Epithelial Subsets Research Articles

Non-canonical IL-22 receptor signaling remodels the mucosal barrier during fungal immunosurveillance.

Mucosal barrier integrity is vital for homeostasis with commensal organisms while preventing pathogen invasion. We unexpectedly found that fungal-induced immunosurveillance enhances resistance to fungal outgrowth and tissue invasion by remodeling the oral mucosal epithelial barrier in mouse models of adult and neonatal Candida albicans colonization. Epithelial subset expansion and tissue remodeling were dependent on interleukin-22 (IL-22) and signal transducer and activator of transcription 3 (STAT3) signaling, through a non-canonical receptor complex composed of glycoprotein 130 (gp130) coupled with IL-22RA1 and IL-10RB. Immunosurveillance-induced epithelial remodeling was restricted to the oral mucosa, whereas barrier architecture was reset once fungal-specific immunity developed. Collectively, these findings identify fungal-induced transient mucosal remodeling as a critical determinant of resistance to mucosal fungal infection during early stages of microbial colonization.

Assessment of the Ferroptosis Regulators: Glutathione Peroxidase 4, Acyl-Coenzyme A Synthetase Long-Chain Family Member 4, and Transferrin Receptor 1 in Patient-Derived Endometriosis Tissue.

Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in various diseases and is gaining considerable attention in the realm of endometriosis. Considering the classical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased iron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective case-control study provides a comprehensive immunohistochemical assessment of the expression and tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis patients. The case group consisted of 38 women with laparoscopically and histologically confirmed endometriosis and the control group consisted of 18 women with other gynecological conditions. Our study revealed a significant downregulation of GPX4 in stromal cells of endometriosis patients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = -2.90, p = 0.005). This finding aligned with slightly, but not significantly, higher iron levels detected in the blood of endometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2-13.5) g/dL versus 12.5 (12.2-13.4) g/dL in the control group; t (54) = -0.897, p = 0.374). Interestingly, there was no concurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014), responsible for iron uptake into cells. Our empirical findings provide support for the involvement of ferroptosis in the context of endometriosis. However, variances in expression patterns within stromal and epithelial cellular subsets call for further in-depth investigations.

Neutrophil-derived PAD4 induces citrullination of CKMT1 exacerbates mucosal inflammation in inflammatory bowel disease.

Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in infection and inflammatory diseases by facilitating the formation of neutrophil extracellular traps (NETs). However, the substrates of PAD4 and its exact role in inflammatory bowel disease (IBD) remain unclear. In this study, we employed single-cell RNA sequencing (scRNA-seq) and substrate citrullination mapping to decipher the role of PAD4 in intestinal inflammation associated with IBD. Our results demonstrated that PAD4 deficiency alleviated colonic inflammation and restored intestinal barrier function in a dextran sulfate sodium (DSS)-induced colitis mouse model. scRNA-seq analysis revealed significant alterations in intestinal cell populations, with reduced neutrophil numbers and changes in epithelial subsets upon PAD4 deletion. Gene expression analysis highlighted pathways related to inflammation and epithelial cell function. Furthermore, we found that neutrophil-derived extracellular vesicles (EVs) carrying PAD4 were secreted into intestinal epithelial cells (IECs). Within IECs, PAD4 citrullinates mitochondrial creatine kinase 1 (CKMT1) at the R242 site, leading to reduced CKMT1 protein stability via the autophagy pathway. This action compromises mitochondrial homeostasis, impairs intestinal barrier integrity, and induces IECs apoptosis. IEC-specific depletion of CKMT1 exacerbated intestinal inflammation and apoptosis in mice with colitis. Clinical analysis of IBD patients revealed elevated levels of PAD4, increased CKMT1 citrullination, and decreased CKMT1 expression. In summary, our findings highlight the crucial role of PAD4 in IBD, where it modulates IECs plasticity via CKMT1 citrullination, suggesting that PAD4 may be a potential therapeutic target for IBD.

