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

Background: Esophageal 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. Methods: We 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)-ß 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. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFß 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.

Successful subtotal gastrectomy and hepatectomy for HER2-positive gastric cancer with liver metastasis after trastuzumab-based chemotherapy: a case report.

BACKGROUND: Conversion surgery (CS) after chemotherapy is weakly recommended as a promising tool for improving prognoses in patients with unresectable gastric cancer. Moreover, several investigators have demonstrated the clinical efficacy of subtotal gastrectomy (sTG) with a small remnant stomach for the nutritional status and surgical outcome compared with total gastrectomy. Here, we report a patient with liver metastasis from human epidermal growth factor receptor 2 (HER2)-positive gastric cancer who underwent sTG and hepatectomy after trastuzumab-based chemotherapy. CASE PRESENTATION: An 84-year-old male patient was diagnosed with HER2-positive gastric cancer with a single liver metastasis. He was treated with eight courses of trastuzumab in combination with S-1 and oxaliplatin as first-line chemotherapy. The primary tumor and liver metastasis shrank significantly. The metastatic liver lesion's reduction rate was 65%. According to the Response Evaluation Criteria in Solid Tumors, the patient had a partial response. Therefore, he underwent an sTG with D2 lymphadenectomy and partial hepatectomy of segment 2. Histopathological examination revealed a grade 3 histological response without lymph node metastases from the primary tumor. No viable cancer cells were observed in the resected liver specimens. The patient received adjuvant chemotherapy with S-1. The postoperative quality of life (QOL) evaluated using the Postgastrectomy Syndrome Assessment Scale-45 was maintained, and the patient was still alive 8 months after the CS without recurrence. CONCLUSIONS: An sTG with a small remnant stomach might be clinically useful for preventing a decline in QOL and improving prognoses in patients with stage IV gastric cancer after chemotherapy.

Epithelial overexpression of IL-33 induces eosinophilic esophagitis dependent on IL-13.

BACKGROUND: Eosinophilic esophagitis (EoE) is an increasingly common inflammatory condition of the esophagus; however, the underlying immunologic mechanisms remain poorly understood. The epithelium-derived cytokine IL-33 is associated with type 2 immune responses and elevated in esophageal biopsy specimens from patients with EoE. OBJECTIVE: We hypothesized that overexpression of IL-33 by the esophageal epithelium would promote the immunopathology of EoE. METHODS: We evaluated the functional consequences of esophageal epithelial overexpression of a secreted and active form of IL-33 in a novel transgenic mouse, EoE33. EoE33 mice were analyzed for clinical and immunologic phenotypes. Esophageal contractility was assessed. Epithelial cytokine responses were analyzed in three-dimensional organoids. EoE33 phenotypes were further characterized in ST2-/-, eosinophil-deficient, and IL-13-/- mice. Finally, EoE33 mice were treated with dexamethasone. RESULTS: EoE33 mice displayed ST2-dependent, EoE-like pathology and failed to thrive. Esophageal tissue remodeling and inflammation included basal zone hyperplasia, eosinophilia, mast cells, and TH2 cells. Marked increases in levels of type 2 cytokines, including IL-13, and molecules associated with immune responses and tissue remodeling were observed. Esophageal organoids suggested reactive epithelial changes. Genetic deletion of IL-13 in EoE33 mice abrogated pathologic changes in vivo. EoE33 mice were responsive to steroids. CONCLUSIONS: IL-33 overexpression by the esophageal epithelium generated immunopathology and clinical phenotypes resembling human EoE. IL-33 may play a pivotal role in the etiology of EoE by activating the IL-13 pathway. EoE33 mice are a robust experimental platform for mechanistic investigation and translational discovery.

Comparison of the subtotal and narrow gastric conduit for cervical esophagogastrostomy after esophagectomy in esophageal cancer patients: a propensity score-matched analysis.

