Single-cell Trajectory Analysis Research Articles

Knocking down of Xkr8 enhances chemotherapy efficacy through modulating tumor immune microenvironment

Scramblase Xk-related protein 8 (Xkr8) regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and a prodrug conjugate of 5-fluorouracil (5-Fu) and oxoplatin (FuOXP) showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with increased infiltration of proliferative NK cells and activated macrophages in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers.

Decreased GATA3 levels cause changed mouse cutaneous innate lymphoid cell fate, facilitating hair follicle recycling.

In mice, skin-resident type 2 innate lymphoid cells (ILC2s) exhibit some ILC3-like characteristics. However, the underlying mechanism remains elusive. Here, we observed lower expression of the ILC2 master regulator GATA3 specifically in cutaneous ILC2s (cILC2s) compared with canonical ILC2s, in line with its functionally divergent role in transcriptional control in cILC2s. Decreased levels of GATA3 enabled the expansion of RORγt fate-mapped (RORγtfm+) cILC2s after postnatal days, displaying certain similarities to ILC3s. Single-cell trajectory analysis showed a sequential promotion of the RORγtfm+ cILC2 divergency by RORγt and GATA3. Notably, during hair follicle recycling, these RORγtfm+ cILC2s accumulated around the hair follicle dermal papilla (DP) region to facilitate the process. Mechanistically, we found that GATA3-mediated integrin α3β1 upregulation on RORγtfm+ cILC2s was required for their positioning around the DP. Overall, our study demonstrates a distinct regulatory role of GATA3 in cILC2s, particularly in promoting the divergence of RORγtfm+ cILC2s to facilitate hair follicle recycling.

Single-Cell Analysis RevealsHistone Deacetylation Factor Guide Intercellular Communication of Tumor Microenvironment that Contribute to Colorectal Cancer Progression and Immunotherapy.

In this study, single-cell RNA-seq data were collected to analyze the characteristics of Histone deacetylation factor (HDF). The tumor microenvironment (TME) cell clusters related to prognosis and immune response were identified by using CRC tissue transcriptome and immunotherapy cohorts from public repository. We explored the expression characteristics of HDF in stromal cells, macrophages, T lymphocytes, and B lymphocytes of the CRC single-cell dataset TME and further identified 4 to 6 cell subclusters using the expression profiles of HDF-associated genes, respectively. The regulatory role of HDF-associated genes on the CRC tumor microenvironment was explored by using single-cell trajectory analysis, and the cellular subtypes identified by biologically characterized genes were compared with those identified by HDF-associated genes. The interaction of HDF-associated gene-mediated microenvironmental cell subtypes and tumor epithelial cells were explored by using intercellular communication analysis, revealing the molecular regulatory mechanism of tumor epithelial cell heterogeneity. Based on the expression of feature genes mediated by HDF-related genes in the microenvironment T-cell subtypes, enrichment scoring was performed on the feature gene expression in the CRC tumor tissue transcriptome dataset. It was found that the feature gene scoring of microenvironment T-cell subtypes (HDF-TME score) has a certain predictive ability for the prognosis and immunotherapy benefits of CRC tumor patients, providing data support for precise immunotherapy in CRC tumors.

Data-driven selection of analysis decisions in single-cell RNA-seq trajectory inference.

Single-cell RNA sequencing (scRNA-seq) experiments have become instrumental in developmental and differentiation studies, enabling the profiling of cells at a single or multiple time-points to uncover subtle variations in expression profiles reflecting underlying biological processes. Benchmarking studies have compared many of the computational methods used to reconstruct cellular dynamics; however, researchers still encounter challenges in their analysis due to uncertainty with respect to selecting the most appropriate methods and parameters. Even among universal data processing steps used by trajectory inference methods such as feature selection and dimension reduction, trajectory methods' performances are highly dataset-specific. To address these challenges, we developed Escort, a novel framework for evaluating a dataset's suitability for trajectory inference and quantifying trajectory properties influenced by analysis decisions. Escort evaluates the suitability of trajectory analysis and the combined effects of processing choices using trajectory-specific metrics. Escort navigates single-cell trajectory analysis through these data-driven assessments, reducing uncertainty and much of the decision burden inherent to trajectory inference analyses. Escort is implemented in an accessible R package and R/Shiny application, providing researchers with the necessary tools to make informed decisions during trajectory analysis and enabling new insights into dynamic biological processes at single-cell resolution.