Abstract 3893: A multimodal spatial-omics atlas of lung precancer and progression to adenocarcinoma

Abstract Understanding the earliest changes during lung adenocarcinoma (LUAD) development can set the stage for discovery of fertile targets for disease interception, thereby mitigating the dire public health burden of LUAD. We previously identified bulk-level molecular and immunological changes that are enriched in normal-appearing tissue (NAT) in the local niche of human LUAD, as well as those that commence in adenomatous premalignant lesions (aPMLs, LUAD precursors) and that are further enriched in LUADs. Yet, in-depth understanding of the identities, states, and properties of specific cell subsets in NAT and precancer that trigger LUAD remain largely elusive due to inherent roadblocks to sampling and characterizing aPMLs that are at the center of this trajectory. Here, we aimed to map molecular profiles, states, and interactions of cell subsets that underlie initiation in lesion-adjacent NAT, and to determine how features of these cells evolve along the aPML-LUAD spectrum. We analyzed an expanding cohort of archived matching NATs, aPMLs and LUADs, including challenging small samples, from up to 17 patients. Samples were analyzed using combined high-resolution, multi-modal Visium spatial transcriptomics (ST, n=17) and proteomics (n=12), as well as single-cell RNA-sequencing of fixed cells (scFFPE-seq) from consecutive sections of the same tissues from 15 patients. We also studied a subset of samples by high-plex spatial proteomics (COMET) or subcellular spatial gene expression analysis (Xenium). In total, we sequenced more than half a million single cells comprising diverse epithelial, immune, and stromal subsets, which were concordant with lineage clusters identified by ST analysis of the same samples. Loss of alveolar differentiation was a hallmark of tumor cell-enriched areas in aPMLs and LUADs, with a more pronounced effect in the LUADs. Alveolar intermediate cells (AICs), which we have previously shown to be involved in LUAD initiation, were found in lesion-adjacent NAT, and their signature was enriched in matching aPMLs and further in LUADs. In line with these findings, trajectory analysis showed that AICs are derived from normal alveolar type 2 cells and likely progress towards tumor cells. Spatial neighborhood analysis pointed towards strong crosstalk between AICs and macrophages. We also noted heterogeneity in immune infiltration including patterns indicative of progressive features along the NAT-aPML-LUAD continuum. For example, B cell lineages were not only increased in abundance in LUADs relative to aPMLs, but they also mobilized into relatively more mature tertiary lymphoid aggregates/structures. While T regulatory cells aggregated close to LUADs, they were rather scattered in matching aPMLs. Our multimodal and spatial atlas of NAT, nearby aPML and LUAD elucidates the earliest cellular events underlying transition of NAT to LUAD and that could inform of targets for interception of this trajectory. Citation Format: Ansam Sinjab, Fuduan Peng, Yunhe Liu, Sujuan Yang, Tieling Zhou, Alejandra G. Serrano, Jiping Feng, Lorena Gomez Bolanos, Guangchun Han, Daniel Gustavo Rosen, Stephen G. Swisher, Avrum Spira, Steven M. Dubinett, Luisa M. Solis Soto, Mingyao Li, Junya Fujimoto, Jared Burks, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A multimodal spatial-omics atlas of lung precancer and progression to adenocarcinoma [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 3893.

Abstract 5840: Identifying drug tolerant persister to neoadjuvant osimertinib in resectable non-small cell lung cancer harbouring EGFR mutations (NORA) via single-cell RNA sequencing

