Subpopulation Of Epithelial Cells Research Articles

Intestinal metaplasia key molecules and UPP1 activation via Helicobacter pylori /NF-kB: drivers of malignant progression in gastric cancer

Gastric cancer (GC) remains a significant global health challenge due to its high morbidity and mortality rates. The development of GC is a multi-hit process and the exploration of precancerous lesions is crucial. To elucidate the molecular and cellular dynamics underlying gastric carcinogenesis, we conducted an integrative single-cell RNA sequencing analysis of 26,028 high-quality cells from gastric antral mucosa biopsies across various stages, including non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, and early gastric cancer. By constructing a detailed single-cell atlas, we identified distinct epithelial cell subpopulations and their corresponding molecular signatures. We focused on the biological link between gastric epithelial cells and cancer cells. Notably, we observed that gland mucous cells acquired an intestinal-like stem cell phenotype during metaplasia, with MUC6, MUC2 and OLFM4 emerging as the specific markers for unique endocrine cells in early malignant lesions. Additionally, our analysis highlighted UPP1 as a key oncogene, with its expression progressively increasing from normal epithelial cells to malignant cells. UPP1 upregulation was shown to promote GC cell proliferation and migration, implicating it in the oncogenic process. Further, we explored the impact of Helicobacter pylori infection on gene expression, revealing that Helicobacter pylori infection upregulates UPP1 via the NF-κB pathway. Our cell-cell communication analysis underscored the significant role of the Macrophage migration inhibitory factor pathway in the tumor microenvironment, contributing to GC progression. Various key molecules involved in intestinal metaplasia, along with UPP1 and the Macrophage migration inhibitory factor pathway, collectively illustrate the multifaceted nature and complexity of gastric cancer evolution, highlighting the cumulative impacts that drive tumorigenesis.

Abstract A038: A targetable tumor-induced wound-healing response is essential for osteosarcoma lung metastasis

Abstract Lung metastasis is responsible for nearly all deaths in children and teenagers affected by osteosarcoma, the most common pediatric sarcoma. The mechanisms by which malignant bone cells manipulate the lung microenvironment to promote the colonization and growth of metastatic tumor cells remain unclear but represent a potential therapeutic opportunity for preventing and treating metastasis. In this study, we sought to understand how osteosarcoma cells educate the lung microenvironment during metastatic progression. To address this gap, we generated several time series datasets of niche-labeled, single-cell RNA-seq of developing metastatic lesions and then used these to characterize the changes occurring in tumor and tumor-associated stromal cells during colonization. After identifying candidate mechanisms, we tested several therapeutic interventions to evaluate their effects on metastasis development in vitro using a tumor-on-lung co-culture system and in vivo using our animal models. Osteosarcoma cells induced acute epithelial injury upon lung dissemination. Shortly after dissemination, a paracrine loop established between a hyper-secretory, hypo-proliferative subpopulation of tumor cells and alveolar epithelial cells triggers the adoption of a chronic, non-resolving wound phenotype within the surrounding tissues that culminates in extensive tissue fibrosis with persistent extracellular matrix deposition, especially fibronectin. Cellular changes observed during this process include the proliferation of type 2 alveolar epithelial cells, the accumulation of highly fibrogenic epithelial intermediates (analogous to those observed in pulmonary fibrosis), and the emergence of a large population of scar-promoting macrophages. The formation of this matrix seems essential for lesion formation—disruption of the matrix-tumor cell interaction eliminated pseudonodules in vivo. Inhibition of pro-fibrotic pathways using nintedanib, an FDA- approved drug used to treat pulmonary fibrosis, or of tumor-epithelial paracrine signals with anakinra, an antagonist of IL1 signaling, effectively blocked metastatic lesion growth in both murine and human xenograft models, showing evidence of microenvironment reprogramming in the single cell data. Combining these therapies, which target primarily the hyper-secretory, hypo-proliferative “anchor” cells, with conventional therapies, which target proliferating tumor cells, proved highly effective in eliminating established metastatic lesions. Our work demonstrates that disseminated osteosarcoma cells trigger an aberrant, non-resolving wound-healing response in tumor-associated lung tissues, similar to that observed in non-malignant pulmonary fibrosis, and that this pathologic wound-healing response promotes metastatic growth. Therapeutic disruption of fibrosis-associated signaling prevents metastasis and treats established metastatic disease in multiple osteosarcoma models. Citation Format: James B. Reinecke, Leyre Jimenez Garcia, Amy C. Gross, Matthew V. Cannon, Yogesh Budhathoki, Ryan D. Roberts. A targetable tumor-induced wound-healing response is essential for osteosarcoma lung metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A038.

