Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Flashner, Samuel; Martin, Cecilia; Matsuura, Nobuo; Shimonosono, Masataka; Tomita, Yasuto; Morimoto, Manabu; Okolo, Ogoegbunam; Yu, Victoria X.; Parikh, Anuraag S.; Klein‐Szanto, Andres J.; Yan, Kelley S.; Gabre, Joel; Lü, Chao; Momen-Heravi, Fatemeh; Rustgi, Anil K.; Nakagawa, Hiroshi

doi:10.3791/64676

Cited by 5 publications

(14 citation statements)

References 24 publications

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“…6A). These organoid cultures have been extensively characterized and faithfully recapitulate the genomic and histological features of human normal esophagus and ESCC (48,49). DNAme profiling by RRBS, as well as CUT&Tag for H3K4me3, H3K27me3 and H2AK119Ub, were performed in six independently generated organoid lines (3 x EN; 3 x ESCC).…”

Section: Resultsmentioning

confidence: 99%

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Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

Gretarsson,

Abini-Agbomson,

Gloor

et al. 2024

Preprint

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During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.

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Section: Resultsmentioning

confidence: 99%

“…Murine organoids were generated as previously (49). Briefly, K5Cre ERT2 transgenic mice were crossed to R26tdTomato lsl/lsl and Trp53 loxP/loxP (Jackson Laboratory) to yield K5Cre ERT2 ;R26tdTomato lsl/lslt ;Trp53 loxP/loxP .…”

Section: Organoid Culture Isolation and Propagationmentioning

confidence: 99%

Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

Gretarsson,

Abini-Agbomson,

Gloor

et al. 2024

Preprint

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“…Che-Hong Chen and Daria Mochly-Rosen, Stanford University) were crossed to generate Aldh2*2/Aldh2*1 (Aldh2*2 HET) mice. Under IACUC-approved protocols, Aldh2 WT and Aldh2*2 HET mice were subjected to treatment with 100 mg/mL 4-nitroquinline-1-oxide (4NQO) (TCI NO250, Tokyo, Japan) in drinking water for 16 weeks to generate preneoplastic esophageal 3D organoids according to detailed protocols published by us [16,27]. In brief, 4NQO-treated mice were euthanized and dissected to collect esophagi and esophageal epithelial sheets.…”

Section: Methodsmentioning

confidence: 99%

“…A portion of the tissue was reserved for histological diagnosis of intraepithelial neoplasia lesions. Following mechanical and enzymatic dissociation, the resulting single cells were suspended in advanced DMEM/F12 (ThermoFisher 12634028)-based Murine Organoid Media (MOM) [27]. 2x10 4 single cells were embedded in Matrigel Basement Membrane Extract (Corning 354234) in a 24 well plate (ThermoFisher 12-556-006) and grown in 500 µL MOM media added into each well.…”

Section: Methodsmentioning

confidence: 99%

“…To address this hypothesis, we leveraged our recently developed three dimensional (3D) esophageal organoid platform. 3D organoids are formed from single cells and capture the functional, morphological, and molecular features of the original tissue [16,[23][24][25][26][27][28]. Key to this study, 3D organoids harbor a distinguishable population of CD44H cells that more readily form tumors as well as organoids compared with their counterparts of low CD44 expression (CD44L) [16,[23][24][25].…”

Section: Introductionmentioning

confidence: 99%

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ALDH2 dysfunction accelerates ESCC pathogenesis

Flashner

Shimonosono

Matsuura

et al. 2023

Preprint

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The alcohol metabolite acetaldehyde is a potent human carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulates and causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent amongst East Asians. Chronic alcohol users with heterozygous ALDH2*2 display an increased risk for the development of esophageal squamous cell carcinoma (ESCC) and other alcohol-related cancers. However, how ALDH2 influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three dimensional organoids, and xenograft models. We find that alcohol exposure results in increased organoid formation and tumor growth concurrent with increased reactive oxygen species (ROS), increased DNA damage, and the enrichment of putative cancer stem cells (CSCs) characterized by high CD44 expression. Pharmacological activation of ALDH2 function by Alda-1 inhibits this phenotype, indicating that acetaldehyde is the primary driver of these changes. ALDH2 dysfunction also affects response to a commonly used chemotherapy for the treatment of ESCC. We find that Aldh2 dysfunction facilitated enrichment of CSCs following cisplatin-induced cell death and oxidative stress in murine organoids. Together, these data provide evidence that alcohol exposure results in more aggressive tumors through enrichment of CSCs, which is augmented by ALDH2 dysfunction.

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Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids

Shimonosono,

Morimoto,

Hirose

et al. 2024

Preprint

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BackgroundEsophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco’s Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state.MethodsWe have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling.ResultsThe formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ receptor-mediated signaling. In optimized HOME0, normal human esophageal organoid formation was improved, whereas IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis.Conclusions:HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

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Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Cited by 5 publications

References 24 publications

Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

ALDH2 dysfunction accelerates ESCC pathogenesis

Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids

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