BACKGROUND: Several reports have compared narrow gastric conduit (NGC) with subtotal gastric conduit (SGC) for cervical esophagogastrostomy after esophagectomy; however, whether which one is more beneficial in terms of postoperative complications remains unclear. To determine the optimal gastric conduit type, we retrospectively investigated and compared the postoperative complications between NGC and SGC used in cervical circular-tapered esophagogastrostomy after esophagectomy through a propensity score-matched analysis. METHODS: Between 2008 and 2022, 577 consecutive esophageal cancer patients who underwent esophagectomy and cervical circular-stapled esophagogastrostomy were enrolled in this study. RESULTS: Of the 577 patients, 77 were included each in the SGC and NGC groups, after propensity score matching. Clinical characteristics did not differ between the two groups. The anastomotic leakage rate was significantly lower in the SGC group than in the NGC group (5% vs. 22%, p < 0.01). The anastomotic stenosis rate was significantly higher in the SGC group (16% vs. 5%, p = 0.03). Multivariate logistic analysis showed that NGC, subcutaneous route, and age were significant independent factors associated with anastomotic leakage (odds ratios, 8.58, 6.49, and 5.21; p < 0.01, < 0.01 and 0.03, respectively) and that SGC was a significant independent factor associated with anastomotic stricture (odds ratios, 4.91; p = 0.04). CONCLUSIONS: In cervical circular-stapled esophagogastrostomy after esophagectomy, SGC was superior to NGC in terms of reducing the risk of anastomotic leakage, although the risk of anastomotic stricture needs to be resolved.

ALDH2 dysfunction and alcohol cooperate in cancer stem cell enrichment.

The alcohol metabolite acetaldehyde is a potent human carcinogen linked to esophageal squamous cell carcinoma (ESCC) initiation and development. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulation causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K dominant negative mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent among East Asians. Heterozygous ALDH2*2 increases the risk for the development of ESCC and other alcohol-related cancers. Despite its prevalence and link to malignant transformation, how ALDH2 dysfunction 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 and ALDH2*2 cooperate to increase putative ESCC cancer stem cells with high CD44 expression (CD44H cells) linked to tumor initiation, repopulation and therapy resistance. Concurrently, ALHD2*2 augmented alcohol-induced reactive oxygen species and DNA damage to promote apoptosis in the non-CD44H cell population. Pharmacological activation of ALDH2 by Alda-1 inhibits this phenotype, suggesting that acetaldehyde is the primary driver of these changes. Additionally, we find that Aldh2 dysfunction affects the response to cisplatin, a chemotherapeutic commonly used for the treatment of ESCC. Aldh2 dysfunction facilitated enrichment of CD44H cells following cisplatin-induced oxidative stress and cell death in murine organoids, highlighting a potential mechanism driving cisplatin resistance. Together, these data provide evidence that ALDH2 dysfunction accelerates ESCC pathogenesis through enrichment of CD44H cells in response to genotoxic stressors such as environmental carcinogens and chemotherapeutic agents.

Evaluation of Quality of Life and Prognosis of Gastric Cancer Patients After Laparoscopic Subtotal Gastrectomy.

BACKGROUND/AIM: The clinical significance of laparoscopic subtotal gastrectomy (LsTG) with a small remnant stomach remains unclear in patients with gastric cancer, including at an advanced stage. The present study assessed postoperative quality of life (QOL) and survival after LsTG compared with laparoscopic total gastrectomy (LTG). PATIENTS AND METHODS: We retrospectively analyzed consecutive patients with gastric cancer who underwent LsTG (n=26) or LTG (n=26). Surgical outcome, postoperative nutritional status, QOL, and prognosis were compared between the LsTG and LTG groups. The Postgastrectomy Syndrome Assessment Scale was used to evaluate postoperative QOL. RESULTS: Operating time was significantly shorter (p<0.01) and postoperative morbidity was significantly lower (p=0.04) in the LsTG than in the LTG group. The reduction in body weight after surgery was significantly greater in the LTG than in the LsTG group (p<0.01). The Postgastrectomy Syndrome Assessment Scale revealed that, compared with LTG, LsTG significantly improved postoperative QOL (p<0.05). There was no significant difference in relapse-free survival and cancer-specific survival between the two groups. Three patients in the LTG group died of pneumonia and overall survival was significantly longer in the LsTG group (p=0.01). CONCLUSION: This study demonstrated the efficacy of LsTG with a small remnant stomach to prevent a decline in postoperative QOL and non-cancer-related death.

