generation-and-genetic-manipulation-of-human-cervical-organoids

Generation and Genetic Manipulation of Human Cervical Organoids

Cervical cancer is the fourth most common cancer in women and most frequently affects the uterine ectocervix. The uterine ectocervix is lined by ...

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Activation of NFE2L2 has been linked to chemoresistance in cell line models. Recently, somatic mutations that activate NFE2L2, including mutations in , or  have been found to be associated with poor outcomes in patients with non-small cell lung cancer (NSCLC). However, the impact of these mutations on chemoresistance remains incompletely explored.

Role of KEAP1/NFE2L2 Mutations in the Chemotherapeutic Response of Patients with Non-Small Cell Lung Cancer.

Adiponectin (Ad) is a representative adipocytokine that regulates energy homeostasis including glucose transport and lipid oxidation through activation of AMP-activated protein kinase (AMPK) pathways. Plasma levels of Ad are reduced in obesity, which contributes to type 2 diabetes. Therefore, agents that activate the Ad signaling pathway could ameliorate metabolic diseases such as type 2 diabetes. Here, we report the identification of a high-affinitive agonist antibody against Ad receptors. The antibody was selected by using phage display of human combinatorial antibody libraries. The selected antibody induced phosphorylation of the acetyl-CoA carboxylase (ACC) and AMPK in skeletal muscle cells and stimulated glucose uptake and fatty-acid oxidation (FAO) in myotubes. In addition, the antibody significantly lowered blood glucose levels during a glucose challenge in normal mice as well as basal blood glucose levels in a type 2 diabetic mouse model. Taken together, these results suggest that the agonist antibody could be a promising therapeutic agent for the treatment of metabolic syndrome such as type 2 diabetes.

An adiponectin receptor agonist antibody stimulates glucose uptake and fatty-acid oxidation by activating AMP-activated protein kinase.

Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway.

A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERβ agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Estrogen activates endothelial exocytosis.

Interferon-γ (IFNG) is one of the key cytokines that regulates both innate and adaptive immune responses in the body. However, the role of IFNG in the regulation of vascularization, especially in the context of Vascular endothelial growth factor A (VEGFa)-induced angiogenesis is not clarified. Here, we report that IFNG shows potent anti-angiogenic potential against VEGFa-induced angiogenesis. IFNG significantly inhibited proliferation, migration, and tube formation of Human umbilical vein endothelial cells (HUVECs) both under basal and VEGFa-treated conditions. Intriguingly, Knockdown (KD) of STAT1 abolished the inhibitory effect of IFNG on VEGFa-induced angiogenic processes in HUVECs. Furthermore, IFNG exhibited potent anti-angiogenic efficacy in the mouse model of oxygen-induced retinopathy (OIR), an in vivo model for hypoxia-induced retinal neovascularization, without induction of functional side effects. Taken together, these results show that IFNG plays a crucial role in the regulation of VEGFa-dependent angiogenesis, suggesting its potential therapeutic applicability in neovascular diseases.

Interferon-γ inhibits retinal neovascularization in a mouse model of ischemic retinopathy.

Extracellular vesicles (EVs) mediate cell-cell crosstalk by carrying bioactive molecules derived from cells. Recently, immune cell-derived EVs have been reported to regulate key biological functions such as tumor progression. CD4 T cells orchestrate overall immunity; however, the biological role of their EVs is unclear. This study reveals that EVs derived from CD4 T cells increase the antitumor response of CD8 T cells by enhancing their proliferation and activity without affecting regulatory T cells (Tregs). Moreover, EVs derived from interleukin-2 (IL2)-stimulated CD4 T cells induce a more enhanced antitumor response of CD8 T cells compared with that of IL2-unstimulated CD4 T cell-derived EVs. Mechanistically, miR-25-3p, miR-155-5p, miR-215-5p, and miR-375 within CD4 T cell-derived EVs are responsible for the induction of CD8 T cell-mediated antitumor responses. In a melanoma mouse model, the EVs potently suppress tumor growth through CD8 T cell activation. This study demonstrates that the EVs, in addition to IL2, are important mediators between CD4 and CD8 T cells. Furthermore, unlike IL2, clinically used as an antitumor agent, CD4 T cell-derived EVs stimulate CD8 T cells without activating Tregs. Therefore, CD4 T cell-derived EVs may provide a novel direction for cancer immunotherapy by inducing a CD8 T cell-mediated antitumor response.