Knocking down of Xkr8 enhances chemotherapy efficacy through modulating tumor immune microenvironment

Scramblase Xkr8 regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and chemo prodrug FuOXP showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with an increase of proliferative NK cells and activated macrophages infiltration in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers.

The effect of sevoflurane exposure on cell-type-specific changes in the prefrontal cortex in young mice.

Sevoflurane, the predominant pediatric anesthetic, has been linked to neurotoxicity in young mice, although the underlying mechanisms remain unclear. This study focuses on investigating the impact of neonatal sevoflurane exposure on cell-type-specific alterations in the prefrontal cortex (PFC) of young mice. Neonatal mice were subjected to either control treatment (60% oxygen balanced with nitrogen) or sevoflurane anesthesia (3% sevoflurane in 60% oxygen balanced with nitrogen) for 2 hours on postnatal days (PNDs) 6, 8, and 10. Behavioral tests and single-nucleus RNA sequencing (snRNA-seq) of the PFC were conducted from PNDs 31 to 37. Mechanistic exploration included clustering analysis, identification of differentially expressed genes (DEGs), enrichment analyses, single-cell trajectory analysis, and genome-wide association studies (GWAS). Sevoflurane anesthesia resulted in sociability and cognition impairments in mice. Novel specific marker genes identified 8 distinct cell types in the PFC. Most DEGs between the control and sevoflurane groups were unique to specific cell types. Re-defining 15 glutamatergic neuron subclusters based on layer identity revealed their altered expression profiles. Notably, sevoflurane disrupted the trajectory from oligodendrocyte precursor cells (OPCs) to oligodendrocytes (OLs). Validation of disease-relevant candidate genes across the main cell types demonstrated their association with social dysfunction and working memory impairment. Behavioral results and snRNA-seq collectively elucidated the cellular atlas in the PFC of young male mice, providing a foundation for further mechanistic studies on developmental neurotoxicity induced by anesthesia.

Single-cell transcriptome sequencing reveals aberrantly activated inter-tumor cell signaling pathways in the development of clear cell renal cell carcinoma

BackgroundAberrant intracellular or intercellular signaling pathways are important mechanisms that contribute to the development and progression of cancer. However, the intercellular communication associated with the development of ccRCC is currently unknown. The purpose of this study was to examine the aberrant tumor cell-to-cell communication signals during the development of ccRCC.MethodsWe conducted an analysis on the scRNA-seq data of 6 ccRCC and 6 normal kidney tissues. This analysis included sub clustering, CNV analysis, single-cell trajectory analysis, cell–cell communication analysis, and transcription factor analysis. Moreover, we performed validation tests on clinical samples using multiplex immunofluorescence.ResultsThis study identified eleven aberrantly activated intercellular signaling pathways in tumor clusters from ccRCC samples. Among these, two of the majors signaling molecules, MIF and SPP1, were mainly secreted by a subpopulation of cancer stem cells. This subpopulation demonstrated high expression levels of the cancer stem cell markers POU5F1 and CD44 (POU5F1hiCD44hiE.T), with the transcription factor POU5F1 regulating the expression of SPP1. Further research demonstrated that SPP1 binds to integrin receptors on the surface of target cells and promotes ccRCC development and progression by activating potential signaling mechanisms such as ILK and JAK/STAT.ConclusionAberrantly activated tumor intercellular signaling pathways promote the development and progression of ccRCC. The cancer stem cell subpopulation (POU5F1hiCD44hiE.T) promotes malignant transformation and the development of a malignant phenotype by releasing aberrant signaling molecules and interacting with other tumor cells.

