen in control lung samples compared with CDH lung samples. To the best of our knowledge, this is the first use of spatial transcriptomics in patients with CDH that identifies the contribution of different lung cellular subpopulations in CDH pathophysiology and highlights sex-specific differences.NEW & NOTEWORTHY This is the first use of spatial transcriptomics in patients with congenital diaphragmatic hernia (CDH) that identifies the contribution of different lung cellular subpopulations in CDH pathophysiology and highlights sex-specific differences.

Clonal hematopoiesis and acute myeloid leukemia

During aging phenotypic changes in the hematopoietic system occur, and possible reason of these changes can be accumulation of gene mutations in hematopoietic stem cells or early blood progenitors. Although these mutations are mostly neutral, some may give hematopoietic stem cells and progenitor cells a proliferative advantage. In this case clonal hematopoiesis will arise, which is characterized by the formation of a genetically distinct subpopulation of blood cells. Clonal hematopoiesis may become a basis for the development of hematologic malignancies, such as acute myeloid leukemia. Clonal hematopoiesis associated genes which are most commonly mutated in acute myeloid leukemia patients are DNMT3A, TET2 and ASXL1. The prognostic significance of these gene mutations currently remains a subject of study.

Single-cell RNA sequencing identifies senescence as therapeutic target in rhabdomyolysis-induced acute kidney injury.

The role of macrophages in the development of rhabdomyolysis-induced acute kidney injury (RM-AKI) has been established, but an in-depth understanding of the changes in the immune landscape could help to improve targeted strategies. Whereas senescence is usually associated with chronic kidney processes, we also wished to explore whether senescence could also occur in AKI and whether senolytics could act on immune cells. Single-cell RNA sequencing was used in the murine glycerol-induced RM-AKI model to dissect the transcriptomic characteristics of CD45+ live cells sorted from kidneys 2days after injury. Public datasets from murine AKI models were reanalysed to explore cellular senescence signature in tubular epithelial cells (TECs). A combination of senolytics (dasatinib and quercetin, DQ) was administered to mice exposed or not to RM-AKI. Unsupervised clustering of nearly 17 000 single-cell transcriptomes identified seven known immune cell clusters. Sub-clustering of the mononuclear phagocyte cells revealed nine distinct cell sub-populations differently modified with RM. One macrophage cluster was particularly interesting since it behaved as a critical node in a trajectory connecting one major histocompatibility complex class IIhigh (MHCIIhigh) cluster only present in Control to two MHCIIlow clusters only present in RM-AKI. This critical cluster expressed a senescence gene signature, that was very different from that of the TECs. Senolytic DQ treatment blocked the switch from a F4/80highCD11blow to F4/80lowCD11bhigh phenotype, which correlated with prolonged nephroprotection in RM-AKI. Single-cell RNA sequencing unmasked novel transitional macrophage subpopulation associated with RM-AKI characterized by the activation of cellular senescence processes. This work provides a proof-of-concept that senolytics nephroprotective effects may rely, at least in part, on subtle immune modulation.

Mesenchymal stromal cells as conductors of adipose tissue remodeling.

Adipose tissue exhibits a remarkable capacity to expand, contract, and remodel in response to changes in physiological and environmental conditions. Here, we describe recent advances in our understanding of how functionally distinct tissue-resident mesenchymal stromal cell subpopulations orchestrate several aspects of physiological and pathophysiological adipose tissue remodeling, with a particular focus on the adaptations that occur in response to changes in energy surplus and environmental temperature. The study of adipose tissue remodeling provides a vehicle to understand the functional diversity of stromal cells and offers a lens through which several generalizable aspects of tissue reorganization can be readily observed.