Clinical significance of intraperitoneal paclitaxel combined with systemic chemotherapy for gastric cancer with peritoneal metastasis.

BACKGROUND: Despite investigations of intraperitoneal paclitaxel as a personalized treatment for peritoneal metastasis of gastric cancer, few studies have evaluated its prognostic impact on conversion surgery for unresectable gastric cancer with peritoneal metastasis. Our study aimed to close this gap in knowledge. METHODS: We retrospectively enrolled 128 patients who underwent chemotherapy for peritoneal metastasis from gastric cancer and assigned them into intraperitoneal (IP) (n = 36) and non-IP (n = 92) groups, based on the use of intraperitoneal paclitaxel plus systemic chemotherapy. RESULTS: Disease control rates were 94% and 69% in the IP and non-IP groups, respectively, with the former having a significantly higher tumor response rate than the latter (p < 0.01). The median survival times in the IP and non-IP groups were 665 and 359 days, respectively, with the former having significantly better prognosis than the latter (p = 0.02). Fifteen (42%) and sixteen (17%) patients underwent conversion surgery after chemotherapy in the IP and non-IP groups, respectively, with the former having a significantly higher conversion surgery induction rate than the latter (p < 0.01). Although the prognosis of the conversion surgery group was significantly better than that of the non-conversion surgery group (p < 0.01), there was no significant difference in prognosis between patients in the IP and non-IP groups who underwent conversion surgery (p = 0.22). Multivariate analysis identified performance status and conversion surgery as independent prognostic factors (all p < 0.01). CONCLUSION: Our study demonstrated that the IP chemotherapy was one of important factors for conversion surgery induction, while it was not a risk factor for prognosis.

Modified pull-through coloanal anastomosis to avoid permanent stomas and reduce postoperative complications for lower rectal tumors.

BACKGROUND: We performed pull-through hand-sewn coloanal anastomosis immediately after sphincter-preserving ultralow anterior resection (ULAR) [pull-through ultra (PTU)] to avoid permanent stoma and reduce postoperative complications of lower rectal tumors. This study aimed to compare the clinical outcomes of PTU versus non-PTU (stapled or hand-sewn coloanal anastomosis with diverting stoma) after sphincter-preserving ULAR for lower rectal tumors. METHODS: This retrospective cohort study analyzed prospectively maintained data from 100 consecutive patients who underwent PTU (n = 29) or non-PTU (n = 71) after sphincter-preserving ULAR for rectal tumors between January 2011 and March 2023. In PTU, hand-sewn coloanal anastomosis was immediately performed using 16 stitches of 4-0 monofilament suture during primary surgery. The clinical outcomes were assessed. The primary outcomes were rates of permanent stomas and overall postoperative complications. RESULTS: The PTU group was significantly less likely to require a permanent stoma than the non-PTU group (P < 0.01). None of the patients in the PTU group required permanent stoma and the rate of overall complications was significantly lower in the PTU group (P = 0.01). The median operative time was comparable between the two groups (P = 0.33) but the median operative time during the second stage was significantly shorter in the PTU group (P < 0.01). The rates of anastomotic leakage and complications of Clavien-Dindo grade III were comparable between the two groups. Diverting ileostomy was performed in two patients with an anastomotic leak in the PTU group. The PTU group was significantly less likely to require a diverting ileostomy than those in the non-PTU group (P < 0.01). The composite length of hospital stay was significantly shorter in the PTU group (P < 0.01). CONCLUSIONS: PTU via immediate coloanal anastomosis for lower rectal tumors is a safe alternative to the current sphincter-preserving ULAR with diverting ileostomy for patients who wish to avoid a stoma.

