Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets.

Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying protumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform for identifying potential therapeutic targets to disrupt the contribution of protumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC and inform the development of therapeutics that target ECM stiffness in EAC.

Long-term outcomes following successful endoscopic treatment of T1 esophageal adenocarcinoma: a multicenter cohort study.

BACKGROUND AND AIMS: Endoscopic eradication therapy (EET) is guideline endorsed for management of early-stage (T1) esophageal adenocarcinoma (EAC). Patients with baseline high-grade dysplasia (HGD) and EAC are at highest risk of recurrence after successful EET, but limited data exist on long-term (>5 year) recurrence outcomes. Our aim was to assess the incidence and predictors of long-term recurrence in a multicenter cohort of patients with T1 EAC treated with EET. METHODS: Patients with T1 EAC achieving successful endoscopic cancer eradication with a minimum of 5 years' clinical follow-up were included. The primary outcome was neoplastic recurrence, defined as dysplasia or EAC, and it was characterized as early (<2 years), intermediate (2-5 years), or late (>5 years). Predictors of recurrence were assessed by time to event analysis. RESULTS: A total of 84 T1 EAC patients (75 T1a, 9 T1b) with a median 9.1 years (range, 5.1-18.3 years) of follow-up were included. The overall incidence of neoplastic recurrence was 2.0 per 100 person-years of follow-up. Seven recurrences (3 dysplasia, 4 EAC) occurred after 5 years of EAC remission. Overall, 88% of recurrences were treated successfully endoscopically. EAC recurrence-related mortality occurred in 3 patients at a median of 5.2 years from EAC remission. Complete eradication of intestinal metaplasia was independently associated with reduced recurrence (hazard ratio, .13). CONCLUSIONS: Following successful EET of T1 EAC, neoplastic recurrence occurred after 5 years in 8.3% of cases. Careful long-term surveillance should be continued in this patient population. Complete eradication of intestinal metaplasia should be the therapeutic end point for EET.

Short and long-term outcomes of prophylactic total gastrectomy in 54 consecutive individuals with germline pathogenic mutations in the CDH1 gene.

BACKGROUND: Germline mutation of CDH1 is rare and leads to hereditary diffuse gastric cancer (DGC). METHODS: Patients (pts) with CDH1 mutation who underwent multidisciplinary counseling followed by open prophylactic total gastrectomy (PTG) by a single surgeon were reviewed. RESULTS: Fifty-four pts with a median age of 41 years (16-70 years) underwent PTG between 2006 and 2021. Median operative time was 161 min, and median hospital stay was 7 days (range 6-12). There were 5 complications (9.2%) within 30 days, and two complications (pulmonary embolism and pancreatitis) required readmission. There were no anastomotic leaks. The pathologic analysis of the first 10 pts included the entire gastric mucosa, revealing a median of 15 foci of DGC (range 5-136). The subsequent 44 pts with more limited analysis had a median of 2 foci (range 0-5), and two pts (3.7%) had no foci identified. Median maximum weight loss was 19%. In long-term follow-up (median 4.6 years) of 20 pts, median global QOL was 2.0 (very good), the majority had persistent difficulty with certain foods or liquids, and all stated they would again elect PTG over surveillance endoscopy. CONCLUSIONS: PTG can be performed safely at high-volume referral centers with very good QOL but nutritional sequelae persist.

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.

Patient-derived organoids as a platform for modeling a patient's response to chemoradiotherapy in esophageal cancer.

3D patient-derived organoids (PDOs) have been utilized to evaluate potential therapies for patients with different cancers. However, the use of PDOs created from treatment-naive patient biopsies for prediction of clinical outcomes in patients with esophageal cancer has not yet been reported. Herein we describe a pilot prospective observational study with the goal of determining whether esophageal cancer PDOs created from treatment naive patients can model or predict clinical outcomes. Endoscopic biopsies of treatment-naive patients at a single tertiary care center were used to generate esophageal cancer PDOs, which were treated with standard-of-care chemotherapy, gamma-irradiation, and newer non-standard approaches, such as proton beam therapy or two small molecule inhibitors. Clinical outcomes of patients following neoadjuvant treatment were compared to their in vitro PDO responses, demonstrating the PDO's ability to mirror clinical response, suggesting the value of PDOs in prediction of clinical response to new therapeutic approaches. Future prospective clinical trials should test the use of pre-treatment PDOs to identify specific, targeted therapies for individual patients with esophageal adenocarcinoma.