Branching Trajectories and Diversification of Clonal Escape in Aplastic Anemia Revealed By Single-Cell Genomics

Acquired aplastic anemia (AA) is a bone marrow failure resulting from an autoimmune T-cell attack against hematopoietic stem and progenitor cells (HSPCs). Several escape mechanisms within the HSPC compartment have been reported: i) adaptive alterations causing loss of high-risk HLA allele of AA development [deletion or uniparental disomy (UPD) of chromosome 6p including HLA cluster region, and loss-of-function (LOF) mutations in HLA gene], ii) clonal expansion of paroxysmal nocturnal hemoglobinuria (PNH) clone resulting from PIGA mutation, and iii) presence of clonal hematopoiesis of indeterminate potential (CHIP) mutations (i.e., BCOR/ BCORL1, DNMT3A, ASXL1). Multiple types of alterations often co-occur, however due to the inherent limitations of conventional methods, it is not known if i) these alterations are in the same or independent clones, ii) what is the mutational hierarchy, and iii) what is the cell of origin. We addressed these questions using single-cell and bulk genomic sequencing. We developed custom single-cell panels covering HLA class I and II genes, along with 14 genes commonly mutated in AA and myelodysplastic syndromes. To identify copy number variations and UPD, we included 197 single nucleotide variants (SNVs) frequent across all ethnic backgrounds on chromosomes 6, 7, 8, 20, and 21. The TotalSeq™-D panel (BioLegend), was included to target 42 surface antigens and the samples were prepared on the Tapestri platform (Missionbio). High-resolution HLA genotyping was done using AlloSeq Tx 17 hybrid capture NGS assay (CareDx), and for confirmatory bulk sequencing we applied a custom duplex UMI NGS panel (TWIST Bioscience). We examined ten patients with severe AA (pediatric: n=6, age range 2-15 years; adult: n=4, 26-67), seven patients with refractory cytopenia of childhood (RCC, age range 2-16 years), who presented a hypocellular marrow and absence of a genetic predisposition syndrome, and one healthy control. All 17 patients had at least one of the HLA-A or HLA-B risk alleles linked to AA development. A total of 136,505 cells (median 4,467/run) were successfully genotyped across the samples. Single-cell trajectory analysis revealed that all identified clones arose independent of each other (Figure 1). UPD6p clones were found in five patients with three having multiple UPD6p clones carrying different breakpoints, pointing to clonal diversification. Additionally, three cases had LOF mutations in HLA-A or HLA-B (two having coexisting UPD6p). In a patient with two UPD6p and two HLA-A mutant clones, we verified that HLA-A risk allele (A*02:01) was lost in each instance. Multi-omic analysis revealed that these clones had both granulocytic and non-granulocytic (incl. lymphocytic) surface phenotypes, suggesting their early origin within hematopoietic ontogeny. Seven patients had nine independent clones with PIGA mutations (0.3-11.3% of the cells), consistent with its tendency to generate clonal mosaicism. Clones with mutations in BCOR/BCORL1, ASXL1, DNMT3A, and TET2 were identified in six, seven, four, and two patients, respectively. We next analyzed how many patients carry multiple clones and found that 65% (11/17) have concomitant clones, with the majority (9/11) belonging to different types of clonal escape (Figure 1). Finally, we found that 4/6 patients with adaptive HLA loss (representing true clonal escape from T-cell autoimmunity) also carried CHIP clones, indicating that adaptive hematopoiesis does not protect from the acquisition of CHIP mutations. In conclusion, we show that patients with AA across age spectrum and some patients with RCC share similar clonal backgrounds, with multiple functionally redundant alterations arising independently from coexisting CHIP clones. For the first time at a single-cell level, we were able to resolve the multifaceted clonal mosaic generated by the immunological forces at play in AA, and precisely unravel their immunoediting consequences.

CSIG-02. SHH PATHWAY ACTIVATION DRIVES NEURAL CREST CELL SIGNATURE IN A SUBGROUP OF MPNSTS

Abstract INTRODUCTION Neurofibromatosis Type 1 is hereditary tumor predisposition syndrome that results in the development of innumerable nerve sheath tumors that fall within the spectrum of benign, premalignant and malignant lesions. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs. METHODS We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, a SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment. RESULTS We observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6 . We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines. CONCLUSION SHH pathway activation drives dedifferentiation into a neural crest cell-like state in a subset of MPNSTs. We confirm that inhibiting SHH pathway activity in a subset of MPNSTS prevent growth and malignant progression, providing a rational for future clinical trials.

Elaboration and validation of a prognostic signature associated with disulfidoptosis in lung adenocarcinoma, consolidated with integration of single-cell RNA sequencing and bulk RNA sequencing techniques.

Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer (NSCLC), remains a pervasive global public health concern. Disulfidoptosis, a nascent form of regulated cell death (RCD), presents an emerging field of inquiry. Currently, investigations into disulfidoptosis are in their initial stages. Our undertaking sought to integrate single-cell RNA sequencing (scRNA-seq) in conjunction with traditional bulk RNA sequencing (bulk RNA-seq) methodologies, with the objective of delineating genes associated with disulfidoptosis and subsequently prognosticating the clinical outcomes of LUAD patients. Initially, we conducted an in-depth examination of the cellular composition disparities existing between LUAD and normal samples using scRNA-seq data sourced from GSE149655. Simultaneously, we scrutinized the expression patterns of disulfidoptosis-associated gene sets across diverse cell types. Subsequently, leveraging the bulk RNA-seq data, we formulated disulfidoptosis-related prognostic risk signatures (DRPS) employing LASSO-Cox regression. This was accomplished by focusing on genes implicated in disulfidoptosis that exhibited differential expression within endothelial cells (ECs). Sequentially, the robustness and precision of the DRPS model were rigorously verified through both internal and external validation datasets. In parallel, we executed single-cell trajectory analysis to delve into the differentiation dynamics of ECs. Concluding our study, we undertook a comprehensive investigation encompassing various facets. These included comparative assessments of enrichment pathways, clinicopathological parameters, immune cell abundance, immune response-associated genes, impacts of immunotherapy, and drug predictions among distinct risk cohorts. The scrutiny of scRNA-seq data underscored discernible disparities in cellular composition between LUAD and normal samples. Furthermore, disulfidoptosis-associated genes exhibited marked discrepancies within endothelial cells (ECs). Consequently, we formulated the Disulfidoptosis-Related Prognostic Signature (DRPS) to facilitate prognostic prediction. The prognostic nomogram based on the risk score effectively demonstrated DRPS's robust capacity to prognosticate survival outcomes. This assertion was corroborated by rigorous assessments utilizing both internal and external validation sets, thus affirming the commendable predictive accuracy and enduring stability of DRPS. Functional enrichment analysis shed light on the significant correlation of DRPS with pathways intrinsic to the cell cycle. Subsequent analysis unveiled correlations between DRPS and gene mutations characteristic of LUAD, as well as indications of an immunosuppressive status. Through drug prediction, we explored potential therapeutic agents for low-risk patients. Concluding our investigation, qRT-PCR experiments confirmed the heightened expression levels of EPHX1, LDHA, SHC1, MYO6, and TLE1 in lung cancer cell lines.

Abstract P3-11-05: Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model

Abstract Background: While estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced suppression of ER+ breast cancer has been clinically observed. Our previous study demonstrated that estrogen increased the percentage of cells expressing IL24, linking to the estrogen-dependent growth inhibition of an ER+ aromatase inhibitor (AI)-resistant tumor (Mori et al. Cancers, 2021). To continue our evaluation, we investigated whether progesterone (P4) and dihydrotestosterone (DHT) would affect the growth of this estrogen-suppressive tumor. Methods: An estrogen-suppressive patient-derived xenograft (PDX) model (named GS3) was established from an AI resistant ER+/HER2– brain metastatic breast cancer. E2 (1mg), P4 (10mg), DHT (12.5mg), or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC) and RNA sequencing of PDX specimens were conducted to decipher molecular changes after each treatment. The single-cell RNAseq analysis was further performed to examine gene expression profiles in individual cells. Results: ERα, ERβ, Progesterone receptor (PR), and androgen receptor (AR) genes in GS3 are wild-type and are not amplified. Measurements of tumor volume showed that E2, E2+P4, and DHT suppressed the growth of GS3. The Tumor growth was not modulated by P4 treatment. IHC indicated that the number of Ki-67+ cells were decreased after E2, E2+P4, and DHT treatments, but were not changed after P4 treatment. PR+ cells appeared after E2 and E2+P4 treatments, and the AR+ cells increased after DHT treatment. Bulk-RNA sequencing indicated that E2 and E2+P4 treatments resulted in comparable gene expression patterns, while those of placebo and P4-treated tumors were similar. GSEA analysis showed that in E2 treatment, the hallmark estrogen response gene sets were upregulated, and the hallmark G2M checkpoint gene set was downregulated. However, in DHT treatment, the hallmark interferon alpha/gamma response gene sets and androgen response gene set were upregulated, and the hallmark TNFA signaling via NFkB gene set was downregulated. Single-cell RNA sequencing analysis of Placebo/E2/DHT samples revealed 9 clusters; cells from E2-treated and DHT-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes, although both E2 and DHT treatments resulted in tumor regression. DHT promoted cell cycle arrest, but it did not increase IL24 expression. The hallmark oxidative phosphorylation and androgen response gene sets were upregulated in all clusters from DHT-treated tumors. Trajectory analysis of single cells revealed that three major branches associated with clusters selective to the treatments of placebo, E2, and DHT were separated from a common branch which are consist of G2M phase cells. Conclusions: E2 and DHT were effective suppressor of GS3, but not P4. Based on the results from IHC, bulk-RNAseq, and Single-cell RNAseq, the mechanism of DHT-induced tumor regression is different from that by E2. Our results suggest that DHT/E2 could be treatment options for patients with relapsed ER+ AI-resistance breast cancer. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Noriko Kanaya, George Somlo, Shiuan Chen. Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-05.