AMH regulates a mosaic population of AMHR2-positive cells in the ovarian surface epithelium

The function and homeostasis of the mammalian ovary depend on complex paracrine interactions between multiple cell types. Using primary mouse tissues and isolated cells, we showed in vitro that ovarian follicles secrete a factor(s) that suppresses growth of ovarian epithelial cells in culture. Most of the growth suppressive activity was accounted for by Anti-Mullerian Hormone/Mullerian Inhibitory Substance (AMH/MIS) secreted by granulosa cells of the follicles, as determined by immune depletion experiments. Additionally, conditioned medium from granulosa cells from wild type control, but not AMH knockout, suppressed epithelial cell growth. Tracing of the AMH regulated cells using AMHR2 (AMH receptor 2)-Cre:ROSA26 mutant mice indicated the presence of populations of AMHR2-positive epithelial cells on the ovarian surface and oviduct epithelia. Cells isolated from the mutant mice indicated that a subpopulation of cells marked by AMHR2-Cre:ROSA26 accounted for most cell growth and expansion in ovarian surface epithelial cells, and the AMHR2 lineage derived cells were regulated by AMH in vitro; whereas, fewer AMHR2-Cre:ROSA26 marked cells accounted for oviduct epithelial cell outgrowth. The results reveal a paracrine pathway in maintaining follicle-epithelial homeostasis in ovary and support a subpopulation of AMHR2 lineage marked epithelial cells as ovarian epithelial stem/progenitor cells with higher proliferative potential regulatable by follicle secreted AMH.

Identification of KLHL17 as a prognostic marker for prostate cancer based on single-cell sequencing and in vitro/in vivo experiments

BackgroundProstate cancer (PCa) is a leading cause of cancer-related mortality among men, characterized by significant heterogeneity that complicates diagnosis and treatment.Methods and resultsTo enhance our understanding of PCa, we utilized single-cell RNA sequencing (scRNA-seq) data to identify distinct malignant epithelial cell subpopulations and their molecular characteristics. By integrating scRNA-seq data with bulk RNA-seq data, we constructed a prognostic risk score model. The influence of key genes identified in the risk score on PCa was validated through both in vitro and in vivo experiments. Our study revealed eight unique malignant epithelial cell clusters, each exhibiting distinct molecular characteristics and biological functions. KEGG and GO enrichment analyses highlighted their involvement in various pathways. The prognostic risk score model demonstrated strong predictive power for patient outcomes, particularly in predicting progression-free survival (PFS). Notably, KLHL17, identified as a high-risk gene, was found to significantly impact PCa cell proliferation, migration, invasion, and apoptosis upon knockdown. This finding was further validated in vivo using a subcutaneous xenograft tumor model, where reduced KLHL17 expression led to decreased tumor growth.ConclusionOur research provides a comprehensive analysis of PCa heterogeneity and highlights KLHL17 as a potential therapeutic target.

Dissecting the Single-Cell Diversity and Heterogeneity Underlying Cervical Precancerous Lesions and Cancer Tissues.

The underlying cellular diversity and heterogeneity from cervix precancerous lesions to cervical squamous cell carcinoma (CSCC) is investigated. Four single-cell datasets including normal tissues, normal adjacent tissues, precancerous lesions, and cervical tumors were integrated to perform disease stage analysis. Single-cell compositional data analysis (scCODA) was utilized to reveal the compositional changes of each cell type. Differentially expressed genes (DEGs) among cell types were annotated using BioCarta. An assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis was performed to correlate epigenetic alterations with gene expression profiles. Lastly, a logistic regression model was used to assess the similarity between the original and new cohort data (HRA001742). After global annotation, seven distinct cell types were categorized. Eight consensus-upregulated DEGs were identified in B cells among different disease statuses, which could be utilized to predict the overall survival of CSCC patients. Inferred copy number variation (CNV) analysis of epithelial cells guided disease progression classification. Trajectory and ATAC-seq integration analysis identified 95 key transcription factors (TF) and one immunohistochemistry (IHC) testified key-node TF (YY1) involved in epithelial cells from CSCC initiation to progression. The consistency of epithelial cell subpopulation markers was revealed with single-cell sequencing, bulk sequencing, and RT-qPCR detection. KRT8 and KRT15, markers of Epi6, showed progressively higher expression with disease progression as revealed by IHC detection. The logistic regression model testified the robustness of the resemblance of clusters among the various datasets utilized in this study. Valuable insights into CSCC cellular diversity and heterogeneity provide a foundation for future targeted therapy.

Landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment

AimsWe sought to reveal the landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment and investigate their parts on esophageal squamous carcinoma (ESCC) development. BackgroundEpithelial cells play an important role in the occurrence and development of ESCC through multiple mechanisms. While the landscape of epithelial cell subpopulations in ESCC, remains unclear. ObjectiveExploring the role of epithelial cell subpopulations in ESCC progression. MethodsSeurat R package was used for single-cell RNA sequencing (scRNA-seq) data filtering, dimensionality reduction, clustering and differentially expressed genes analysis. Cellmarker database was adopted for cell cluster annotation. Functional enrichment analysis was carried out by Gene Ontology (GO) analysis. InferCNV package was conducted for copy number variation (CNV) of epithelial cell subpopulations in all chromosomal regions. Pseudotime trajectory analysis was implemented for exploring differentiation trajectory of epithelial cells subgroups during the cancer progression. CellChat analysis was used for probing the interactions between epithelial cells and NK/T cells. cellular experiments were performed using Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR), Wound-Healing Assay and transwell. Results11 major cell subpopulations were identified in ESCC and adjunct tissues. Further reclassification of epithelial cells uncovered 4 subpopulations. Enrichment analysis revealed that highly expressed genes in 4 epithelial cell subpopulations were related to cell proliferation, immune response and angiogenesis. CNV analysis found that UBD + epithelial cells and GAS2L3+ epithelial cells had a higher proportion of CNV. Cell differentiation trajectories disclosed that KRT6C+ and GSTA1+ epithelial cells were in an intermediate state of differentiation, while UBD+ and GAS2L3+ epithelial cells are in an end state of differentiation during ESCC progression. Finally, we found that four epithelial cell subpopulations all inhibited NK/T cells through NECTIN2-TIGIT and CLEC2B-KLRB1. Low ATF3 and DDIT3 mRNA expression inhibited ESCC cell migration and invasion. ConclusionHere, we obtained a through epithelial cell atlas of ESCC at single-cell resolution, explored the role of epithelial cell in ESCC progression, and unveiled immunosuppressive signals to NK/T cells in promoting ESCC. Our findings expand the comprehension of epithelial cells and offer a theoretical guidance for future anti-epithelial cell treatment of ESCC.

Abstract A046: Spatial transcriptomics reveals heterogeneity of epithelial and stromal compartments in healthy and tumor pancreas

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal cancers, with a 5-year survival rate of only 13%. PDAC develops from precursor lesions, most commonly Pancreatic Intraepithelial Neoplasia (PanIN). Studying PanIN in human patients is difficult due to its microscopic nature and the lack of indication for sampling the pancreas in the absence of disease. In collaboration with Gift of Life - Michigan, a local organ and tissue donor organization, we obtained over 80 pancreata for research. Surprisingly, we found PanIN in the vast majority of the organs, regardless of the donor’s age; the prevalence of PanIN compared to the relatively low incidence of PDAC indicates that most lesions are destined not to progress. To gather insight into factors that might distinguish lesions designed to progress from lesions that are likely to remain dormant, we sought to characterize both epithelial cells and their supporting microenvironment. Using spatial transcriptomics, we determined that PanIN epithelial cells are transcriptionaally similar to pancreatic cancer; further, limited genetic characterization revealed that they contain similar oncogenic KRAS mutations as pancreatic malignancy. We then sought to determine whether factors in the microenvironment distinguish sporadic PanIN from those associated with overt malignancy. We used 10x Genomic CytAssist Visium technology and profiled an initial batch of 7 tissue sections from donor pancreata and an equal number from tumors. We employed the BayesPrism algorithm to deconvolute the Visium data (which lacked single-cell resolution), using our single-cell RNAseq atlas to define stroma- and epithelial-specific expression profiles. We then segmented the tissue into epithelial and stromal compartments. We further identified various stromal subpopulations, with macrophages and fibroblasts being the most abundant cell types. We defined subpopulations of epithelial cells, fibroblasts, and macrophages through unsupervised clustering. In both donor samples and tumors, we identified heterogeneous populations of stromal cells present in specific geographic patterns, with PanIN-adjacent stroma clearly distinct from stroma surrounding healthy ducts. Cell-cell neighborhood analysis revealed a distinct geographic arrangement of epithelial and stromal compartments, indicating potential communication patterns. Independent unsupervised statistical analysis using Non-negative Matrix Factorization (NMF) using Coordinated Gene Association in Pattern Sets (CoGAPS) R package also revealed similar patterns. Our next steps involve validating gene expression signatures that define each subpopulation and identifying tumor-specific or PanIN-specific genes. Ultimately, we aim to investigate interactions between epithelial cells, fibroblasts, and macrophages to identify potential tumor-promoting or tumor-restraining pathways. Citation Format: Ahmed M. Elhossiny, Jude Okoye, Imade Williams, Yaqing Zhang, Hannah Watkoske, Atul Deshpande, Elana Fertig, Arvind Rao, Eileen S. Carpenter, Timothy L. Frankel, Marina Pasca di Magliano. Spatial transcriptomics reveals heterogeneity of epithelial and stromal compartments in healthy and tumor pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A046.