Abstract Introduction: Adjuvant osimertinib is the standard of care for resected, stage IB-IIIA non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutation. Yet, the role of neoadjuvant osimertinib is not clear. In the NORA trial (NCT04816838), we aimed to identify the drug tolerant persister (DTP) after neoadjuvant osimertinib for surgically resectable, EGFR-mutated stage IA-IIIA NSCLC. Methods: Patients harboring EGFR Exon 19 deletion or L858R at clinical stage IA-IIIA were given 80 mg of neoadjuvant osimertinib daily for two 28-day cycles followed by surgical resection and adjuvant osimertinib for 3 years. The key exploratory endpoint was to characterize the DTP via single-cell RNA sequencing (scRNA-seq) with pre and post-osimertinib samples. Results: We performed scRNA-seq on twenty-five patients enrolled at Yonsei Cancer Center from June 2021 to March 2022. Patients with stage IA (n=8, 32%), IB (n=7, 28%), IIA (n=4, 16%), IIB (n=4, 16%), and IIIA (n=2, 8%) were included. For EGFR mutation, exon 19 del and L858R accounted for 10 (40%) patients and 15 (60%) patients, respectively. The ORR (all partial response) was 44% (n=11), major pathologic response (MPR) rate was 24% (n=6), and pathologic complete response rate was 0%. ScRNA-seq revealed a total of 252,339 cells (60 clusters) annotated as epithelial cells (10 clusters), fibroblasts (7 clusters), immune cells (17 clusters), myeloid (15 clusters), endothelial cells (11 clusters) that showed distinct enrichment in post-osimertinib samples, non-MPR, and L858R subset. We found that a distint epithelial subset, namely EP8-AGER, identified as alveolar type 1 (AT1) cells was enriched in the non-MPR subset showing activation of Wnt, Hippo, and YAP/TAZ pathway. Pseudotemporal ordering of this cluster showed activation of signaling pathway for COL4A2/COL4A3 related to structural consistuent of extracellular matrix, and RTKN2 associated with NF-κB. In the fibroblast subset, the proportion of FB6-CD74 associated with chemokine activity and NF-κB was higher in the non-MPR and L858R group. In the myeloid subset, CD8-C1-GZMK identified as CD8+ T cells with activation/effector and cytotoxic signatures was also enriched in the post-osimertinib samples. Among macrophages, 3 clusters (annotated as SELENOP+, SPP1+, PLXDC2+) showed enrichment of cholesterol metabolism with activation of apolipoprotein E that promotes conversion of proinflammatory macrophages into anti-inflammatory phenotypes. Lastly, the proportion of EC9-LYZ, annotated as scavenging endothelial cells was higher in the non-MPR and L858R group. Conclusion: Neoadjuvant osimertinib resulted in the cellular reprogramming in the tumor microenvironment, notably AT1 epithelial cell which may lead to a loop between cancer cells and fibroblasts for the generation of DTP cells. Citation Format: Jii Bum (Joy) Lee, Su-Jin Choi, Young Joon Park, Kyoung-Ho Park, Seung Hyun Yong, Hyo Sup Shim, Byung Jo Park, Chang Young Lee, Min Hee Hong, Byoung Chul Cho, Sun Min Lim. Identifying drug tolerant persister to neoadjuvant osimertinib in resectable non-small cell lung cancer harbouring EGFR mutations (NORA) via single-cell RNA 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 5840.

Single‐cell RNA sequencing reveals telocytes subsets of human lung

AbstractBackgroundTelocyte (TC) is a recently defined renewed cell and its dominant intercommunication with other cells displays multiple functions in tissue homeostasis and diseases. Alveolar epithelial cells and immune cells were in the lung cancer heterogeneity, progression, and metastasis, and further associated with antitumor therapeutic strategies. However, few studies focus on the roles of TCs in lung cancer.MethodsIn this article, we used the public scRNA‐Seq data (including healthy control, chronic obstructive pulmonary disease, non–small cell lung cancer, lymph node metastases from non–small cell lung cancer, and systemic sclerosis–associated interstitial lung disease patients) to analyze the cellular dynamics in human lung and distinct types of TCs and their communication networks with the variety of cell types.ResultsSix subclusters of TCs were identified by expression of specific biological function markers, which demonstrated the diversity of TCs subsets in lung tissue. Further results showed TCs had communication with epithelial cells or immune cells subsets by the ligand–receptor interaction, including TIMP metallopeptidase inhibitor 1CD63, fibulin 1integrin subunit beta 1, vimentinCD44, macrophage migration inhibitory factorCD74, and amyloid beta precursor proteinCD74. Ligand–receptor interaction heterogeneity was revealed in lung tissue of healthy or diseases. Enhanced specific signals in ligandreceptor interaction were revealed, including integrin beta 1 and CD44 were appraised in the communication of TCs with epithelial cells, NK cells, NKT cells, CD4+ exhausted T cells, CD4+ memory/effector T cells, CD4+ naïve T cells, CD8+ exhausted T cells, CD8+ memory/effector T cells, and CD8+ naïve T cells. CD63, a marker identified in TCs exosomes was emphasized in our current analysis which is closely related to communication of TCs with other cell types.ConclusionThese results will provide us with new insight into the mechanisms of TCs‐dominated communication and promise therapy of TC exosomes in lung diseases.