Intestinal fibrosis in aganglionic segment of Hirschsprung's disease revealed by single-cell RNA sequencing.

Hirschsprung's disease (HSCR) is a relatively common congenital disability. Accumulating extracellular matrix (ECM) prompts intestinal fibrosis remodelling in the aganglionic segments of HSCR. The contributions of various cellular subsets in the fibrogenesis of HSCR segments are poorly understood. Single-cell transcriptomics from 8 aganglionic segments and 5 normal segments of 7 HSCR subjects and 26 healthy segments of seven healthy donors were analysed. Fibrotic phenotype and alterations were explored using differential expression analysis and single-cell trajectory analysis. Fibrosis-related transcription factors were inferred through single-cell regulatory network inference. Bulk transcriptomic data, proteomic data, immunohistochemistry (IHC) and real-time polymerase chain reaction were used to validate the alterations in the HSCR intestine. Various collagen, fibronectin and laminin protein-coding genes expression were up-regulated in the stromal and glial cells of the HSCR intestine. The number of fibroblasts and myofibroblasts in the aganglionic segments increased, and more myofibroblasts were activated at an earlier stage in HSCR segments, which infers that there is an intestinal fibrosis phenotype in HSCR segments. The fibrotic regulators POSTN, ANXA1 and HSP70 were highly expressed in the ECM-related cellular subsets in the transitional segments and aganglionic segments. The transcription factor regulatory network revealed that fibrosis-related and megacolon-related NR2F1 in the fibroblasts and glial subsets was up-regulated in the aganglionic segment. This work identifies intestinal fibrosis and related regulators in aganglionic segments of HSCR; hence, anti-fibrotic therapy may be considered to prevent HSCR-associated enterocolitis (HAEC), relieve intestinal stricture and improve cell therapy.

Dynamic single cell analysis in a proximal-tubule-on-chip reveals heterogeneous epithelial colonization strategies of uropathogenic Escherichia coli under shear stress.

The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic Escherichia coli (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work in vivo revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow high-resolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient and mediated by P pili at the earliest time-points. These bound bacteria initiated a founder population which rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.

Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

Abstract 2630: Heterogeneity inducible tumor stem cells relate distant metastasis and the treatment resistance

Abstract Regarding metastatic potential of cancer cells and the refractory status, regulatory and epigenetic factors underlie the cancer treatment resistance by inducing cellular heterogeneity. Circulating tumor cells (CTCs) and their stemness are critical for metastases formation associated with poor survival rates. In colorectal cancer (CRC), several stem-cell markers, such as CD44v9 and LGR5, the positive cells cause intertumoral heterogeneity and promote refractory potential. However, the mastermind of the cells which is critical for the treatment resistance remains unknown. Here, we aimed to identify the mechanisms of the treatment resistance of the CRC by focusing on typical stem cells. We selected several stem cell-related genes such as pluripotency related genes, based on the expression profile analysis of CRC patients along with drug susceptibility assays using patient-derived organoids. Despite the small number of cancer stem cells, the single cell produced heterogeneous cell populations in vitro and in vivo, expressing various differentiation and stem biomarkers, and confirmed by the knockdown analysis. Single-cell trajectory analysis suggested that heterogeneous populations including cancer stem and some persister cells were derived from the stem cells following administration of anti-cancer drugs. Furthermore, the stem cells formed heterogeneous liver metastases at a high rate, and these unique cells were observed in the bloodstream of CRC patients. This present study unveils a mechanism of CRC treatment resistance and highlights the potential of stem marker-expressing cells as CTCs. Citation Format: Norikatsu Miyoshi, Shiki Fujino, Yuki Sekido, Tsuyoshi Hata, Takayuki Ogino, Hidekazu Takahashi, Mamoru Uemura, HIrofumi Yamamoto, Yuichiro Doki, Hidetoshi Eguchi. Heterogeneity inducible tumor stem cells relate distant metastasis and the treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2630.