Abstract PR005: An atlas of cellular heterogeneity in primary and metastatic renal cell carcinomas

Abstract Renal cell carcinoma (RCC) tumors harbor various layers of intra-tumor heterogeneity at the genetic, transcriptomic, and cellular levels. A series of recent studies have underlined the importance of charting this cellular variability to understand the cell of origin of various RCCs and the mechanisms that underlie their response to therapy. However, the regulatory factors driving intra-tumor heterogeneity in RCCs, the role of this heterogeneity in driving metastasis, and its association with clinical outcomes are mostly unknown. To address these questions, we have generated a cellular map of transcriptional heterogeneity in RCCs, which consists of &amp;gt;100,000 single-cell expression profiles from six patient-derived xenografts (PDX) models, including models derived from primary and brain metastatic tumors, as well as ten primary and six metastatic patient tumor tissues. We also developed a computational method for Gene Expression Decomposition and Integration (GEDI) that enables seamless integration of single-cell transcriptomic data across heterogeneous cancer samples while providing interpretable axes of variation. Using GEDI, we performed a fine-grained analysis of the heterogeneity of cancer cells and found that, despite patient-specific differences, common sources of intra-tumor heterogeneity exist across samples, driven by variable activity of pathways such as TNF-α/NF-κB signaling and oxidative phosphorylation (OxPhos). Analysis of data from our PDX models confirmed that this heterogeneity was stable and reproducible. A major source of intra-tumor heterogeneity was hypoxia signaling, even in VHL-deficient clear cell RCC samples (ccRCC), challenging the traditional view that ccRCC cells have a uniform pseudo-hypoxic status due to VHL inactivation. Regulatory network activity projection allowed us to disentangle the contributions of Hypoxia Inducible Factors HIF1A and HIF2A, revealing their divergent regulatory programs: while HIF1A is associated with cell-cycle and proliferation signatures, the activity of HIF2A correlates with epithelial-mesenchymal transition (EMT) within tumors, which we validated by RNA sequencing of HIF1A and EPAS1 knockdown in VHL-negative cells. Next, we used GEDI to study the cell state changes that occur during metastasis in both neoplastic and non-neoplastic tumor-infiltrating cells. By examining the differences in gene expression patterns between primary and metastatic samples, we identified various pathways that are transcriptionally activated during this transition, including EMT and Oxphos. We also uncovered distinct subpopulations of cancer cells with high gene expression changes associated with a metastatic profile. Analysis of the ligand-receptor interactions in the tumor microenvironment revealed that interactions from malignant cells to immune cells were over-represented in metastatic tumors. Overall, our cellular atlas has uncovered a complex continuum of cell states in RCCs, highlighting various drivers of the intra-tumor heterogeneity and establishing various cell states associated with metastasis. Citation Format: Ariel Madrigal, Minjun Kim, Adrien Osakwe, Tianyuan Lu, Zohreh Mehrjoo, Elham Moslemi, Rick Farouni, Larisa Morales-Soto, Yu Chang Wang, Matthew Dankner, Haig Djambazian, Kevin Petrecca, Jonathan Spicer, Fadi Brimo, Peter Siegel, Morag Park, Jiannis Ragoussis, Simon Tanguay, Yasser Riazalhosseini, Hamed S. Najafabadi. An atlas of cellular heterogeneity in primary and metastatic renal cell carcinomas [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR005.

SDPS-32 A CLINICALLY COMPATIBLE IN VITRO DRUG-SCREENING PLATFORM IDENTIFIES THERAPEUTIC VULNERABILITIES IN PRIMARY CULTURES OF BRAIN METASTASES

Abstract Approximately 20% of cancer patients (including lung, melanoma, breast, renal, and colorectal cancer patients) develop brain metastases. Current systemic therapies often show limited efficacy for brain metastasis treatment and support palliative care only. In this preclinical study, short-term cultured primary cancer cells from brain metastasis patients were molecularly characterized using next-generation sequencing and functionally evaluated using high-throughput in vitro drug screening to identify pharmacological treatment sensitivities. Next-generation sequencing identified matched genetic alterations between brain metastasis tissues and corresponding patient-derived cultured cancer cells. Mutant allele frequencies indicated that short-term cultures of cancer cells reflect the existence of distinct subpopulations of cancer cells within brain metastases. This indicates that our short-term cultured primary brain metastasis cells are suitable models for recapitulating heterogeneity in the sensitivity of tumor cells to anti-cancer drugs. Employing our in-house high-throughput in vitro drug screening platform, we screened the primary cultures from the brain metastasis patients for response to 267 anticancer compounds, and integrated the molecular and high-throughput drug screening data sets. Our preclinical study provides proof-of-concept for combining molecular profiling with in vitro drug screening for improved prediction of therapeutic vulnerabilities in brain metastasis patients. This approach could advance the use of patient-derived cancer cells in clinical practice and might eventually facilitate decision-making for personalized drug treatment.