Single-cell atlas of healthy vocal folds and cellular function in the endothelial-to-mesenchymal transition.

The vocal fold is an architecturally complex organ comprising a heterogeneous mixture of various layers of individual epithelial and mesenchymal cell lineages. Here we performed single-cell RNA sequencing profiling of 5836 cells from the vocal folds of adult Sprague-Dawley rats. Combined with immunostaining, we generated a spatial and transcriptional map of the vocal fold cells and characterized the subpopulations of epithelial cells, mesenchymal cells, endothelial cells, and immune cells. We also identified a novel epithelial-to-mesenchymal transition-associated epithelial cell subset that was mainly found in the basal epithelial layers. We further confirmed that this subset acts as intermediate cells with similar genetic features to epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma. Finally, we present the complex intracellular communication network involved homeostasis using CellChat analysis. These studies define the cellular and molecular framework of the biology and pathology of the VF mucosa and reveal the functional importance of developmental pathways in pathological states in cancer.

6-Phosphogluconate dehydrogenase promotes glycolysis and fatty acid synthesis by inhibiting the AMPK pathway in lung adenocarcinoma cells

Abnormal metabolism has emerged as a prominent hallmark of cancer and plays a pivotal role in carcinogenesis and progression of lung adenocarcinoma (LUAD). In this study, single-cell sequencing revealed that the metabolic enzyme 6-phosphogluconate dehydrogenase (PGD), which is a critical regulator of the pentose phosphate pathway (PPP), is significantly upregulated in the malignant epithelial cell subpopulation during malignant progression. However, the precise functional significance of PGD in LUAD and its underlying mechanisms remain elusive. Through the integration of TCGA database analysis and LUAD tissue microarray data, it was found that PGD expression was significantly upregulated in LUAD and closely correlated with a poor prognosis in LUAD patients. Moreover, in vitro and in vivo analyses demonstrated that PGD knockout and inhibition of its activity mitigated the proliferation, migration, and invasion of LUAD cells. Mechanistically, immunoprecipitation-mass spectrometry (IP-MS) revealed for the first time that IQGAP1 is a robust novel interacting protein of PGD. PGD decreased p-AMPK levels by competitively interacting with the IQ domain of the known AMPKα binding partner IQGAP1, which promoted glycolysis and fatty acid synthesis in LUAD cells. Furthermore, we demonstrated that the combination of Physcion (a PGD-specific inhibitor) and metformin (an AMPK agonist) could inhibit tumor growth more effectively both in vivo and in vitro. Collectively, these findings suggest that PGD is a potential prognostic biomarker and therapeutic target for LUAD.

Single-cell RNA sequencing and spatial transcriptome reveal potential molecular mechanisms of lung cancer brain metastasis

BackgroundLung cancer is a highly aggressive and prevalent disease worldwide. By the time it is first diagnosed, distant metastases have usually already occurred. Among them, the prognosis of patients with brain metastasis from lung cancer is very poor. Therefore, it is particularly important to identify the evolutionary status of tumor cells during lung cancer brain metastases and discover the underlying mechanisms of lung cancer brain metastases. MethodsIn this study, we analysed three types of data: single-cell RNA sequencing, bulk RNA sequencing, and spatial transcriptome. Firstly, we identified early metastatic epithelial cell clusters (EMEC) using CNV and trajectory analysis in scRNA-seq data. Secondly, we integrated scRNA-seq and spatial transcriptome data with the help of MIA (Multimodal intersection analysis) to explore the biological characteristics of EMEC. Finally, we used bulk RNA-seq data to validate the molecular characteristics of EMEC. ResultA total of 55,763 single cells were obtained and divided into 9 cell types. In brain metastasis, we found a significantly higher proportion of epithelial cells. In addition, we identified a specific subpopulation of epithelial cells, which was named as “early metastatic epithelial cell clusters (EMEC)”. It is enriched in oxidative phosphorylation, coagulation, complement. Moreover, we also found that EMEC underwent cellular communication with other immune cells through ligand-receptor pairs such as MIF-(CD74 + CXCR4) and MIF-(CD74 + CD44). Next, we validated that EMEC were associated with poor clinical prognosis using three independent external datasets. Finally, spatial transcriptome analysis revealed specificity in the spatial distribution of EMEC, which shifted from the peripheral regions to the central regions of the tumour as the depth of tumor invasion progressed. ConclusionThis study reveals the potential molecular mechanisms of lung cancer brain metastasis from both single-cell and spatial transcriptomic perspectives, providing biological insights and clinical reference value for detecting patients suffering from lung cancer brain metastasis.