Pseudo-Temporal Analysis of Single-Cell RNA Sequencing Reveals Trans-Differentiation Potential of Greater Epithelial Ridge Cells Into Hair Cells During Postnatal Development of Cochlea in Rats.

The hair cells of the cochlea play a decisive role in the process of hearing damage and recovery, yet knowledge of their regeneration process is still limited. Greater epithelial ridge (GER) cells, a type of cell present during cochlear development that has the characteristics of a precursor sensory cell, disappear at the time of maturation of hearing development. Its development and evolution remain mysterious for many years. Here, we performed single-cell RNA sequencing to profile the gene expression landscapes of rats’ cochlear basal membrane from P1, P7, and P14 and identified eight major subtypes of GER cells. Furthermore, single-cell trajectory analysis for GER cells and hair cells indicated that among the different subtypes of GER, four subtypes had transient cell proliferation after birth and could transdifferentiate into inner and outer hair cells, and two of them mainly transdifferentiated into inner hair cells. The other two subtypes eventually transdifferentiate into outer hair cells. Our study lays the groundwork for elucidating the mechanisms of the key regulatory genes and signaling pathways in the trans-differentiation of GER cell subtypes into hair cells and provides potential clues to understand hair cell regeneration.

Construction and Validation of a Ferroptosis-Related Prognostic Signature for Melanoma Based on Single-Cell RNA Sequencing.

Melanoma, the deadliest type of skin cancer, is on the rise globally. The generally poor prognosis makes melanoma still an enormous public health problem. Ferroptosis is a newly emerging form of iron-dependent regulated cell death, which has been implicated in the development and treatment of several tumors. However, whether there is a connection between ferroptosis-related genes and the prognosis of melanoma patients remains an enigma. In the present study, we identified a ferroptosis-related genes signature to predict the prognosis of melanoma patients by analyzing single-cell RNA-sequencing data from the Gene Expression Omnibus (GEO). Single-cell trajectory analysis was performed to explore malignant differentiation. CellChat was used to investigate intercellular communications in melanoma. Collectively, a novel four-gene signature (CP, MAP1LC3A, transferrin, and TP53) was constructed for prognosis prediction. COX proportional hazards regression analysis showed that the established ferroptosis-associated risk model was an independent prognostic predictor for melanoma patients (HR = 2.3293; 95%CI 1.1528–4.706) (p < 0.018). Patients with low-risk scores had significantly better overall survival (OS) than those with high-risk scores in The Cancer Genome Atlas, GSE59455, and GSE22153 dataset (p = 0.0015, p = 0.031, p = 0.077). Furthermore, the gene expression level of the four genes were verified in multistrain melanoma cell lines and normal human epidermal melanocytes (NHEM). The protein expression level of the four genes in clinical samples were further verified in the Human Protein Atlas (HPA) databases. Taken together, our study identified the prognostic significance of the ferroptosis-related genes in melanoma and developed a novel four-gene prognostic signature, which may shed light on the prognostic assessment and clinical decision making for melanoma patients.

