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.

Clinical Impact of Early Tumour Shrinkage in Metastatic or Unresectable Oesophageal Cancer Treated with Pembrolizumab plus Chemotherapy.

INTRODUCTION: Metastatic or unresectable locally advanced oesophageal cancer remains a disease with high mortality. More recently, pembrolizumab plus chemotherapy has been indicated as the first-line treatment for those patients, but the predictive factors for treatment efficacy remain controversial. This study investigated the clinical utility of early tumour shrinkage (ETS) and depth of response (DpR) in metastatic or unresectable oesophageal cancer treated with pembrolizumab plus CF therapy. METHODS: ETS and DpR, defined as the percent decreases at the second evaluation and the percentage of the maximal tumour shrinkage during treatment, were measured in 53 eligible patients. The ETS and DpR cut-off values were 20% and 30%, respectively, based on survival outcomes. RESULTS: Twenty-seven patients (51%) were treatment-naïve, while 26 (49%) had received any treatment before initiating pembrolizumab plus CF therapy. The median progression-free survival (PFS) and overall survival (OS) for ETS ≥20% and <20% were 12.7 and 5.5 months and 14.4 and 8.2 months, and 12.7 and 4.9 months and 14.4 and 8.0 months for DpR ≥30% and <30%, respectively. ETS <20% showed early tumour growth, whereas ETS ≥20% had a good response rate with sufficient longer response duration. In addition, an ETS cut-off of 20% predicted the best overall response and was not associated with prior treatment. In multivariable analysis, ETS ≥20% and DpR ≥30% were independent factors of longer PFS. CONCLUSION: Our findings suggest that an ETS is a promising on-treatment marker for early prediction of further sensitivity to pembrolizumab plus CF therapy.

Protein-enhanced feeds after esophagectomy for esophageal cancer attenuate postoperative catabolism: a prospective observational study.

PURPOSE: To evaluate if nutritional supplements can attenuate postoperative catabolism in esophageal cancer patients after radical esophagectomy. METHODS: We analyzed data prospectively from 30 patients who underwent curative esophagectomy for esophageal cancer between August and December, 2019. We compared postoperative nitrogen balance and changes in body composition between patients who received protein-enhanced enteral feeds (PEF) or other enteral feeds (OF). RESULTS: Postoperative enteral feeding was administered safely to all patients. The average nitrogen balance was significantly higher in the PEF group than in the OF group on postoperative days (PODs) 3 (2.41 vs. - 1.50 g, P = 0.002), 4 (3.74 vs. - 0.08 g, P = 0.006), and 5 (3.27 vs. 0.11 g, P = 0.031). The cumulative nitrogen balance in the 7 days after surgery was significantly higher in the PEF group than in the OF group (6.12 vs. - 8.40 g, P = 0.025). The bodyweight loss and lean body mass loss on POD 14 were equivalent in the two groups (bodyweight loss 3.70 vs. 2.87%, P = 0.25; lean body mass loss, 4.34 vs. 1.91%, P = 0.33). CONCLUSIONS: PEF improved the postoperative nitrogen balance significantly in patients who underwent esophagectomy.

Are Incidental Minute Pulmonary Nodules Ultimately Determined to Be Metastatic Nodules in Esophageal Cancer Patients?

PURPOSE: Esophageal cancer patients may simultaneously have resectable esophageal cancer and undiagnosable incidental minute solid pulmonary nodules. While the latter is rarely metastatic, only a few studies have reported on the outcomes of such nodules after surgery. In this retrospective study, we assessed the incidence of such nodules, the probability that they are ultimately metastatic nodules, and the prognosis of patients after esophagectomy according to the metastatic status of the nodules. METHODS: Data of 398 patients who underwent esophagectomy for resectable esophageal cancer between January 2012 and December 2016 were collected. We reviewed computed tomography (CT) images from the first visit and searched for incidental minute pulmonary nodules <10 mm in size. We followed the outcomes of these nodules and compared the characteristics of metastatic and nonmetastatic nodules. We also assessed the prognosis of patients whose minute pulmonary nodules were metastatic. RESULTS: Among the patients who underwent esophagectomy, 149 (37.4%) had one or more minute pulmonary nodules, with a total of 285 nodules. Thirteen (4.6%) of these nodules in 12 (8.1%) patients were ultimately diagnosed as being metastatic. Thirteen (8.7%) patients experienced recurrence at a different location from where the nodules were originally identified. Characteristics of the metastatic nodules were not unique in terms of size, SUVmax, or location in the lungs. Two-year and 5-year overall survival rates of patients whose nodules were metastatic were 64.2 and 32.1%, respectively. CONCLUSION: The rate of minute pulmonary nodules which were ultimately metastatic was 4.6%. Our findings suggest that esophagectomy followed by the identification of minute pulmonary nodules is an acceptable strategy even if the nodules cannot be diagnosed as being metastatic on the first visit CT due to their small size.