Three-dimensional genome architecture coordinates key regulators of lineage specification in mammary epithelial cells

Although lineage-specific genes have been identified in the mammary gland, little is known about the contribution of the 3D genome organization to gene regulation in the epithelium. Here, we describe the chromatin landscape of the three major epithelial subsets through integration of long- and short-range chromatin interactions, accessibility, histone modifications, and gene expression. While basal genes display exquisite lineage specificity via distal enhancers, luminal-specific genes show widespread promoter priming in basal cells. Cell specificity in luminal progenitors is largely mediated through extensive chromatin interactions with super-enhancers in gene-body regions in addition to interactions with polycomb silencer elements. Moreover, lineage-specific transcription factors appear to be controlled through cell-specific chromatin interactivity. Finally, chromatin accessibility rather than interactivity emerged as a defining feature of the activation of quiescent basal stem cells. This work provides a comprehensive resource for understanding the role of higher-order chromatin interactions in cell-fate specification and differentiation in the adult mouse mammary gland.

OP05 High-dimensional single-cell analysis identifies cellular signatures associated with response to vedolizumab therapy in ulcerative colitis

Abstract Background The unpredictability of response to biological therapy in ulcerative colitis (UC) patients, including vedolizumab (VDZ), an anti-α4β7 integrin antibody, poses a serious challenge for medical care. This study aims to identify the relevant cellular and molecular signatures that mark response to VDZ by combining single-cell transcriptomics (scRNAseq) and high-dimensional flow cytometry (Cytek). Methods Gut biopsies and peripheral blood mononuclear cells (PBMCs) from patients with active UC were collected 2 weeks prior and 14 weeks after starting VDZ therapy. Response was evaluated at week 14 based on endoscopy and Physician Global Assessment. Transcriptome profiles of 159.188 and 95.057 high-quality single cells were generated from biopsies and PBMCs, respectively. Additional 797.670 CD45+ mucosal cells and 900.000 PBMCs were processed for validating scRNAseq results by Cytek. Results From a total of 25 UC patients (mean age 41±12.7 years, 52% female, 36% anti-TNFα naive), 15 patients (60%) reached response at week 14. Patients with no prior use of anti-TNFα were more likely to achieve remission with VDZ compared to anti-TNFα exposed patients (89% vs 44% response rate, respectively). Analysis of cell abundances revealed a partial recovery of the epithelial and stromal cell mucosal compartments in responders at week 14. Both scRNAseq and Cytek show that in responders VDZ prevented T cells (CD8+IL17+ and CD4+PD1+ subsets) and innate immune cells (NKs, DCs and inflammatory monocytes) from entering mucosa, resulting in their enrichment in PBMCs. In contrast, non-response to VDZ was marked by an expansion of inflammatory fibroblasts and activated endothelial cells, and depletion of most epithelial subsets. VDZ retained CD4 T cells in PBMCs in non-responders, but failed to halt active mucosal inflammation at week 14. Strikingly, involvement of the innate immune cells in UC inflammation prior to VDZ treatment was much more prominent in non-responders who showed higher baseline abundance of NKs, ILCs, γδ-T cells, DCs and inflammatory monocytes in their peripheral blood and mucosa compared to responders. While VDZ inhibited integrin β7 expression on circulating T cells in all patients, only in responders it coincided with the lower mucosal abundance of lymphocytes, suggesting that T cells in non-responders might employ alternative, β7-independent trafficking routes. Conclusion This is the largest prospective scRNAseq study addressing the response to biological therapy in UC. We showed that active, innate immune cell-mediated inflammation marks the primary non-response to VDZ that persists prior to and throughout the treatment, accompanied by β7-independent lymphocyte migration.

A novel lncRNA-mediated epigenetic regulatory mechanism in periodontitis.