Clinical Significance of Recurrence Risk Score for Conversion Surgery in Patients With Advanced Gastric Cancer.

BACKGROUND/AIM: To develop a recurrence risk score for determining the clinical indication for adjuvant chemotherapy in patients with initially unresectable advanced gastric cancer who underwent conversion surgery after chemotherapy. PATIENTS AND METHODS: A total of 65 patients with stage IV gastric cancer who underwent conversion surgery after chemotherapy were retrospectively enrolled. We established a risk score based on clinicopathological factors related to recurrence after conversion surgery. RESULTS: Out of 65 patients, 40 (62%) had recurrence after conversion surgery. The 5-year overall survival rates in patients with and without recurrence were 14.4% and 87.1%, respectively (p<0.01). Multivariate logistic regression analysis identified the depth of tumor invasion (pT2-4) and histological tumor response (grade 0-1a) as an independent risk factor for disease recurrence (p=0.033 and p=0.048, respectively). A scoring system determined by these two factors was created; total score ranged from 0 to 2 points, and patients were categorized into three groups (scores of 0 vs. 1 vs. 2 points). This scoring system showed that 12 (18%), 15 (23%), and 38 (58%) patients had recurrence risk scores of 0, 1, and 2 points, respectively. There was a close relationship between a high score and the presence of tumor recurrence (p<0.01). Moreover, our model system had a high sensitivity for the prediction of recurrence, compared with the pathological stage. CONCLUSION: Recurrence risk score is a promising tool for assessing the need for adjuvant chemotherapy in patients with initially unresectable advanced gastric cancer after conversion surgery.

Clinical Significance of Eligibility Criteria Determined by the SPIRITS Trial in Patients with Advanced Gastric Cancer.

INTRODUCTION: This study aimed to assess the clinical significance of eligibility criteria determined by phase 3 clinical trials in the clinical practice of patients with advanced gastric cancer who underwent chemotherapy. METHODS: Patients with stage IV gastric cancer who received chemotherapy between February 2002 and December 2021 were retrospectively enrolled and divided into two groups (the eligible vs. ineligible group) based on eligibility criteria determined by the SPIRITS (S-1 vs. S-1 plus cisplatin) trial. RESULTS: Among the 207 patients, 103 (49.8%) and 104 (50.2%) patients were classified into eligible and ineligible groups, respectively. Eligibility criteria were significantly correlated with age, the first-line regimen of chemotherapy, the presence or absence of conversion surgery, and tumor response to the first-line chemotherapy (all p < 0.01). The eligible group had a significantly higher induction of post-progression chemotherapy after first- and second-line chemotherapy than did the ineligible group (all p < 0.01). The ineligible group had significantly poorer prognoses than the eligible group (p < 0.0001). Multivariate analysis showed that peritoneal dissemination, tumor response, conversion surgery, and eligibility criteria were independent prognostic factors (all p < 0.05). CONCLUSION: Eligibility criteria determined by the SPIRITS trial may have clinical utility for predicting tumor response, the induction of conversion surgery, and prognosis in patients with advanced gastric cancer who underwent chemotherapy.

Clinical Outcome After Esophagectomy or Definitive Chemoradiotherapy in Elderly Patients (≥80 Years) With Esophageal Cancer.