Single-Cell Multi-Omic Analysis Uncovers Comprised Immune Function and Primary Resistance Mechanism in Acute Myeloid Leukemia

Background: Approximately 20% of AML patients do not respond to induction chemotherapy (primary resistance) and 40-60% of patients develop secondary resistance, eventually leading to relapse followed by refractory disease (RR-AML). Diversified molecular mechanisms have been proposed for drug resistance and RR phenotype. However, we still cannot predict when relapse will occur, nor which patients will become resistant to therapy.Single-cell multi-omic (ScMo) profiling may provide new insights into our understanding of hematopoietic stem cell (HSC) differentiation trajectories, tumor heterogeneity and clonal evolution. Here we applied ScMo to profile bone marrow (BM) from AML patients and healthy controls.Methods: AML samples were collected at diagnosis with institutional IRB approval. Cells were stained with a panel of 62 DNA barcoded antibodies and 10x Genomics Single Cell 3' Library Kit v3 was used to generate ScMo data. After normalization, clusters were identified using Uniform Manifold Approximation and Projection (UMAP) and annotated using MapCell (Koh and Hoon, 2019). We analyzed 23,933 cells from 4 adult AML BM samples, and 39,522 cells from 2 healthy adults and 3 sorted CD34+ normal BM samples. Gene set enrichment analysis (GSEA) and Enrichr program were used to examine underlying pathways among differentially expressed genes between healthy and AML samples.Results: We identified 16 cell types between the AML and normal samples (Fig 1a) amongst 45 clusters in the UMAP projection (Fig 1b). Comparative analysis of the T cell clusters in AML samples with healthy BM cells identified an “AML T-cell signature” with over-expression of genes such as granzymes, NK/T cell markers, chemokine and cytokine, proteinase and proteinase inhibitor (Fig 2a). Among them, IL32 is known to be involved in activation-induced cell death in T cells and has immunosuppressive role, while CD8+ GZMB+ and CD8+ GZMK+ cells are considered as dysfunctional or pre-dysfunctional T cells. Indeed, Enrichr analysis showed the top rank of phenotype term - “decreased cytotoxic T cell cytolysis”.We next examined whether NK cells, are similarly dysfunctional in the AML ecosystem. The “AML NK cell signature” includes Fc Fragment family, IFN-stimulated genes (ISGs), the effector protein-encoding genes and other genes when compared to normal NK cells (Fig 2b). GSEA analysis revealed “PD-1 signalling” among the top 5 ranked pathways in AML-NK cells, though no increase in PD-1 protein nor PDCD1 gene were identified in these cells. Inhibitory receptor CD160 was expressed higher in AML samples along with exhaustion (dysfunction) associated genes TIGIT, PRF1 and GZMB (Fig 2c). Enrichr analysis uncovered enrichment of “abnormal NK cell physiology and ”impaired natural killer cell mediated cytotoxicity“. Similarly, the ”AML monocyte signature“ was significantly enriched with genes in ”Tumor Infiltrating Macrophages in Cancer Progression and Immune Escape“ and ”Myeloid Derived Suppressor Cells in Cancer Immune Escape“.We also analyzed HSPC component in one pair of cytogenetically matched, untreated complete remission (CR) /RR AML pair (Fig 2d). Notably, half of the 10 genes overexpressed in RR-AML, CXCR4, LGALS1, S100A8, S100A9, SRGN (Serglycin), regulate cell-matrix interaction and play pivotal roles in leukemic cells homing bone marrow niche. The first 4 of these genes have been demonstrated as prognostic indicators of poor survival and associated with chemo-resistance and anti-apoptotic function. Furthermore, single-cell trajectory analysis of this CR/RR pair illustrated a change in differentiation pattern of HSPCs in CR-AML to monocytes in RR-AML. We are currently analyzing more AML samples to validate these findings.Conclusions: Our ScMo analysis demonstrates that the immune cells are systematically reprogrammed and functionally comprised in the AML ecosystem. Upregulation of BM niche factors could be the underlying mechanism for RR-AML. Thus, reversing the inhibited immune system is an important strategy for AML therapy and targeting leukemic cell-BM niche interaction should be considered for cases with high expression of these molecules on AML HSPCs.Note: J.Z. and J.A.S. share co-first authorship. [Display omitted] DisclosuresScolnick: Proteona Pte Ltd: Current holder of individual stocks in a privately-held company. Xu: Proteona Pte Ltd: Current Employment. Ooi: Jansen: Honoraria; Teva Pharmaceuticals: Honoraria; GSK: Honoraria; Abbvie: Honoraria; Amgen: Honoraria. Lovci: Proteona Pte Ltd: Current Employment. Chng: Aslan: Research Funding; Takeda: Honoraria; Johnson & Johnson: Honoraria, Research Funding; BMS/Celgene: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria.