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 significance of recurrent gastroesophageal junction intestinal metaplasia after endoscopic eradication of Barrett's esophagus.

BACKGROUND AND AIMS: After endoscopic eradication of Barrett's esophagus (BE), recurrence of intestinal metaplasia at the gastroesophageal junction (GEJIM) is common. The clinical significance of this finding is unclear. We assessed whether recurrent GEJIM is associated with increased risk of subsequent dysplasia and whether endoscopic treatment lowers this risk. METHODS: A retrospective, multicenter, cohort study was performed of treated BE patients who achieved complete eradication of intestinal metaplasia (IM). Postablation follow-up was performed at standard intervals. Recurrent GEJIM was defined as nondysplastic IM on gastroesophageal junction biopsy specimens without endoscopic evidence of BE. Patients were categorized as "never-GEJIM," "GEJIM-observed," or "GEJIM-treated." Endoscopic treatment for recurrent GEJIM was at the endoscopists' discretion. The primary outcome was dysplasia recurrence. Analyses were performed using log-rank tests and Cox proportional hazards modeling. RESULTS: Six hundred thirty-three patients were analyzed; median follow-up was 47 months (interquartile range, 24-69). Most patients (81%) had high-grade dysplasia or intramucosal adenocarcinoma before treatment. Dysplasia recurrence was 2.2% per year. GEJIM-observed patients had the lowest rate of recurrence (.6%/y) followed by GEJIM-treated (2.2%/y) and never-GEJIM (2.6%/y) (log-rank P = .07). In multivariate analyses, compared with never-GEJIM, the risk of dysplasia recurrence was significantly lower in GEJIM-observed patients (adjusted hazard ratio, .19; 95% confidence interval, .05-.81) and not different in GEJIM-treated patients (adjusted hazard ratio, .81; 95% confidence interval, .39-1.67). Older age and longer initial BE length were independently associated with recurrence. CONCLUSIONS: Recurrent GEJIM after endoscopic eradication of BE was not associated with an increased risk of subsequent dysplasia. Future studies are warranted to determine if observation is appropriate for this finding.

Generation and Characterization of Patient-Derived Head and Neck, Oral, and Esophageal Cancer 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 (IC50 ) Support Protocol: Production of RN in HEK293T cell conditioned medium.

Depletion of CD4 and CD8 Positive T Cells Impairs Venous Thrombus Resolution in Mice.

Resolution of deep venous thrombosis involves coordinated inflammatory processes. T cells regulate inflammation in vivo and modulate vascular remodeling in other settings, but their role in venous thrombus resolution remains undefined. To determine the role of T cells in venous thrombus resolution in vivo, stasis induced thrombi were created by vena cava ligation in outbred CD-1 mice. CD4 and CD8 positive T cells, as determined by flow cytometry, were present in thrombi both during thrombus formation and resolution. Depletion of the CD4 and CD8 positive T cells by antibody treatment selectively impaired thrombus resolution compared to animals treated with isotype control antibodies, without an effect on venous thrombus formation. Quantitation of intra-thrombus macrophage numbers, fibrinolytic marker expression, and gelatinolytic activity by zymography revealed that T cell depletion decreased the number of macrophages, reduced the expression of fibrinolytic marker urokinase plasminogen activator (uPA), and decreased the activity of matrix metalloprotinease-9 (MMP-9). These data implicate CD4 and CD8 positive T cells in functionally contributing to venous thrombus resolution, thus representing a potential therapeutic target, but also underscoring potential risks involved in T cell depletion used clinically for solid organ and hematopoietic transplantation procedures.

Oncogenic Transformation of Human-Derived Gastric Organoids.