BACKGROUND/AIM: Given the increased incidence of esophageal cancer (EC) in individuals over 80 years old, the optimum therapeutic strategy for elderly patients is needed to be established with scientific evidence. Here, we studied the short-term and long-term outcomes after treatment of patients aged 80 years old or older with EC. PATIENTS AND METHODS: Eighty patients with EC aged 80 years old or older, who underwent esophagectomy (n=23), definitive chemoradiotherapy (dCRT) (n=46) or best supportive care (n=11) between January 2010 and March 2019 were included in this study, and clinical data were compared among these groups. RESULTS: Surgery had a great benefit on the 3-year overall survival (OS) compared to dCRT (68.4% vs. 29.3%, p<0.01). The cure rates of treatment were 86.9% in surgery and 34.8% in dCRT. dCRT led to a better 3-year OS compared to BSC (29.3% vs. 0%, p<0.01); however, dCRT did not improved OS in patients with T4. Patients with T4 had high frequency of adverse events and treatment-related death in dCRT; CTCAE Grade 3-5 was observed in 100% of all T4 patients and Grade 5 in 57.1%. Multivariate analysis revealed that T4 was an independent risk factor of treatment-related death after dCRT (p<0.01). CONCLUSION: Surgery is the first treatment option for resectable EC even in elderly patients, and dCRT can be considered as an alternative. However, dCRT may induce severe toxicity especially in T4 EC patients.

Additional Effects of Docetaxel on Neoadjuvant Radiotherapy With Cisplatin/5-Fluorouracil for Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: To further improve the prognosis of locally advanced esophageal cancer patients, investigating new perioperative treatment strategies is necessary. The current study aimed to retrospectively investigate neoadjuvant radiotherapy with cisplatin and 5-fluorouracil (CF-RT) and radiotherapy with docetaxel and CF (DCF-RT) and compare their treatment outcomes in patients with locally advanced esophageal squamous cell carcinoma (ESCC). PATIENTS AND METHODS: We retrospectively reviewed 95 patients with ESCC who received CF-RT or DCF-RT followed by esophagectomy. The CF-RT group received chemotherapy consisting of two courses of CF repeated every 4 weeks. The DCF-RT group received chemotherapy consisting of two courses of DCF repeated every 2 weeks. A radiotherapy dose of 1.8-2 Gy was administered per session, up to a total of 40-41.4 Gy. Adverse events of neoadjuvant chemoradiotherapy, surgical outcomes, pathological responses, prognosis, and recurrence patterns were evaluated. RESULTS: Both the CF-RT and DCF-RT groups had equivalent pathological complete response rates of the primary tumor at 31.6% and 38.6%, respectively. However, the DCF-RT group had significantly better 5-year disease-free survival and 5-year overall survival than (HR=0.50, 95%CI=0.26-0.97, p=0.0392) than the CF-RT group. CONCLUSION: DCF-RT may be a candidate neoadjuvant therapy for locally advanced ESCC.

A comparison of the surgical invasiveness and short-term outcomes between thoracoscopic and pneumatic mediastinoscopic esophagectomy for esophageal cancer.

PURPOSE: Minimally invasive esophagectomy (MIE) has been widely accepted as a treatment for esophageal cancer. This retrospective study compared the short-term outcomes and surgical invasiveness between thoracoscopic esophagectomy (TE) and mediastinoscopic esophagectomy with pneumomediastinum (pneumatic mediastinoscopic esophagectomy [PME]). METHODS: A total of 72 patients who underwent TE or PME were included and assessed for their surgical findings, postoperative complications, and inflammatory responses on postoperative day (POD) 1, 3, 5, and 7. RESULTS: The PME group exhibited a significantly shorter operative time and fewer lymph nodes retrieved than the TE group. Furthermore, the PME group tended to have greater incidences of recurrent laryngeal nerve palsy and lower incidences of atelectasis than the TE group. The PME group had significantly lower white blood cell counts on POD 5, serum C-reactive protein (CRP) levels on POD 3 than the TE group. CONCLUSION: PME seems to be less invasive than TE and can be considered the preferred option for patients with lower-stage esophageal cancer expected to have severe pleural adhesion or who cannot tolerate TE.

A prognostic scoring system for conversion surgery after trastuzumab-based chemotherapy for human epidermal growth factor receptor 2-positive advanced gastric cancer.

