modeling-oral-esophageal-squamous-cell-carcinoma-in-3d-organoids

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Columbia University

Columbia College

Advanced gastric cancer in the lower third of the stomach often results in stricture of the gastric cavity and digestive symptoms. Gastrojejunostomy has been suggested to improve such symptoms, and the advent of new anticancer agents for gastric cancer has improved the response rate of the disease, which makes it possible to perform R0 gastrectomy in part of patients with stage IV gastric cancer. We experienced a rare case in which a patient with stage IV gastric cancer and cancerous pyloric stenosis was treated with R0 surgery after undergoing a gastrojejunal bypass procedure and multidisciplinary treatment. There have not been any previous reports about cases in which a previous gastrojejunostomy was utilized as a reconstruction route during distal gastrectomy in a patient with gastric cancer that had been treated with chemotherapy and/or CRT.

A case report of curative distal gastrectomy for stage IV gastric cancer after chemoradiotherapy in a patient with a gastrojejunal gastric bypass.

The homeostatic proliferation-differentiation gradient in the esophageal epithelium is perturbed under inflammatory disease conditions such as gastroesophageal reflux disease and eosinophilic esophagitis. Herein we describe the protocols for rapid generation (<14 days) and characterization of single-cell-derived, three-dimensional (3D) esophageal organoids from human subjects and mice with normal esophageal mucosa or inflammatory disease conditions. While 3D organoids recapitulate normal epithelial renewal, proliferation, and differentiation, non-cell autonomous reactive epithelial changes under inflammatory conditions are evaluated in the absence of the inflammatory milieu. Reactive epithelial changes are reconstituted upon exposure to exogenous recombinant cytokines. These changes are modulated pharmacologically or genetically ex vivo. Molecular, structural, and functional changes are characterized by morphology, flow cytometry, biochemistry, and gene expression analyses. Esophageal 3D organoids can be translated for the development of personalized medicine in assessment of individual cytokine sensitivity and molecularly targeted therapeutics in esophagitis patients © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal organoids from biopsy or murine esophageal epithelial sheets Basic Protocol 2: Propagation and cryopreservation of esophageal organoids Basic Protocol 3: Harvesting of esophageal organoids for RNA isolation, immunohistochemistry, and evaluation of 3D architecture Basic Protocol 4: Modeling of reactive epithelium in esophageal organoids.

Modeling Epithelial Homeostasis and Reactive Epithelial Changes in Human and Murine Three-Dimensional Esophageal Organoids.

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal cancer PDO Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5: PDO content analysis by flow cytometry Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC ) Support Protocol: Production of RN in HEK293T cell conditioned medium.

Generation and Characterization of Patient-Derived Head and Neck, Oral, and Esophageal Cancer Organoids.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

We have previously constructed a novel microRNA (miRNA)-based prognostic model and cancer-specific mortality risk score formula to predict survival outcome in oral squamous cell carcinoma (OSCC) patients who are already categorized into "early-stage" by the TNM staging system. A total of 836 early-stage OSCC patients were assigned the mortality risk scores. We evaluated the efficacy of various treatment regimens in terms of survival benefit compared to surgery only in patients stratified into high (risk score ≥0) versus low (risk score <0) mortality risk categories. For the high-risk group, surgery with neck dissection significantly improved the 5-year survival to 75% from 46% with surgery only ( < 0.001); a Cox proportional hazard model on time-to-death demonstrated a hazard ratio of 0.37 for surgery with neck dissection (95% CI: 0.2-0.6; =0.0005). For the low-risk group, surgery only was the treatment of choice associated with 5-year survival benefit. Regardless of treatment selected, those with risk score ≥2 may benefit from additional therapy to prevent cancer relapse. We also identified hTERT (human telomerase reverse transcriptase) as a gene target common to the prognostic miRNAs. There was 22-fold increase in the hTERT expression level in patients with risk score ≥2 compared to healthy controls ( < 0.0005). Overexpression of hTERT was also observed in the patient-derived OSCC organoid compared to that of normal organoid. The DNA cancer vaccine that targets hTERT-expressing cells currently undergoing rigorous clinical evaluation for other tumors can be repurposed to prevent cancer recurrence in these high-risk early-stage oral cancer patients.

MicroRNA-Based Cancer Mortality Risk Scoring System and hTERT Expression in Early-Stage Oral Squamous Cell Carcinoma.

Three-dimensional (3D) organoids are a novel tool to model epithelial cell biology and human diseases of the esophagus. 3D organoid culture systems have been utilized to investigate the pathobiology of esophageal cancer, including both squamous cell carcinoma and adenocarcinoma. Additional organoid-based approaches for study of esophageal development and benign esophageal diseases have provided key insights into esophageal keratinocyte differentiation and mucosal regeneration. These investigations have implications for the identification of esophageal cancer stem cells, as well as the potential to halt malignant progression through induction of differentiation pathways. Patient-derived organoids (PDOs) from human tissue samples allow for unique and faithful in vitro modeling of esophageal cancers, and provide an exciting platform for investigation into personalized medicine and targeted treatment approaches, as well as new models for understanding therapy resistance and recurrent disease. Future directions include high-throughput genomic screening using PDOs, and study of tumor-microenvironmental interactions through co-culture with immune and stromal cells and novel extracellular matrix complexes.

Understanding the cellular origin and progression of esophageal cancer using esophageal organoids.

Alcohol (ethanol) consumption is a major risk factor for head and neck and esophageal squamous cell carcinomas (SCCs). However, how ethanol (EtOH) affects SCC homeostasis is incompletely understood.

Alcohol Metabolism Enriches Squamous Cell Carcinoma Cancer Stem Cells That Survive Oxidative Stress via Autophagy.

Although basal cell hyperplasia is a histologic hallmark of eosinophilic esophagitis (EoE), little is known about the capabilities of epithelial renewal and differentiation in the EoE inflammatory milieu. In murine esophageal epithelium, there are self-renewing and slowly proliferating basal stem-like cells characterized by concurrent expression of CD73 (5'-nucleotidase ecto) and CD104 (integrin β4). Here, we investigated CD73CD104 cells within the basal population of human esophageal epithelium and clarified the biological significance of these cells in the EoE epithelium.

CD73 Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCF E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCF-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCF leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCF-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCF-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.

Article	Year
A case report of curative distal gastrectomy for stage IV gastric cancer after chemoradiotherapy in a patient with a gastrojejunal gastric bypass. Surgical case reports\| PubMed ID: 27837525	2016
Modeling Epithelial Homeostasis and Reactive Epithelial Changes in Human and Murine Three-Dimensional Esophageal Organoids. Current protocols in stem cell biology\| PubMed ID: 32105412	2020
Generation and Characterization of Patient-Derived Head and Neck, Oral, and Esophageal Cancer Organoids. Current protocols in stem cell biology\| PubMed ID: 32294323	2020
Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes. PloS one\| PubMed ID: 32966340	2020
MicroRNA-Based Cancer Mortality Risk Scoring System and hTERT Expression in Early-Stage Oral Squamous Cell Carcinoma. Journal of oncology\| PubMed ID: 33510789	2021
Understanding the cellular origin and progression of esophageal cancer using esophageal organoids. Cancer letters\| PubMed ID: 33838281	2021
Alcohol Metabolism Enriches Squamous Cell Carcinoma Cancer Stem Cells That Survive Oxidative Stress via Autophagy. Biomolecules\| PubMed ID: 34680112	2021
CD73 Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 35108658	2022
Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation. Nature communications\| PubMed ID: 36329064	2022

Masataka Shimonosono

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