Periodontitis is a highly prevalent chronic inflammatory disease with an exaggerated host immune response, resulting in periodontal tissue destruction and potential tooth loss. The long non-coding RNA, LncR-ANRIL, located on human chromosome 9p21, is recognized as a genetic risk factor for various conditions, including atherosclerosis, periodontitis, diabetes, and cancer. LncR-APDC is an ortholog of ANRIL located on mouse genome chr4. This study aims to comprehend the regulatory role of lncR-APDC in periodontitis progression. Our experimental findings, obtained from lncR-APDC gene knockout (KO) mice with induced experimental periodontitis (EP), revealed exacerbated bone loss and disrupted pro-inflammatory cytokine regulation. Downregulation of osteogenic differentiation occurred in bone marrow stem cells harvested from lncR-APDC-KO mice. Furthermore, single-cell RNA sequencing of periodontitis gingival tissue revealed alterations in the proportion and function of immune cells, including T and B cells, macrophages, and neutrophils, due to lncR-APDC silencing. Our findings also unveiled a previously unidentified epithelial cell subset that is distinctively presenting in the lncR-APDC-KO group. This epithelial subset, characterized by the positive expression of Krt8 and Krt18, engages in interactions with immune cells through a variety of ligand-receptor pairs. The expression of Tff2, now recognized for its role in chronic inflammatory conditions, exhibited a notable increase across various tissue and cell types in lncR-APDC deficient mice. Additionally, our investigation revealed the potential for a direct binding interaction between lncR-APDC and Tff2. Intra-gingival administration of AAV9-lncR-APDC was shown to have therapeutic effects in the EP model. In conclusion, our results suggest that lncR-APDC plays a critical role in the progression of periodontal disease and holds therapeutic potential for periodontitis. Furthermore, the presence of the distinctive epithelial subpopulation and significantly elevated Tff2 levels in the lncR-APDC-silenced EP model offer new perspectives on the epigenetic regulation of periodontitis pathogenesis.

Activation of a transient progenitor state in the epicardium is required for zebrafish heart regeneration

The epicardium, a mesothelial cell tissue that encompasses vertebrate hearts, supports heart regeneration after injury through paracrine effects and as a source of multipotent progenitors. However, the progenitor state in the adult epicardium has yet to be defined. Through single-cell RNA-sequencing of isolated epicardial cells from uninjured and regenerating adult zebrafish hearts, we define the epithelial and mesenchymal subsets of the epicardium. We further identify a transiently activated epicardial progenitor cell (aEPC) subpopulation marked by ptx3a and col12a1b expression. Upon cardiac injury, aEPCs emerge from the epithelial epicardium, migrate to enclose the wound, undergo epithelial-mesenchymal transition (EMT), and differentiate into mural cells and pdgfra+hapln1a+ mesenchymal epicardial cells. These EMT and differentiation processes are regulated by the Tgfβ pathway. Conditional ablation of aEPCs blocks heart regeneration through reduced nrg1 expression and mesenchymal cell number. Our findings identify a transient progenitor population of the adult epicardium that is indispensable for heart regeneration and highlight it as a potential target for enhancing cardiac repair.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

Abstract 1586: An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma

Abstract Lung adenocarcinomas (LUAD) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [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 1586.

Abstract PR006: An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma

Abstract Lung adenocarcinomas (LUADs) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR006.

NAIP—NLRC4 inflammasome activation in tuft cells contributes to host defense against bacteria

Abstract The small intestinal epithelium is exposed to various microbes, and intestinal epithelial cells (IECs) have developed an array of host defense mechanisms to distinguish commensals from pathogenic microbes and initiate an immune response against the later. NAIP—NLRC4 inflammasome induced-pyroptosis, which is activated when bacterial flagellin or type III secretion systems are detected in the cytosol of the host cell, is one such mechanism. Activation of NAIP—NLRC4 induces extrusion of IECs and release of the inflammatory mediators prostaglandin E2 and IL-18. Work on epithelial inflammasome activation thus far has not delineated if different subtypes of IECs have specific roles in the ensuing immune response. Tuft cells are a rare subset of IEC that is primarily known for its role in sensing parasites, and it is unknown whether they are involved in promoting antibacterial host responses. Here we show that upon activation of the NAIP—NLRC4 inflammasome specifically in tuft cells, they uniquely among IECs release prostaglandin D2, and within the small intestine, tissue IL-22 increases. NLRC4 expression only in tuft cells leads to better control of Salmonella Typhimurium compared to NLRC4 null controls. Taken together, these data suggest that NAIP— NLRC4 inflammasome activation in tuft cells leads to lipid mediator induced downstream inflammatory responses that are complementary to signaling pathways that are activated by other IEC subsets. Understanding how NAIP—NLRC4 activation in different epithelial subsets affects downstream inflammatory responses will inform on the different signaling pathways that synergize together to mount pathogen specific host defense mechanisms.