PURPOSE: To investigate the clinical indications and prognostic significance of surgical interventions after chemotherapy using trastuzumab-containing regimens for patients with human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (AGC). METHODS: A total of 146 patients with AGC who underwent chemotherapy were enrolled in this retrospective study. Tumors with an immunohistochemistry (IHC) score of 3 + or an IHC score of 2 + plus fluorescence in situ hybridization positivity were defined as HER2-positive AGC. We devised a scoring system for predicting prognosis associated with conversion surgery. RESULTS: Thirty-three patients received trastuzumab-based chemotherapy for HER2-positive tumors. Multivariate analyses identified advanced age, peritoneal dissemination, histologically undifferentiated tumors, and tumor response of progressive disease as independent prognostic factors for a worse prognosis. Twelve patients with HER2-positive AGC underwent conversion surgery. The conversion surgery group of patients with HER2-positive AGC had a better prognosis than the chemotherapy-alone group. A prognostic scoring system based on age, peritoneal dissemination, and histological type was significantly correlated with the presence or absence of conversion surgery and the prognosis of patients with HER2-positive AGC. CONCLUSIONS: Our scoring system has the clinical potential to predict prognosis associated with conversion surgery after trastuzumab-containing chemotherapy for patients with HER2-positive AGC.

Esophageal plexiform fibromyxoma: A case report with molecular analysis for MALAT1-GLI1 fusion.

Plexiform fibromyxoma (PFM) is a rare gastrointestinal tract tumor that develops in the stomach in most cases. Here, we report an extremely rare case of esophageal PFM. A female in her mid-30 s presented with difficulty in swallowing and breathing. Endoscopic examination revealed a submucosal tumor measuring approximately 45 × 50 mm in the upper thoracic esophagus. The biopsied specimen did not show definite histological evidence of gastrointestinal stromal tumors (GISTs). Since imatinib administration based on a clinical diagnosis of GIST did not show a therapeutic effect for tumor reduction, tumor resection was performed. The resected tumor exhibited proliferation of spindle tumor cells with abundant myxoid and vascular stroma separated by a muscular layer, indicating a plexiform arrangement. Immunohistochemical analysis demonstrated that the tumor cells diffusely expressed vimentin and alpha-smooth muscle actin, but not desmin, c-kit, DOG1, and CD34. MALAT1-GLI1 fusion was detected in formalin-fixed paraffin-embedded tissue using RT-PCR and Sanger sequencing. The results suggested that a fibromyxoid tumor can develop in the esophagus, showing an identical histology and MALAT1-GLI1 fusion to gastric PFM.

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

BACKGROUND & AIMS: 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 ß4). Here, we investigated CD73+CD104+ cells within the basal population of human esophageal epithelium and clarified the biological significance of these cells in the EoE epithelium. METHODS: We performed flow cytometry on esophageal biopsy samples from EoE and non-EoE patients to determine the quantity of CD73+CD104+ cells in the epithelium. Simulating the EoE milieu we stimulated primary patient-derived and immortalized cell line-derived esophageal organoids with interleukin (IL)4 and IL13 and analyzed by flow cytometry, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. We performed single-cell RNA sequencing on primary organoids in the setting of IL13 stimulation and evaluated the CD73+CD104+ population. We performed fluorescent-activated cell sorting to purify CD73+CD104+ and CD73- CD104+ populations and seeded these groups in organoid culture to evaluate the organoid formation rate and organoid size. We used RNA interference to knock down CD73 in esophageal organoids to evaluate organoid formation rates and size. We evaluated the effects of signal transducer and activator of transcription 6 (STAT6) signaling inhibition by RNA interference, a STAT6 inhibitor, AS1517499, as well as the proton pump inhibitor omeprazole. RESULTS: EoE patients showed decreased epithelial CD73+CD104+ cell content. IL4 and IL13 stimulation depleted this population in 3-dimensional organoids with a recapitulation of basal cell hyperplasia as corroborated by single-cell RNA sequencing of the organoids, which suggests depletion of CD73+CD104+ cells. The CD73+CD104+ population had enhanced organoid formation compared with the CD73-CD104+ population. Similarly, knock-down of CD73 resulted in decreased organoid formation rate. Genetic and pharmacologic inhibition of STAT6 prevented T helper 2 cytokine-induced depletion of CD73+CD104+ cells. Lastly, omeprazole treatment prevented the effects of IL4 and IL13 on the CD73+CD104+ population. CONCLUSIONS: This study addressed the role of CD73+CD104+ cells in epithelial renewal and homeostasis in the context of EoE. The depletion of the CD73+CD104+ self-renewal population by helper T cell 2 cytokines in EoE milieu may be perpetuating epithelial injury. Future therapies targeting epithelial restitution in EoE could decrease the need for immune modulation and steroid therapy.