Cancer-Associated Fibroblast-Like Tumor Cells Remodel the Ewing Sarcoma Tumor Microenvironment.

Despite limited genetic and histologic heterogeneity, Ewing sarcoma (EwS) tumor cells are transcriptionally heterogeneous and display varying degrees of mesenchymal lineage specification in vitro. In this study, we investigated if and how transcriptional heterogeneity of EwS cells contributes to heterogeneity of tumor phenotypes in vivo. Single-cell proteogenomic-sequencing of EwS cell lines was performed and integrated with patient tumor transcriptomic data. Cell subpopulations were isolated by FACS for assessment of gene expression and phenotype. Digital spatial profiling and human whole transcriptome analysis interrogated transcriptomic heterogeneity in EwS xenografts. Tumor cell subpopulations and matrix protein deposition were evaluated in xenografts and patient tumors using multiplex immunofluorescence staining. We identified CD73 as a biomarker of highly mesenchymal EwS cell subpopulations in tumor models and patient biopsies. CD73+ tumor cells displayed distinct transcriptional and phenotypic properties, including selective upregulation of genes that are repressed by EWS::FLI1, and increased migratory potential. CD73+ cells were distinguished in vitro and in vivo by increased expression of matrisomal genes and abundant deposition of extracellular matrix (ECM) proteins. In epithelial-derived malignancies, ECM is largely deposited by cancer-associated fibroblasts (CAF), and we thus labeled CD73+ EwS cells, CAF-like tumor cells. Marked heterogeneity of CD73+ EwS cell frequency and distribution was detected in tumors in situ, and CAF-like tumor cells and associated ECM were observed in peri-necrotic regions and invasive foci. EwS tumor cells can adopt CAF-like properties, and these distinct cell subpopulations contribute to tumor heterogeneity by remodeling the tumor microenvironment. See related commentary by Kuo and Amatruda, p. 5002.

A Microfluidic Liquid Biopsy Platform to Monitor Protein Biomarker Heterogeneity in Single Circulating Therapy‐Resistance Cancer Cell

AbstractTumor cells display heterogenous molecular signatures during the course of cancer and create distinct tumor cell subpopulations which challenge effective therapeutic decisions. Detection and monitoring of these heterogenous molecular events at single cell level are imperative to identify tumor cell subpopulations and to engage the best therapeutic options for the individual patient. Herein, a microfluidic liquid biopsy platform to analyze circulating tumor cells (CTCs) at single cell level is reported. The individual CTCs are captured in an alternating current‐induced microfluidic platform and analyzed by using surface‐enhanced Raman scattering spectroscopy. This platform selectively captures single CTCs from the patient's peripheral blood mononuclear cells. Using cell line models and patient samples, it is shown that the assay can simultaneously detect multiple protein biomarkers on a single CTC. The platform can stratify the CTCs into different subpopulations based on their cancer‐associated protein signature changes in response to drug treatment. This enables the identification of CTC subpopulations that are probably not responding to treatment and may assist clinicians in specifically monitoring and eliminating therapy‐resistant cancer cells within a lesion. This single CTC monitoring chip will likely have high clinical importance in disease diagnosis and treatment monitoring, and advance the knowledge of cancer heterogeneity.

Pro-fibrogenic and adipogenic aspects of chronic muscle degeneration are contributed by distinct stromal cell subpopulations.