Cellular and transcriptional diversity over the course of human lactation

Human breast milk (hBM) is a dynamic fluid that contains millions of cells, but their identities and phenotypic properties are poorly understood. We generated and analyzed single-cell RNA-sequencing (scRNA-seq) data to characterize the transcriptomes of cells from hBM across lactational time from 3 to 632 d postpartum in 15 donors. We found that the majority of cells in hBM are lactocytes, a specialized epithelial subset, and that cell-type frequencies shift over the course of lactation, yielding greater epithelial diversity at later points. Analysis of lactocytes reveals a continuum of cell states characterized by transcriptional changes in hormone-, growth factor-, and milk production-related pathways. Generalized additive models suggest that one subcluster, LC1 epithelial cells, increases as a function of time postpartum, daycare attendance, and the use of hormonal birth control. We identify several subclusters of macrophages in hBM that are enriched for tolerogenic functions, possibly playing a role in protecting the mammary gland during lactation. Our description of the cellular components of breast milk, their association with maternal–infant dyad metadata, and our quantification of alterations at the gene and pathway levels provide a detailed longitudinal picture of hBM cells across lactational time. This work paves the way for future investigations of how a potential division of cellular labor and differential hormone regulation might be leveraged therapeutically to support healthy lactation and potentially aid in milk production.

Effects of hyperinsulinemia on pancreatic cancer development and the immune microenvironment revealed through single-cell transcriptomics

BackgroundHyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that genetically reduced insulin production resulted in ~ 50% suppression of pancreatic intraepithelial neoplasia (PanIN) precancerous lesions in mice. However, only female mice remained normoglycemic, and only the gene dosage of the rodent-specific Ins1 alleles was tested in our previous model. Moreover, we did not delve into the molecular and cellular mechanisms associated with modulating hyperinsulinemia.MethodsWe studied how reduced Ins2 gene dosage affects PanIN lesion development in both male and female Ptf1aCreER;KrasLSL-G12D mice lacking the rodent-specific Ins1 gene (Ins1-/-). We generated control mice having two alleles of the wild-type Ins2 gene (Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/+) and experimental mice having one allele of Ins2 gene (Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/-). We then performed thorough histopathological analyses and single-cell transcriptomics for both genotypes and sexes.ResultsHigh-fat diet–induced hyperinsulinemia was transiently or modestly reduced in female and male mice, respectively, with only one allele of Ins2. This occurred without dramatically affecting glucose tolerance. Genetic reduction of insulin production resulted in mice with a tendency for less PanIN and acinar-to-ductal metaplasia (ADM) lesions. Using single-cell transcriptomics, we found hyperinsulinemia affected multiple cell types in the pancreas, with the most statistically significant effects on local immune cell types that were highly represented in our sampled cell population. Specifically, hyperinsulinemia modulated pathways associated with protein translation, MAPK-ERK signaling, and PI3K-AKT signaling, which were changed in epithelial cells and subsets of immune cells.ConclusionsThese data suggest a potential role for the immune microenvironment in hyperinsulinemia-driven PanIN development. Together with our previous work, we propose that mild suppression of insulin levels may be useful in preventing pancreatic cancer by acting on multiple cell types.

An Integrated Taxonomy for Monogenic Inflammatory Bowel Disease

Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis. To build a taxonomy model, we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by (1) overlapping syndromic features, (2) response to hematopoietic stem cell transplantation, (3) bulk RNA-sequencing of 32 tissues, (4) single-cell RNA-sequencing of >50 cell subsets from the intestine of healthy individuals and patients with IBD (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4). Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8+ T cells, neutrophils, epithelial subsets, and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mammalian target of rapamycin activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD. Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.

Quality-Controlled Sputum Analysis by Flow Cytometry.