The role of adjuvant chemotherapy in esophageal cancer patients after neoadjuvant chemotherapy plus surgery.

BACKGROUND: Esophageal cancer typically has a poor prognosis. While neoadjuvant chemotherapy (NAC) is reported to be effective for esophageal cancer patients, the prognosis of patients for whom NAC is ineffective remains poor. METHODS: In total, 113 patients with thoracic esophageal squamous cell carcinoma who were treated between January 2006 and December 2015 were enrolled. These patients received NAC followed by radical surgery and had three or more pathologic positive lymph nodes. The effectiveness and feasibility of adjuvant chemotherapy (AC) were evaluated. RESULTS: Forty patients received AC (AC(+) group) and 73 patients did not (AC(-) group). Two-year relapse-free survival (RFS) rates of the AC(+) and AC(-) groups were 30.0% and 28.8%, respectively (p = 0.47). These patients were further divided into two subgroups, i.e., those with 3-6 positive lymph nodes (3-6 subgroup) and those with ≥ 7 positive lymph nodes (≥ 7 subgroup). Within the 3-6 subgroup (72 patients), 2-year RFS rates of the AC(+) and AC(-) groups were 38.5% and 33.9%, respectively (p = 0.31). Within the ≥ 7 subgroup (41 patients), 2-year RFS rates of the AC(+) and AC(-) groups were 25.9% and 7.1%, respectively (p = 0.04). CONCLUSIONS: AC may offer a significant additional benefit to the prognosis of esophageal cancer patients who have many positive lymph nodes even after NAC.

Correlation between Skeletal Muscle Mass and Adverse Events of Neoadjuvant Chemotherapy in Patients with Gastric Cancer.

PURPOSE: Neoadjuvant chemotherapy (NAC) followed by surgery is a promising treatment strategy for patients with advanced gastric cancer. Severe toxicity associated with the treatment may reduce the dose intensity of chemotherapy, resulting in the effect of chemotherapy being attenuated. Recently, skeletal muscle mass has been reported to be associated with the treatment outcomes of cancer patients. The purpose of this study was to clarify whether pretreatment skeletal muscle mass is a predictor of adverse events as well as the relationship between changes in skeletal muscle mass and adverse events during NAC. METHODS: This study included 41 advanced gastric cancer patients who were treated with NAC followed by surgery. Body composition was assessed before and after NAC. The relationship between the pre-NAC body composition and adverse events was investigated as well as the relationship between changes in body composition and adverse events. RESULTS: Univariate and multivariate analyses revealed that low pre-NAC skeletal muscle mass was the only factor significantly associated with severe diarrhea (p = 0.03). There was no significant difference in body weight before and after NAC, but skeletal muscle mass was significantly reduced after NAC (-5.93 ± 7.69%, p < 0.01). Furthermore, patients who experienced severe diarrhea showed significantly greater relative skeletal muscle decrease than those who did not (p = 0.03). CONCLUSIONS: Pre-NAC low skeletal muscle mass was a useful predictor of severe diarrhea. Prevention of severe adverse events may contribute to maintaining the skeletal muscle mass.

[Interventional Radiology for Intra-Abdominal Abscess after Gastrectomy].

Approximately 20% of patients develop some complications after gastrectomy. These complications should be treated appropriately to achieve a positive outcome. The records of 6 patients with postoperative intra-abdominal abscesses treated with interventional radiology (IVR) were analyzed. The cause of abscess was anastomotic leakage in 4 patients and contaminated surgery after gastric perforation in 2 patients. Intra-abdominal abscesses were detected on postoperative day 12 (median), and an IVR-guided drainage tube was inserted with a median interval of 1 day. The drainage tube was kept in place for 26 days (median), and patients were discharged 6.5 days (median) after drainage tube removal. No patients were converted to open surgery. Early IVR-guided drainage was essential and effective for intra-abdominal abscess treatment after gastrectomy.

[A Case of Resected Gastric Cancer Occurring Simultaneously with Intrahepatic Cholangiocarcinoma after S-1 plus Cisplatin Chemotherapy].