Chronic skeletal muscle degeneration is characterized by fiber atrophy accompanied by deposition of extracellular matrix (ECM) components and fatty infiltration. Excessive accumulation of ECM leads to fibrosis via the contribution of fibro-adipogenic precursors (FAPs). Fibrosis also accompanies disuse atrophy and sarcopenia without significant inflammation. The present study aimed to comparatively analyze heterogeneous population of FAPs during acute injury and immobilization (tenotomy and denervation). The comparative analysis was accomplished based on the following 3 stromal cell subpopulations: i) CD140a(+)/Sca1(+); ii) CD140a(+)/Sca1(-); iii) CD140a(-)/Sca1(+). RNASeq analysis was employed to characterize and compare their quiescent and activated states. Whereas CD140a(-)/Sca1(+) was the most predominant activated subpopulation in tenotomy, denervation stimulated the CD140a(+)/Sca1(+) subpopulation. Immobilization models lacked myofiber damage and exhibited a minute increase in CD45(+) cells, as compared to acute injury. Transcriptome analysis showed common and discordant regulation of ECM components, without profound proliferative activation. Herein, we suggest unique surface markers for further identification of the investigated cell subpopulations. FAP subpopulations show similar activation kinetics in an inflammatory environment but the present findings highlight the fact that inflammation may not be a prerequisite for FAP activation. Delayed proliferation kinetics indicate that signals beyond inflammation might trigger FAP activation, leading to irreversible stromal changes.

Signaling pathways governing the maintenance of breast cancer stem cells and their therapeutic implications.

Breast cancer stem cells (BCSCs) represent a distinct subpopulation of cells with the ability to self-renewal and differentiate into phenotypically diverse tumor cells. The involvement of CSC in treatment resistance and cancer recurrence has been well established. Numerous studies have provided compelling evidence that the self-renewal ability of cancer stem cells is tightly regulated by specific signaling pathways, which exert critical roles to maintain an undifferentiated phenotype and prevent the differentiation of CSCs. Signaling pathways such as Wnt/β-catenin, NF-κB, Notch, Hedgehog, TGF-β, and Hippo have been implicated in the promotion of self-renewal of many normal and cancer stem cells. Given the pivotal role of BCSCs in driving breast cancer aggressiveness, targeting self-renewal signaling pathways holds promise as a viable therapeutic strategy for combating this disease. In this review, we will discuss the main signaling pathways involved in the maintenance of the self-renewal ability of BCSC, while also highlighting current strategies employed to disrupt the signaling molecules associated with stemness.

Disruption of cortical cell type composition and function underlies diabetes-associated cognitive decline

Aims/hypothesisType 2 diabetes is associated with increased risk of cognitive decline although the pathogenic basis for this remains obscure. Deciphering diabetes-linked molecular mechanisms in cells of the cerebral cortex could uncover novel therapeutic targets.MethodsSingle-cell transcriptomic sequencing (scRNA-seq) was conducted on the cerebral cortex in a mouse model of type 2 diabetes (db/db mice) and in non-diabetic control mice in order to identify gene expression changes in distinct cell subpopulations and alterations in cell type composition. Immunohistochemistry and metabolic assessment were used to validate the findings from scRNA-seq and to investigate whether these cell-specific dysfunctions impact the neurovascular unit (NVU). Furthermore, the behavioural and cognitive alterations related to these dysfunctions in db/db mice were assessed via Morris water maze and novel object discrimination tests. Finally, results were validated in post-mortem sections and protein isolates from individuals with type 2 diabetes.ResultsCompared with non-diabetic control mice, the db/db mice demonstrated disrupted brain function as revealed by losses in episodic and spatial memory and this occurred concomitantly with dysfunctional NVU, neuronal circuitry and cerebral atrophy. scRNA-seq of db/db mouse cerebral cortex revealed cell population changes in neurons, glia and microglia linked to functional regulatory disruption including neuronal maturation and altered metabolism. These changes were validated through immunohistochemistry and protein expression analysis not just in the db/db mouse cerebral cortex but also in post-mortem sections and protein isolates from individuals with type 2 diabetes (74.3 ± 5.5 years) compared with non-diabetic control individuals (87.0 ± 8.5 years). Furthermore, metabolic and synaptic gene disruptions were evident in cortical NVU cell populations and associated with a decrease in vascular density.Conclusions/interpretationTaken together, our data reveal disruption in the cellular and molecular architecture of the cerebral cortex induced by diabetes, which can explain, at least in part, the basis for progressive cognitive decline in individuals with type 2 diabetes.Data availabilityThe single-cell sequencing data that supports this study are available at GEO accession GSE217665 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE217665).Graphical

Engineering tandem CD33xCD146 CAR CIK (cytokine-induced killer) cells to target the acute myeloid leukemia niche.