Integrative analysis of bulk and single-cell RNA sequencing reveals the gene expression profile and the critical signaling pathways of type II CPAM

BackgroudType II congenital pulmonary airway malformation (CPAM) is a rare pulmonary microcystic developmental malformation. Surgical excision is the primary treatment for CPAM, although maternal steroids and betamethasone have proven effective in reducing microcystic CPAM. Disturbed intercellular communication may contribute to the development of CPAM. This study aims to investigate the expression profile and analyze intercellular communication networks to identify genes potentially associated with type II CPAM pathogenesis and therapeutic targets.MethodsRNA sequencing (RNA-seq) was performed on samples extracted from both the cystic area and the adjacent normal tissue post-surgery in CPAM patients. Iterative weighted gene correlation network analysis (iWGCNA) was used to identify genes specifically expressed in type II CPAM. Single-cell RNA-seq (scRNA-seq) was integrated to unveil the heterogeneity in cell populations and analyze the communication and interaction within epithelial cell sub-populations.ResultsA total of 2,618 differentially expressed genes were identified, primarily enriched in cilium-related biological process and inflammatory response process. Key genes such as EDN1, GPR17, FPR2, and CHRM1, involved in the G protein-coupled receptor (GPCR) signaling pathway and playing roles in cell differentiation, apoptosis, calcium homeostasis, and the immune response, were highlighted based on the protein-protein interaction network. Type II CPAM-associated modules, including ciliary function-related genes, were identified using iWGCNA. By integrating scRNA-seq data, AGR3 (related to calcium homeostasis) and SLC11A1 (immune related) were identified as the only two differently expressed genes in epithelial cells of CPAM. Cell communication analysis revealed that alveolar type 1 (AT1) and alveolar type 2 (AT2) cells were the predominant communication cells for outgoing and incoming signals in epithelial cells. The ligands and receptors between epithelial cell subtypes included COLLAGEN genes enriched in PI3K-AKT singaling and involved in epithelial to mesenchymal transition.ConclusionsIn summary, by integrating bulk RNA-seq data of type II CPAM with scRNA-seq data, the gene expression profile and critical signaling pathways such as GPCR signaling and PI3K-AKT signaling pathways were revealed. Abnormally expressed genes in these pathways may disrupt epithelial-mesenchymal transition and contribute to the development of CPAM. Given the effectiveness of prenatal treatments of microcystic CPAM using maternal steroids and maternal betamethasone administration, targeting the genes and signaling pathways involved in the development of CPAM presents a promising therapeutic strategy.