It is sometimes difficult to differentiate between metastatic and primary liver tumors, when the liver tumor occurs simultaneously with a gastric cancer. We encountered a case of resected gastric cancer, which occurred concomitantly with intrahepatic cholangiocarcinoma after S-1 plus cisplatin chemotherapy, in a patient who was previously diagnosed with metastatic liver tumor before treatment. An 80-year-old man was admitted to our hospital because of epigastralgia. Endoscopic study of the upper gastrointestinal tract showed a type 3 tumor at the upper body of the stomach. A plain CT scan showed an irregular, low-density area, which was enhanced by contrast medium in the lateral segment of the liver. We performed an ultrasound- guided needle biopsy, because it was impossible to make a definitive diagnosis by dynamic CT, contrast-enhanced ultrasonography, and MRI. Immunohistochemical analysis for cytokeratin 7/20 resulted in 7 (+)/20 (-) for both the gastric cancer and the liver tumor. Therefore, we diagnosed the patient with gastric cancer, which occurred concomitantly with the metastatic liver tumor, and administered chemotherapy with S-1 plus cisplatin. After 3 courses of the regimen, a reduction in the size of mass was observed in the stomach and the liver. We subsequently performed left hepatectomy and total gastrectomy with lymph node dissection. Microscopic examination revealed the gastric cancer, which occurred simultaneously with the intrahepatic cholangiocarcinoma. The postoperative course was uneventful, and the patient remains well without recurrences.

[Analysis of Esophageal Cancer Patients Undergoing Non-Curative Resection].

The prognosis of esophageal cancer patients who undergo non-curative resection is very poor. Here, we retrospectively analyzed the factors associated with non-curative resection. Thirty-five patients with cT3 or T4 thoracic esophageal cancer treated with neoadjuvant chemotherapy or chemoradiotherapy followed by esophageal resection were included in this study. Among the 35 patients, 27 underwent curative resection (R0 group), while 8 underwent non-curative resection (R1R2 group). A comparison of the clinicopathological factors between groups revealed no significant differences in presurgical factors. The pathological T factor was significantly deeper in the R1R2 group than in the R0 group (p=0.0086). Histopathological response tended to be higher in the R0 group (p=0.055). An accurate preoperative diagnosis of T factor is important.

[Two Cases of Pathological Complete Response with Neoadjuvant Therapy for Advanced Rectal Cancer].

We report the cases of 2 patients with advanced rectal cancer who achieved pathological complete response to treatment with preoperative therapy. Case 1 was a 72-year-old man diagnosed with cT3N1M0 advanced rectal cancer. Neoadjuvant chemoradiotherapy with S-1 (120 mg/day) and radiation (50 Gy/25 Fr) was administered. Eight weeks after the last chemoradiation therapy, we performed laparoscopic Hartmann's operation. Case 2 was a 59-year-old man diagnosed with cT4aN0M0 advanced rectal cancer. He was treated with XELOX plus bevacizumab as neoadjuvant chemotherapy. Four weeks after 4 courses of treatment, he underwent laparoscopic Mile's operation. Neoadjuvant therapy was performed without any complications, and a pathological complete response was achieved in both patients. These cases demonstrated that neoadjuvant therapy can be a promising option for locally advanced rectal cancer.

[Chemotherapy for gastric cancer patients over 75 years of age].

PURPOSE: The efficacy of chemotherapy for advanced or recurrent gastric cancer in patients who were aged over 75 years was investigated. MATERIALS AND METHODS: Progression free survival (PFS) and overall survival (OS) of advanced gastric cancer patients who received first-line chemotherapy with TS-1 plus cisplatin or TS-1 in our hospital from 2009 to 2013 were determined. The patients were divided into two groups: H and L. H group patients were aged over 75 years, and L group patients were aged less than 75 years. RESULTS: Median PFS and median OS of patients in the H and L groups who received TS-1 plus cisplatin chemotherapy were not significantly different. PFS was 77[range, 13-211] days and 139[range, 53-211]days for the H and L groups, respectively(p=0.141), while OS was 523[range, 22-1,030] days and 402 [range, 322-623] days, respectively (p=0.620). Similarly, median PFS and median OS of patients who received TS-1 chemotherapy were not significantly different between the H and L groups. PFS was 103[range, 51-156]days and 152.5[range, 85-278]days for the H and L groups, respectively (p=0.230), while OS was 414 [range, 224-714]days and 605[range, 452-1,077] days, respectively ( p=0.1337). CONCLUSION: PFS and OS were not significantly different in younger patients with advanced gastric cancer who received TS-1 plus cisplatin or TS-1 chemotherapy compared to that in similarly treated elderly patients with advanced gastric cancer.

Survival Impact of Inflammation-based Prognostic Scores in Metastatic or Unresectable Esophageal Cancer Treated With Pembrolizumab Plus Chemotherapy.