Moxifloxacin induces random migration in human corneal fibroblasts via the protein kinase C epsilon/zonula occludens-1 signaling pathway

Moxifloxacin (MOX) suppresses cell movement in human corneal fibroblasts (HCFs). Zonula occludens-1 (ZO-1) is localized to the leading edge of migrating HCFs. This study explored the role of ZO-1 in MOX-suppressed cell migration in HCFs. A single-cell trajectory analysis revealed that MOX negatively regulated the migratory properties of HCFs including migration distance, migration velocity, and directionality (P < 0.001, P < 0.001, and P = 0.018, respectively). MOX increased endogenous ZO-1 in HCFs in a concentration-dependent manner (P = 0.083, P = 0.005, and P = 0.001 at 10, 50, and 100 μg/ml, respectively), but decreased the phosphorylation of endogenous ZO-1 at serines, threonines, and tyrosines. In contrast, MOX did not alter the expression of protein kinase C epsilon (PKCε), Rac-1, Cdc42, and MRCKβ. However, MOX did also reduce the phosphorylation level of PKCε at serines and threonines (P < 0.001 at 100 μg/ml). In addition, MOX increased the phosphorylation level of Rac-1 in a concentration-dependent manner (P < 0.001 at 100 μg/ml). Compared with the mock cells, the directionality of cell movement increased significantly in ZO-1-expressing HCFs (P = 0.012) and decreased significantly in ZO-1-silenced HCFs (P = 0.002). The directionality did not change significantly in Rac-1-silenced HCFs. ZO-1-expressing HCFs moved faster than mock cells. PKCε, Cdc42, Rac-1, and phosphorylated Rac-1 were decreased in ZO-1-overexpressing HCFs, but increased in ZO-1-silenced HCFs. Finally, silencing ZO-1 blocked MOX hyperactivation of Rac-1. These suggest that MOX might trigger random migration in human corneal stromal cells through PKCε-modulated ZO-1 inactivation and Rac-1 hyperactivation.

Abstract 3201: Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen

Abstract Growing evidence suggests that tumor progression can interfere with normal hematopoiesis and skew the host system to undergo myeloid biased changes. Hematopoietic stem and progenitor cells (HSPCs) are a rare population of precursor cells giving rise to progeny that replenish blood cells throughout life. HSPCs are mainly localized in the bone marrow niche in healthy adults, however pathophysiological conditions such as cancer can cause extramedullary hematopoiesis in organs like the spleen. In this study, we investigated tumor-driven phenotypic and molecular alterations in the HSPC compartment at a single-cell level during extramedullary hematopoiesis using single-cell multiomics. We used a B16-F10 melanoma mouse model to uncover the tumor-mediated changes. Mice with tumor-burden showed splenomegaly and alterations in the composition of the HSPC compartment in the spleen consistent with extramedullary hematopoiesis. We then isolated four HSPC populations, including Lin- Sca1+ cKit+ (LSK), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocyte-erythrocyte progenitor (MEP) cells from the spleen and bone marrow of melanoma-burdened mice using the BD FACSMelody™ Cell Sorter. Next, we used an integrated workflow on the BD Rhapsody™ Single-Cell Analysis System for the downstream single-cell multiomics analysis. We utilized oligo-conjugated antibodies for NGS-based sample multiplexing and protein detection (BD® Single-Cell Multiplexing Kit, BD® AbSeq Assay), along with whole transcriptome analysis for a comprehensive analysis of mRNA expression. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization enabled delineation of the developmental trajectories of HSPC populations and exploration of phenotypic and transcriptomic changes across different hematopoietic subpopulations and across distinct anatomic sites. The analysis also identified unique hematopoietic cell clusters present during melanoma-driven extramedullary hematopoiesis in the spleen. Overall, our findings highlight melanoma-driven perturbations in hematopoiesis by utilizing advanced single-cell multiomics technology. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD-23277 (v1.0) 1120 BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2020 BD. All rights reserved. Citation Format: Nihan Kara, Nikolay Samusik, Xiaoshan Shi, Chip Lomas, Stephanie Widmann, Aaron J. Tyznik. Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3201.