Abstract PO1-25-04: PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer

Abstract Background Inflammatory breast cancer (IBC) is a lethal subtype of breast cancer characterized by rapidly arising diffuse erythema and edema of the overlying skin that was thought to contribute to rapid metastasis. Dissecting the intricate cellular ecosystems within tumors and affected skin and their complex interplay may be pivotal in unraveling the aggressive nature of IBC. Methods Integrative analysis containing single-cell RNA sequencing, bulk RNA sequencing, and whole exome sequencing, was performed on paired tumor and skin samples from 17 treatment-naive IBC patients and 5 non-IBC patients from multiple centers. Meanwhile, paraffin sections and serum specimens from two centers were conducted to validate the omics results. Additionally, public single-cell transcriptomic data of non-IBC tumors and normal mammary glands, RNA microarray data from IBC cases, and the Cancer Genome Atlas (TCGA) database were utilized to validate the characteristics of IBC microenvironment. The regulatory mechanism was verified by in vitro experiments. Results We delineated the landscapes of IBC tumor and affected skin microenvironment at the single-cell level and demonstrated that the luminal progenitors (LP) cells highly expressing pleiotrophin (PTN) were most significantly enriched in IBC tumors compared to non-IBC tumors and normal mammary glands, in two independent cohorts. Moreover, the serum concentrations of PTN were significantly higher in IBC patients compared with non-IBC patients. Notably, PTN secreted by PTN+ LP cells interacted with Neuropilin 1(NRP1) receptor on endothelial tip cells in both tumor and affected skin of IBC. Moreover, in vitro assay demonstrated knocking down the expression of NRP1 on human umbilical vein endothelial cells significantly blocked PTN-induced migration, tube formation and sprouting, which might be independent of VEGFA. In addition, two immature phenotypes of PVL_MMP9 and PVL_CCL19 cells recruited by endothelial tip cells via PDGFB/PDGFRB axis were accumulated in affected skin of IBC, which might be associated with promotion of malignant cell metastasis. Finally, we revealed that malignant cells in affected skin of IBC had a higher ability of epithelial-mesenchymal transition than IBC intratumoral malignant cells and identified enhanced crosstalk between immature PVL cells and malignant cells in the skin affected with IBC , through TNFSF12-TNFRSF12A, TGFB-TGFBR1/TGFBR2, MDK-SDC1/4/NCL/(ITGA6+ITGB1). Conclusion We identified a unique, non-malignant epithelial cell subpopulation PTN+ LP cells, which induce massive angiogenesis via the PTN-NRP1 axis, recruit the immature phenotype PVL cells accumulated in affected skin of IBC thus promoting tumor progression. Our findings offer comprehensive insights into the pathogenesis of IBC and uncover a previously unknown, promising therapeutic target. Patient of discovery cohort details Clinical and pathology details for inflammatory breast cancer(IBC) and non-IBC patients analysed by single-cell RNA sequencing(scRNA-seq), bulk RNA sequencing(bulk RNA-seq) and whole exome sequencing(WES) in this study. Clinicalpathological feature of validation cohort Citation Format: Mengmeng Zhang, Kaiwen Zhou, Ting Liu, Filipa Lynce, Filipa Lynce, Wendy Chen, Qianjun Chen, Xiaoping Li, Zhongyu Yuan, Juan Xu, Zhenhai Cai, Jianping Guo, Nan Shao, Ying Lin. PTN-positive luminal progenitors induce angiogenesis and metastasis in inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-04.

Abstract 6997: Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis

Abstract Originating from within the distal lung, lung adenocarcinoma (LUAD) is the most common pathologic subtype of non-small cell lung carcinoma (NSCLC) and encompasses an assortment of histologic presentations. Colocalized within the distal alveolar epithelium are two alveolar epithelial cell (AEC) subpopulations: alveolar epithelial type 1 (AT1) and alveolar epithelial type 2 (AT2) cells. We previously demonstrated that signatures unique to each AEC subpopulation were detected in LUAD tumors formed by oncogenic KRASG12D activation in mice and were associated with disparate survival outcomes, transcriptomic patterning, and histologic presentation. Epithelial growth factor receptor (EGFR) mutations are among the most frequent oncogenic drivers in thoracic oncology. Therefore, we set out to determine if EGFR oncogenic activation resulted in differential LUAD presentation and outcomes based on the cell of origin in which it was activated. To activate EGFR oncogenic mutations in AT1 or AT2 cells, we employed triple transgenic mouse models (AT1: Gramd2-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790 and AT2: Sftpc-CreERT2:EF1-LSL-tTA:TetO-EGFRdel20/T790), which allowed for EGFR activation in either Gramd2+ AT1 or Sftpc+ AT2 cells. Longitudinal analysis of hematoxylin and eosin-stained lungs over the course of 20 weeks was performed. Blinded pathology review demonstrated that EGFR oncogenic activation in Gramd2+ AT1 cells resulted in histologically defined LUAD with lepidic properties at early time points and solid adenocarcinoma properties at later time points. This differed from EGFR activation in Sftpc+ AT2 cells, which resulted in pre-carcinogenic atypical adenomatous hyperplasia (AAH) formation that did not progress to adenocarcinoma. While preliminary, our results suggest that cell of origin strongly influences EGFR-mediated lung oncogenesis and concomitant pathology and histologic presentation. This work may advance our understanding while informing patient stratification and treatment strategies for LUAD patients. Citation Format: Aaron M. Neely, Andrea Lanz, William Dean Wallace, Anthony W. Kim, Zea Borok, Crystal N. Marconett. Elucidating the tumorigenic properties of alveolar epithelial type 1 cells in EGFR-mediated LUAD tumorigenesis [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 6997.