Novel antitumor therapeutic strategy using CD4 T cell-derived extracellular vesicles.

Tongue epithelium is one of the most proliferative and regenerative epithelia in our body. However, tongue stem cell research is hampered partly by the lack of optimal animal models to study tongue injury, repair, and regeneration. Here, we establish a novel chemically induced tongue injury-recovery mouse model. Focal application of sodium hydroxide for a limited time led to the denudation of suprabasal layers, leaving the basal layer. Time course study revealed that tongue epithelial cells robustly proliferate over one week after the tongue injury. Importantly, we demonstrated that our novel mouse model could be employed in the lineage tracing of the tongue stem cells under the injury and repair process and further showed that tongue stem cells proliferate faster and generate larger clones in the injury condition than in the steady state condition. Our data indicate the development of a novel chemically induced tongue injury-recovery mouse model for tongue stem cell research, which will significantly facilitate the preclinical study for the pathogenesis and treatment of caustic ingestion.

Establishment of a chemical tongue injury-recovery mouse model.

Esophageal epithelium is one of the most proliferative and regenerative epithelia in our body, indicating robust stem cell activity. However, the underlying mechanisms regulating the self-renewal and differentiation of esophageal stem cells need to be more elucidated. Here, we identify the role of YAP1 in esophageal stem cells. YAP1 is differentially expressed in the nuclei of esophageal basal cells. Furthermore, the treatment of verteporfin, a YAP1 inhibitor, interfered with esophageal organoid formation. Consistently, YAP1 deletion decreased esophageal organoid formation and the expression of basal genes while increasing the expression of suprabasal genes. Finally, global transcriptomic analysis revealed that YAP1 inhibition induced a significant enrichment of gene sets related to keratinization and cornification, while depleting gene sets related to DNA repair and chromosome maintenance. Our data uncover a novel regulatory mechanism for esophageal stem cells, which could provide a potential strategy for esophageal regenerative medicine.

YAP1 regulates esophageal stem cells' self-renewal and differentiation.

Natural killer (NK) cells are gaining growing attention due to their promise for immunotherapy. A fast and accurate system is needed to test NK cell biology and their therapeutic application. Here, we report a lung cancer organoid-based system to evaluate NK cells' cytotoxicity. We first established the lung cancer organoids on top of Matrigel, which allows the co-culture with NK cells. When co-cultured, NK cells moved close to and inside the lung cancer organoids. When we analyzed by flow cytometry, co-culture of NK cells induced a significantly higher ratio of cell death of lung cancer organoids, suggesting that lung cancer organoids can be employed to test the cytotoxicity of NK cells. Finally, the pre-treatment of NK cells with A83-01, a TGF inhibitor, significantly enhanced the cell death of lung cancer organoids by NK cells, indicating that lung cancer organoid-based system faithfully recapitulates cell line-based system in evaluating the  cytotoxicity of NK cells. These data represent that cancer organoid-based NK cell co-culture system is a reliable platform for studying NK cell biology and evaluating their cytotoxicity for screening for NK cell immunotherapy.

Lung Cancer Organoid System to Evaluate the Cytotoxicity of Natural Killer Cells.

IL2-mediated modulation of small extracellular vesicles secretion and PD-L1 expression: a novel perspective for neutralizing immune suppression within cancer cells.