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed using cytology-based methodologies. Its utility is limited because reading the slides is time-consuming and requires highly specialized personnel. Moreover, extensive debris and the presence of too many squamous epithelial cells (SECs), or cheek cells, often renders a sample inadequate for diagnosis. In contrast, flow cytometry allows for high-throughput phenotyping of cellular populations while simultaneously excluding debris and SECs. The protocol presented here describes an efficient method to dissociate sputum into a single cell suspension, antibody stain and fix cellular populations, and acquire samples on a flow cytometric platform. A gating strategy that describes the exclusion of debris, dead cells (including SECs) and cell doublets is presented here. Further, this work also explains how to analyze viable, single sputum cells based on a cluster of differentiation (CD)45 positive and negative populations to characterize hematopoietic and epithelial lineage subsets. A quality control measure is also provided by identifying lung-specific macrophages as evidence that a sample is derived from the lung and is not saliva. Finally, it has been demonstrated that this method can be applied to different cytometric platforms by providing sputum profiles from the same patient analyzed on three flow cytometers; Navios EX, LSR II, and Lyric. Furthermore, this protocol can be modified to include additional cellular markers of interest. A method to analyze an entire sputum sample on a flow cytometric platform is presented here that makes sputum amenable for developing high-throughput diagnostics of lung disease.

Enhanced Susceptibility of Galectin-1 Deficient Mice to Experimental Colitis.

Galectin-1 is a β-galactoside-binding lectin, ubiquitously expressed in stromal, epithelial, and different subsets of immune cells. Galectin-1 is the prototype member of the galectin family which shares specificity with β-galactoside containing proteins and lipids. Immunomodulatory functions have been ascribed to endogenous galectin-1 due to its induction of T cell apoptosis, inhibitory effects of neutrophils and T cell trafficking. Several studies have demonstrated that administration of recombinant galectin-1 suppressed experimental colitis by modulating adaptive immune responses altering the fate and phenotype of T cells. However, the role of endogenous galectin-1 in intestinal inflammation is poorly defined. In the present study, the well-characterized acute dextran sulfate sodium (DSS)-induced model of ulcerative colitis was used to study the function of endogenous galectin-1 during the development of intestinal inflammation. We found that galectin-1 deficient mice (Lgals1−/− mice) displayed a more severe intestinal inflammation, characterized by significantly elevated clinical scores, than their wild type counterparts. The mechanisms underlying the enhanced inflammatory response in colitic Lgals1−/− mice involved an altered Th17/Th1 profile of effector CD4+ T cells. Furthermore, increased frequencies of Foxp3+CD4+ regulatory T cells in colon lamina propria in Lgals1−/− mice were found. Strikingly, the exacerbated intestinal inflammatory response observed in Lgals1− / − mice was alleviated by adoptive transfer of wild type Foxp3+CD4+ regulatory T cells at induction of colitis. Altogether, these data highlight the importance of endogenous galectin-1 as a novel determinant in regulating T cell reactivity during the development of intestinal inflammation.

Abstract PO023: Single-cell transcriptomics identifies gene expression networks driving differentiation and tumorigenesis in the human fallopian tube

Abstract The human fallopian tube harbors the cell-of-origin for the majority of high-grade serous ‘ovarian’ cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We performed single-cell transcriptomic profiling in 12 primary fallopian specimens from 8 patients, analyzing around 53,000 individual cells to map the major immune, fibroblastic and epithelial cell types present in this organ. We identified 10 epithelial sub-populations, characterized by diverse transcriptional programs including SOX17 (enriched in secretory epithelial cells), TTF3 and RFX3 (enriched in ciliated cells) and NR2F2 (enriched in early, partially differentiated secretory cells). Based on transcriptional signatures, we reconstructed a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3high intermediate. Computational deconvolution of the cellular composition of advanced HGSCs based on epithelial subset signatures identified the ‘early secretory’ population as a likely precursor state for the majority of HGSCs. The signature of this rare population of cells comprised both epithelial (EPCAM, KRT) and mesenchymal (THY1, ACTA2) features, and was enriched in mesenchymal-type HGSCs, a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia. Further computational analyses on fibroblast and immune cells together with tumor single-cell data reveals extended contribution of non-epithelial microenvironment and cell-cell interactions and communications in fallopian tube that potentially drive HGSC progression. Citation Format: Huy Q. Dinh, Xianzhi Lin, Forough Abbasi, Robbin Nameki, Marcela Haro, Claire Olingy, Heidi Chang, Lourdes Hernandez, Simon Gayther, Kelly N. Wright, Paul-Joseph Aspuria, Beth Karlan, Rosario I. Corona, Andrew Li, B.J. Rimel, Matthew Siedhoff, Fabiola Medeiros, Kate Lawrenson. Single-cell transcriptomics identifies gene expression networks driving differentiation and tumorigenesis in the human fallopian tube [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO023.