Abstract 4356: An isoform-resolution transcriptomic atlas of colorectal cancer from long-read single-cell sequencing

Abstract Colorectal cancer (CRC), a complex and molecularly heterogenous disease that lacks robust predictive markers and targeted therapies, is the second leading cause of cancer death worldwide. In recent years, technological advances in short-read single-cell RNA sequencing (scRNA-seq) have been instrumental in deciphering tumor cell heterogeneities. However, most studies have focused on gene-level expression quantification without considering alterations in transcript structures arising from widespread alternative end processing and/or splicing which are frequently dysregulated in cancer. Here, we combined short- and long-read scRNA-seq of CRC patient samples to build the first isoform-resolution CRC transcriptomic atlas. We identified 394 dysregulated transcript structures in tumor epithelial cells, including 299 derived from the coupling of multiple alternative splicing events. Additionally, we characterized genes and isoforms associated with different colon epithelial lineages and tumor cell subpopulations exhibiting varying levels of stemness and differentiation, as well as their distinct prognostic implications. Finally, we built an algorithm by integrating mass spectrometry data and novel peptides derived from predicted open reading frames of recurrent tumor-specific transcripts to curate a panel of recurring neoepitopes. Overall, this study unveils the transcriptomic landscape of CRC and may drive the identification of neoantigens based on full-length transcriptomic atlases to aid future development of more universal neoantigen-based cancer vaccines. Citation Format: Zhongxiao Li, Bin Zhang, Jia Jia Chan, Hossein Tabatabaeian, Qing Yun Tong, Xiao Hong Chew, Xiaonan Fan, Charlene Chan, Patrick Driguez, Faith Cheong, Shi Wang, Bei En Siew, Ian Jse-Wei Tan, Kai-Yin Lee, Bettina Lieske, Wai-Kit Cheong, Dennis Kappei, Ker-Kan Tan, Xin Gao, Yvonne Tay. An isoform-resolution transcriptomic atlas of colorectal cancer from long-read single-cell sequencing [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 4356.

Single nuclei transcriptomics of the in situ human limbal stem cell niche

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

Mutation of SOCS2 induces structural and functional changes in mammary development.

Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or control mice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare.

Identification of estrogen response-associated STRA6+ granulosa cells within high-grade serous ovarian carcinoma by single-cell sequencing

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is a pathologic subtype of ovarian cancer (OC) with a more lethal prognosis. Extensive heterogeneity results in HGSOC being more susceptible to treatment resistance and adverse treatment effects. Revealing the heterogeneity involved is crucial. MethodsWe downloaded the single-cell RNA-seq (scRNA) data from GEO database and performed a scRNA analysis for cell landscape of HGSOC by using the Seurat package. The highly expressed genes were uploaded into the DAVID and KEGG database for enrichment analysis, and the AUCell package was used to calculate cancer-associated hallmark score. The SCENIC analysis was used for key regulons, the estrogen response enrichment scores in TCGA-OV RNA-seq dataset were calculated by using the GSVA package. Besides, the expression of STRA6 and IRF1 and the cell invasion and migration in si-STRA6 OC cells were detected by using the quantitative reverse transcription (qRT)-PCR method and Transwell assay respectively. ResultsWe successfully constructed a single-cell atlas of HGSOC and delineated the heterogeneity of epithelial cells therein. There were five epithelial cell subpopulations, GLDC + Epithelial cells, PEG3+ leydig cells, STRA6+ granulosa cells, POLE2+ Epithelial cells, and AURKA + Epithelial cells. STRA6+ granulosa cells have the potential to promote tumor growth as well as the highest estrogen response early activity through the biological pathways analysis of highly expressed genes and estrogen response score of ssGSEA. We found that IRF1 and STRA6 expression was remarkably upregulated in the OC cancer cell line HEY. Silencing of STRA6 markedly decreased the invasion and migration ability of the OC cancer cell line HEY. ConclusionThere is extreme heterogeneity of epithelial cells in HGSOC, and STRA6+ granulosa cells may be able to promote cancer progression. Our findings are benefit to the heterogeneity identification of HGSOC and develop targeted therapy strategy for HGSOC patients.

Decoding the tumor microenvironment and molecular mechanism: unraveling cervical cancer subpopulations and prognostic signatures through scRNA-Seq and bulk RNA-seq analyses.