Cervical cancer is the fourth most common female cancer, with the uterine ectocervix being the most commonly affected site. However, cervical stem cells, their differentiation, and their regulation remain poorly understood. Here, we report the isolation of a population enriched for human cervical stem cells and their regulatory mechanisms. Using single-cell RNA sequencing, we characterize the cellular heterogeneity of the human ectocervix and identify cluster-specific cell surface markers. By establishing normal and precancerous cervical organoids and an intralingual transplantation system, we show that ITGB4 and CD24 enable enrichment of human and murine ectocervical stem cells. We discover that Lactobacilli-derived lactic acid regulates cervical stem cells' self-renewal and early tumorigenesis through the PI3K-AKT pathway and YAP1. Finally, we show that D-lactic acid suppresses growth of normal and precancerous organoids, while L-lactic acid does not. Our findings reveal roles of human cervical stem cells and microbial metabolites in cervical health and diseases.

Microbial metabolites control self-renewal and precancerous progression of human cervical stem cells.

Iron is vital to living cells, playing a key role in cellular respiration, DNA synthesis, and various metabolic functions. Importantly, cancer cells have a higher dependency on iron compared to normal cells to support their rapid growth and survival. Due to this fact, tumors are more vulnerable to ferroptosis, an iron-dependent form of regulated cell death. Radiation therapy (RT), a standard treatment for many cancer patients, is known to induce ferroptosis. Ultra-high dose rate FLASH RT offers an improved therapeutic window by minimizing damage to normal tissues while preserving tumor control. However, the precise biological mechanisms behind the protective effects of FLASH RT on normal tissues remain unclear. In this study, we propose that variations in lipid peroxidation and ferroptosis, driven by intrinsic differences in iron levels between normal and cancerous tissues, contribute to this effect. Our findings show that FLASH RT increases lipid peroxidation and induces ferroptosis in tumor cells but does not significantly elevate lipid peroxidation and ferroptosis in normal tissues compared to conventional RT. To determine whether raising iron levels in normal tissues could abrogate the protective effects of FLASH, mice were fed a high-iron diet before RT. A high-iron diet before and after RT reversed the protective effect of FLASH, resulting in increased intestinal damage and lipid peroxidation. This suggests that baseline iron levels and iron-driven lipid peroxidation are critical factors in mediating the protective outcomes of FLASH RT. Overall, our study sheds light on the role of iron in modulating RT responses and provides new mechanistic insights into how FLASH RT influences normal and cancerous tissues.

Article	Year
Role of KEAP1/NFE2L2 Mutations in the Chemotherapeutic Response of Patients with Non-Small Cell Lung Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 31548347	2020
An adiponectin receptor agonist antibody stimulates glucose uptake and fatty-acid oxidation by activating AMP-activated protein kinase. Cytokine\| PubMed ID: 31629112	2020
A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk. Genome biology\| PubMed ID: 32381040	2020
Estrogen activates endothelial exocytosis. Biochemical and biophysical research communications\| PubMed ID: 33895548	2021
Interferon-γ inhibits retinal neovascularization in a mouse model of ischemic retinopathy. Cytokine\| PubMed ID: 33926775	2021
Novel antitumor therapeutic strategy using CD4 T cell-derived extracellular vesicles. Biomaterials\| PubMed ID: 36067566	2022
Establishment of a chemical tongue injury-recovery mouse model. Biochemical and biophysical research communications\| PubMed ID: 36099782	2022
YAP1 regulates esophageal stem cells' self-renewal and differentiation. Biochemical and biophysical research communications\| PubMed ID: 38909534	2024
Lung Cancer Organoid System to Evaluate the Cytotoxicity of Natural Killer Cells. International journal of stem cells\| PubMed ID: 38919124	2025
IL2-mediated modulation of small extracellular vesicles secretion and PD-L1 expression: a novel perspective for neutralizing immune suppression within cancer cells. Cancer communications (London, England)\| PubMed ID: 39440703	2024
Microbial metabolites control self-renewal and precancerous progression of human cervical stem cells. Nature communications\| PubMed ID: 40057497	2025
Tissue-specific iron levels modulate lipid peroxidation and the FLASH radiotherapy effect. Cell death & disease\| PubMed ID: 40897687	2025

Youngtae Jeong

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