Pembrolizumab plus chemotherapy has been indicated as the first-line treatment for metastatic or unresectable locally advanced esophageal cancer. However, pretreatment biomarkers for predicting clinical outcomes remain unclear. We investigated the predictive value of inflammation-based prognostic scores in patients treated with pembrolizumab and chemotherapy. The Prognostic Nutritional Index (PNI), C-reactive protein/albumin ratio (CAR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) were calculated before initial treatment in 65 eligible patients with metastatic or unresectable locally advanced esophageal cancer receiving pembrolizumab plus CF therapy, and the relationship between these biomarkers and clinical outcomes was analyzed. The objective response rate (ORR) and progression disease (PD) were observed in 51% and 21% of all patients. Patients with PNI<39 have significantly worse treatment responses than those with PNI≥39 (ORR; 28% vs. 60%, PD; 44% vs. 13%, P=0.020). Progression-free survival (PFS) is significantly associated with the PNI and CAR (P<0.001 and P=0.004, respectively). Overall survival (OS) is associated with PNI, CAR, and PLR (P<0.001, P=0.008, and P=0.018, respectively). The PNI cutoff value of 39 is identified as an independent factor for PFS (odds ratio=0.27, 95% CI: 0.18-0.81, P=0.012) and OS (odds ratio=0.22, 95% CI: 0.08-0.59, P=0.003). Patients with PNI<39 have significantly worse 6-month PFS and 1-year OS than those with PNI≥39 (27.8% vs. 66.7%, 27.2% vs. 81.1%, respectively). In conclusion, inflammation-based prognostic scores are associated with survival in patients treated with pembrolizumab plus CF therapy. Pretreatment PNI is a promising candidate for predicting treatment response and survival.

Short-term Outcomes of Adjuvant Nivolumab After Neoadjuvant Chemotherapy in Patients With Resected Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIM: CheckMate 577 evaluated adjuvant nivolumab therapy after neoadjuvant chemoradiotherapy and surgery for esophageal cancers. However, the efficacy of this treatment in patients who received neoadjuvant chemotherapy remains unknown. This study investigated the short-term outcomes of adjuvant nivolumab therapy in patients with advanced esophageal squamous cell carcinoma post-neoadjuvant chemotherapy. PATIENTS AND METHODS: Out of 956 patients with thoracic esophageal cancer who underwent radical esophagectomy, 227 who exhibited ypN1-3 after neoadjuvant chemotherapy and surgery were included in this study. RESULTS: Among 227 patients, 30 received adjuvant nivolumab and 197 received non-nivolumab adjuvant therapy. The nivolumab group displayed a higher number of lymph node metastases compared to the control group. Patients with ypN1-2 tended to have longer recurrence-free survival (RFS) in the nivolumab group than in the non-nivolumab group (p=0.095). In the propensity score-matched cohort, no differences in patient characteristics were observed. Adjuvant nivolumab therapy significantly prolonged RFS in patients who received neoadjuvant chemotherapy (p=0.013). Patients with ypN1-2 in the nivolumab group had significantly longer RFS than their counterparts in the non-nivolumab group (p=0.001), but not in ypN3 (p=0.784). The 1-year postoperative recurrence rates were 59% for the non-nivolumab group and 24% for the nivolumab group (p=0.007). Nivolumab-related adverse events in patients receiving neoadjuvant chemotherapy were mostly consistent across all grades, while the frequency of increased aspartate aminotransferase (AST) levels was relatively higher compared to CheckMate577. CONCLUSION: Adjuvant nivolumab was more likely to prolong 1-year RFS in patients receiving neoadjuvant chemotherapy, especially in those with ypN1-2, and had acceptable adverse events.

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.

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.

NOTCH3 limits the epithelial-mesenchymal transition and predicts a favorable clinical outcome in esophageal cancer.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human squamous cell carcinomas and is characterized by chemotherapy resistance and poor prognosis associated with the epithelial-mesenchymal transition (EMT). A subset of ESCC displays loss-of-function mutations in genes encoding Notch receptor family members, including NOTCH3. Although Notch signaling regulates EMT in ESCC cells, the role of NOTCH3 in EMT and chemotherapy resistance remains elusive. This study aimed to examine the role of NOTCH3 in EMT and chemotherapy resistance, and determine whether NOTCH3 expression can be used to predict the response to chemotherapy. METHODS: In vitro and in vivo assays were conducted to clarify the contribution of NOTCH3 to chemotherapy resistance. Using specimens from 120 ESCC patients treated with neoadjuvant chemotherapy, we compared the expression levels of NOTCH3 and genes involved in EMT according to the degree of chemotherapy sensitivity. RESULTS: In ESCC cells, chemotherapy resistance was associated with NOTCH3 downregulation and concurrent activation of EMT. RNA interference to silence NOTCH3 resulted in induction of the EMT marker Vimentin (VIM), leading to chemotherapy resistance in ESCC cells. Conversely, ectopic expression of the activated form of NOTCH3 suppressed EMT and sensitized cells to chemotherapy. Results of chromatin immunoprecipitation assays suggested that NOTCH3 may repress transcription of the VIM. CONCLUSIONS: Our findings suggest that NOTCH3 may control chemotherapy sensitivity by regulating EMT. NOTCH3 may serve as a novel biomarker to predict better clinical outcomes in ESCC patients.

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.