In acute myeloid leukemia (AML), malignant stem cells hijack the normal bone marrow niche where they are largely protected from the current therapeutic approaches. Thus, eradicating these progenitors is the ultimate challenge in the treatment of this disease. Specifically, the development of chimeric antigen receptors (CARs) against distinct mesenchymal stromal cell subpopulations involved in the maintenance of leukemic stem cells within the malignant bone marrow microenvironment could represent a new strategy to improve CAR T-cell therapy efficacy, which is still unsuccessful in AML. As a proof of concept, we generated a novel prototype of Tandem CAR, with one specificity directed against the leukemic cell marker CD33 and the other against the mesenchymal stromal cell marker CD146, demonstrating its capability of simultaneously targeting two different cell types in a 2D co-culture system. Interestingly, we could also observe an in vitro inhibition of CAR T cell functionality mediated by stromal cells, particularly in later effector functions, such as reduction of interferon-gamma and interleukin-2 release and impaired proliferation of the CAR+ effector Cytokine-Induced Killer (CIK) cells. Taken together, these data demonstrate the feasibility of a dual targeting model against two molecules, which are expressed on two different target cells, but also highlight the immunomodulatory effect on CAR CIK cells exerted by stromal cells, confirming that the niche could be an obstacle to the efficacy of CAR T cells. This aspect should be considered in the development of novel CAR T cell approaches directed against the AML bone marrow niche.

Regulation by cyclic di-GMP attenuates dynamics and enhances robustness of bimodal curli gene activation in Escherichia coli.

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

Resolving the Pathogenesis of Anaplastic Wilms Tumors through Spatial Mapping of Cancer Cell Evolution.

While patients with intermediate-risk (IR) Wilms tumors now have an overall survival (OS) rate of almost 90%, those affected by high-stage tumors with diffuse anaplasia have an OS of only around 50%. We here identify key events in the pathogenesis of diffuse anaplasia by mapping cancer cell evolution over anatomic space in Wilms tumors. We spatially mapped subclonal landscapes in a retrospective cohort of 20 Wilms tumors using high-resolution copy-number profiling and TP53 mutation analysis followed by clonal deconvolution and phylogenetic reconstruction. Tumor whole-mount sections (WMS) were utilized to characterize the distribution of subclones across anatomically distinct tumor compartments. Compared with non-diffuse anaplasia Wilms tumors, tumors with diffuse anaplasia showed a significantly higher number of genetically distinct tumor cell subpopulations and more complex phylogenetic trees, including high levels of phylogenetic species richness, divergence, and irregularity. All regions with classical anaplasia showed TP53 alterations. TP53 mutations were frequently followed by saltatory evolution and parallel loss of the remaining wild-type (WT) allele in different regions. Morphologic features of anaplasia increased with copy-number aberration (CNA) burden and regressive features. Compartments demarcated by fibrous septae or necrosis/regression were frequently (73%) associated with the emergence of new clonal CNAs, although clonal sweeps were rare within these compartments. Wilms tumors with diffuse anaplasia display significantly more complex phylogenies compared with non-diffuse anaplasia Wilms tumors, including features of saltatory and parallel evolution. The subclonal landscape of individual tumors was constrained by anatomic compartments, which should be considered when sampling tissue for precision diagnostics.

Accurate integration of multiple heterogeneous single-cell RNA-seq data sets by learning contrastive biological variation.

For most biological and medical applications of single-cell transcriptomics, an integrative study of multiple heterogeneous single-cell RNA sequencing (scRNA-seq) data sets is crucial. However, present approaches are unable to integrate diverse data sets from various biological conditions effectively because of the confounding effects of biological and technical differences. We introduce single-cell integration (scInt), an integration method based on accurate, robust cell-cell similarity construction and unified contrastive biological variation learning from multiple scRNA-seq data sets. scInt provides a flexible and effective approach to transfer knowledge from the already integrated reference to the query. We show that scInt outperforms 10 other cutting-edge approaches using both simulated and real data sets, particularly in the case of complex experimental designs. Application of scInt to mouse developing tracheal epithelial data shows its ability to integrate development trajectories from different developmental stages. Furthermore, scInt successfully identifies functionally distinct condition-specific cell subpopulations in single-cell heterogeneous samples from a variety of biological conditions.