Cervical carcinoma (CC) represents a prevalent gynecological neoplasm, with a discernible rise in prevalence among younger cohorts observed in recent years. Nonetheless, the intrinsic cellular heterogeneity of CC remains inadequately investigated. We utilized single-cell RNA sequencing (scRNA-seq) transcriptomic analysis to scrutinize the tumor epithelial cells derived from four specimens of cervical carcinoma (CC) patients. This method enabled the identification of pivotal subpopulations of tumor epithelial cells and elucidation of their contributions to CC progression. Subsequently, we assessed the influence of associated molecules in bulk RNA sequencing (Bulk RNA-seq) cohorts and performed cellular experiments for validation purposes. Through our analysis, we have discerned C3 PLP2+ Tumor Epithelial Progenitor Cells as a noteworthy subpopulation in cervical carcinoma (CC), exerting a pivotal influence on the differentiation and progression of CC. We have established an independent prognostic indicator-the PLP2+ Tumor EPCs score. By stratifying patients into high and low score groups based on the median score, we have observed that the high-score group exhibits diminished survival rates compared to the low-score group. The correlations observed between these groups and immune infiltration, enriched pathways, single-nucleotide polymorphisms (SNPs), drug sensitivity, among other factors, further underscore their impact on CC prognosis. Cellular experiments have validated the significant impact of ATF6 on the proliferation and migration of CC cell lines. This study enriches our comprehension of the determinants shaping the progression of CC, elevates cognizance of the tumor microenvironment in CC, and offers valuable insights for prospective CC therapies. These discoveries contribute to the refinement of CC diagnostics and the formulation of optimal therapeutic approaches.

Single-cell analysis reveals the spatial-temporal expression of genes associated with esophageal malformations

Understanding the molecular mechanisms of congenital diseases is challenging due to their occurrence within specific developmental stages. Esophageal malformations are examples of such conditions, characterized by abnormalities in the development of esophagus during embryogenesis. These developmental malformations encompass a range of anomalies, including esophageal atresia, and tracheoesophageal fistula. Here, we investigated the preferential expression of 29 genes that are implicated in such malformations and their immediate interactome (a total of 67 genes). We conducted our analyses across several single-cell atlases of embryonic development, encompassing approximately 150,000 cells from the mouse foregut, 180,000 cells from human embryos, and 500,000 cells from 24 human organs. Our study, spanning diverse mesodermal and endodermal cell populations and early developmental stages, shows that the genes associated with esophageal malformations show their highest cell-type specific expression in lateral plate mesoderm cells and at the developmental stage of E8.75–E9.0 days. In human embryos, these genes show a significant cell-type specific expression among subpopulations of epithelial cells, fibroblasts and progenitor cells including basal cells. Notably, members of the forkhead-box family of transcription factors, namely FOXF1, FOXC1, and FOXD1, as well as the SRY-box transcription factor, SOX2, demonstrate the most significant preferential expression in both mouse and human embryos. Overall, our findings provide insights into the temporal and cellular contexts contributing to esophageal malformations.

Single-cell RNA sequencing reveals the heterogeneity of epithelial cell and fibroblast cells from non- to metastatic lymph node OTSCC.

Lymph node metastasis (LNM) is one of the common features of oral tongue squamous cell carcinoma (OTSCC). LNM is also taken as a sign of advanced OTSCC and poor survival rate. Recently, single-cell RNA sequencing has been applied in investigating the heterogeneity of tumor microenvironment and discovering the potential biomarkers for helping the diagnosis and prognosticating. Pathogenesis of LNM in OTSCC remains unknown. Specifically, cancer-associated fibroblasts (CAFs) and epithelial tumor cells could foster the progression of tumors. Thus, in this study, we aimed to comprehensively analyze the roles of subpopulations of CAFs and epithelial tumor cells in lymph node metastatic OTSCC using the integration of OTSCC single-cell RNA sequencing datasets. Four distinct subtypes of CAFs, namely vascular CAFs, myofibroblast CAFs, inflammatory CAFs, and growth arrest CAFs were successfully discovered in LNM tumor and confirmed the roles of GAS and PTN pathways in the progression of tumor metastasis. In addition, NKAIN2+ epithelial cells and FN1+ epithelial cells specifically exhibited an upregulation of PTN, NRG, MIF, and SPP1 signaling pathways in the metastatic OTSCC. In doing so, we put forth some potential biomarkers that could be utilized for the purpose of diagnosing and prognosticating OTSCC during its metastatic phase and tried to confirm by immunofluorescence assays.