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 human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

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

BACKGROUND: 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. METHODS: We utilized three-dimensional (3D) organoids and xenograft tumor transplantation models to investigate how EtOH exposure influences intratumoral SCC cell populations including putative cancer stem cells defined by high CD44 expression (CD44H cells). RESULTS: Using 3D organoids generated from SCC cell lines, patient-derived xenograft tumors, and patient biopsies, we found that EtOH is metabolized via alcohol dehydrogenases to induce oxidative stress associated with mitochondrial superoxide generation and mitochondrial depolarization, resulting in apoptosis of the majority of SCC cells within organoids. However, CD44H cells underwent autophagy to negate EtOH-induced mitochondrial dysfunction and apoptosis and were subsequently enriched in organoids and xenograft tumors when exposed to EtOH. Importantly, inhibition of autophagy increased EtOH-mediated apoptosis and reduced CD44H cell enrichment, xenograft tumor growth, and organoid formation rate. CONCLUSIONS: This study provides mechanistic insights into how EtOH may influence SCC cells and establishes autophagy as a potential therapeutic target for the treatment of EtOH-associated SCC.

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

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.

Clinical impact of creatine phosphokinase and c-reactive protein as predictors of postgastrectomy complications in patients with gastric cancer.

BACKGROUND: Postoperative complications have been linked to the morbidity and mortality of several cancers. However, predicting whether complications will occur in the early period after surgery or not is challenging. Hence, this study aimed to examine the diagnostic accuracy of serum creatine phosphokinase (CPK) and c-reactive protein (CRP) in predicting the development of postgastrectomy complications. METHODS: We retrospectively analyzed 188 patients with gastric cancer (GC) who underwent gastrectomy. The diagnostic accuracy of serum CPK and CRP was investigated using the areas under the curves (AUC). The CPK ratio was defined as the CPK on postoperative day (POD) 1 to the CPK on a preoperative day. RESULTS: Out of 188 patients, 48 (25.5%) developed postoperative complications. The complications group had a greater operative time (p = 0.037), higher CPK ratio on POD1 (p < 0.0001), and a higher serum CRP level on POD3 (p = 0.001). The AUC for the CPK ratio was 0.772, with an optimal cutoff value of 7.05, whereas that for CRP was 0.659, with an optimal cutoff value of 11.4 mg/L. The CPK ratio on POD1 (p < 0.0001) and the CRP on POD3 (p = 0.007) were independent factors for predicting the development of postgastrectomy complications. The CPK ratio on POD1 and the CRP on POD3 predicted postgastrectomy complications in 41 patients (85.4%). According to combined value of both CPK ratio and CRP level, the positive predictive value and the negative predictive value was 0.70 and 0.829. And sensitivity and specificity were 0.438 and 0.936. CONCLUSION: The CPK ratio on POD1 and the CRP on POD3 after gastrectomy for GC were predictive factors for complication development and may be employed to prevent the development of such complications and improve the prognosis of patients with GC.