Study of Gene Expression in Glioblastoma Hypoxic Model

Inspite of multi model cure therapy the mortality rate among the patients of brain tumor always remain high.The low survival rate is due to the ability of these tumors to recur quickly and aggressively. The cellular, genetic, and epigenetic heterogeneity of Glioblastoma multiforme tumors makes designing targeted treatments difficult. Within the tumor there are distinct subpopulations of cells that are stem-like in their behavior and are critical as therapeutic target. The regulation of these cells at molecular level is not yet properly studied. The presence of hypoxic environment controls the stem cells and slower down the progression of tumor. The goal of this work is to use PCR to determine gene expression in glial cells that have been exposed to hypoxia.

Cross-Dataset Single-Cell Analysis Identifies Temporal Alterations in Cell Populations of Primary Pancreatic Tumor and Liver Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) has a unique tumor microenvironment composed of various cell populations such as cancer cells, cancer-associated fibroblasts (CAFs), immune cells, and endothelial cells. Recently, single-cell RNA-sequencing analysis (scRNA-seq) has systemically revealed the genomic profiles of these cell populations in PDAC. However, the direct comparison of cell population composition and genomic profile between primary tumors (at both early- and late-stage) and metastatic tumors of PDAC is still lacking. In this study, we combined and analyzed recent scRNA-seq datasets of transgenic KPC mouse models with autochthonous PDAC and matched liver metastasis, revealing the unique tumor ecosystem and cell composition of liver metastasis in contrast to primary PDAC. Metastatic PDAC tumors harbor distinct cancer cell subpopulations from primary tumors. Several unique markers, including HMGA1, were identified for metastasis-enriched cancer cell subpopulations. Furthermore, metastatic tumors reveal significantly enriched granulocytic myeloid-derived suppressor cells (G-MDSCs), mature neutrophils, and granulocyte-myeloid progenitors (GMPs). A common GMP population across primary tumors, liver metastases, and healthy bone marrow was identified as the putative cell origin of tumor-associated neutrophils/granulocytes.

Single-cell transcriptomic analysis uncovers diverse and dynamic senescent cell populations

Senescence is a state of enduring growth arrest triggered by sublethal cell damage. Given that senescent cells actively secrete proinflammatory and matrix-remodeling proteins, their accumulation in tissues of older persons has been linked to many diseases of aging. Despite intense interest in identifying robust markers of senescence, the highly heterogeneous and dynamic nature of the senescent phenotype has made this task difficult. Here, we set out to comprehensively analyze the senescent transcriptome of human diploid fibroblasts at the individual-cell scale by performing single-cell RNA-sequencing analysis through two approaches. First, we characterized the different cell states in cultures undergoing senescence triggered by different stresses, and found distinct cell subpopulations that expressed mRNAs encoding proteins with roles in growth arrest, survival, and the secretory phenotype. Second, we characterized the dynamic changes in the transcriptomes of cells as they developed etoposide-induced senescence; by tracking cell transitions across this process, we found two different senescence programs that developed divergently, one in which cells expressed traditional senescence markers such as p16 (CDKN2A) mRNA, and another in which cells expressed long noncoding RNAs and splicing was dysregulated. Finally, we obtained evidence that the proliferation status at the time of senescence initiation affected the path of senescence, as determined based on the expressed RNAs. We propose that a deeper understanding of the transcriptomes during the progression of different senescent cell phenotypes will help develop more effective interventions directed at this detrimental cell population.

Heterogeneity of glycan biomarker clusters as an indicator of recurrence in pancreatic cancer.

Outcomes following tumor resection vary dramatically among patients with pancreatic ductal adenocarcinoma (PDAC). A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block sections of PDAC tumors collected from curative resections. The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.