The culture of organoids has represented a significant advancement in the gastrointestinal research field. Previous research studies have described the oncogenic transformation of human intestinal and mouse gastric organoids. Here we detail the protocol for the oncogenic transformation and orthotopic transplantation of human-derived gastric organoids.

CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium.

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44-deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7-days post-injury. Human-derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14-20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG-SGM3 (NRGS) mice harboring human-derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7-days post-injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9-expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human-derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prospective assessment of the CryoMaze procedure with continuous outpatient telemetry in 136 patients.

BACKGROUND: Only 40% of patients with atrial fibrillation (AF) undergoing cardiac surgery are treated with surgical AF correction. We prospectively studied endocardial cryoablation of the Cox-maze III lesion set following prespecified rhythm assessment with outpatient telemetry. METHODS: Between 2007 and 2011, 136 patients underwent surgical AF correction using an argon-powered cryoablation device. Patients wore continuous electrocardiogram monitoring prior to and at 6, 12, and 24 months after surgery. The average length of monitoring was 6.5±1 days prior to surgery and 11±4 days at each time point after surgery. Patients were assessed for cardiac rhythm, interval cardioversion or ablation procedures, pacemaker placement, and the use of warfarin or antiarrhythmic medications. The primary endpoint of this study was freedom from AF at 1 year. RESULTS: Mean patient age was 66±12 years, 50% (69 of 138) were male and 41% (55 of 134) had persistent AF. CryoMaze was done in conjunction with mitral valve operation in 95% (131 of 138) and other procedures in 41% (56 of 138). Follow-up was 96% complete at 1 year and 90% at 2 years. Freedom from AF was 76% at 1 year. Perioperative mortality and stroke rates were both 1.5% (2 of 138). Perioperative pacemaker implantation was required in 7% (9 of 136). In univariate analysis, younger age, female gender, decreased height and weight, smaller preoperative and postoperative left atrial diameter, intermittent AF, and freedom from AF at discharge were associated with freedom from AF at 1 year. Actuarial 2- and 4-year (Kaplan-Meier) survival were 93% and 80%, respectively. CONCLUSIONS: The CryoMaze procedure is safe and is associated with 76% freedom from AF at 1 year.

Pregnancy among women surgeons: trends over time.

BACKGROUND: Women compose half of all medical students but are underrepresented in the field of general surgery. Concerns about childbirth and pregnancy during training and practice are factors that may dissuade women from electing a career in surgery. OBJECTIVE: To assess experiences related to childbirth and pregnancy among women general surgeons. DESIGN: Survey questionnaire. SETTING: Self-administered survey sent individually to women surgeons in training and practice. PARTICIPANTS: Women members of the Association for Women Surgeons or the American College of Surgeons who graduated from medical school and practice general surgery or a general surgery subspecialty. MAIN OUTCOME MEASURES: Descriptive data on the timing of pregnancy and perception of stigma attending childbirth and pregnancy as experienced by women surgeons, according to date of medical school graduation (0-9 years since graduation, 10-19 years, 20-29 years, and ≥ 30 years). The survey response rate was 49.6%. Trends over time were evaluated using comparisons of proportions and the Cochrane-Armitage trend tests across age cohorts. RESULTS: The perception of stigma associated with pregnancy during training remained large but decreased from 76% in the most remote cohort to 67% in the most recent graduation cohort (P<.001). External influences, even women resident colleagues, were perceived as evincing negative instead of encouraging attitudes toward childbearing during residency, though less so than men, both resident colleagues and faculty. Frequency of pregnancy and pregnancies earlier in training increased over the time cohorts. CONCLUSIONS: The number of women general surgeons becoming pregnant during training has increased in recent years; however, substantial negative bias persists. Although the overall magnitude of perceived negative attitudes is greater among male peers than female peers and among faculty than peers, even women residents hold negative views of pregnancy among their colleagues during training. More than half of all women surgeons delay childbearing until they are in independent practice, post-training. Surgical residents and faculty of both sexes exerted negative influences with regard to consideration of childbearing. There was also a trend toward increased childbearing in more recent graduates.